Datasets:

claran

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modular-s2orc

like 1

2020-12-10T09:04:12.441Z

1972-04-01T00:00:00.000Z

237235376

s2

Ruminal Bacterial Degradation of Benzo(b)-thien-4-yl Methylcarbamate (Mobam) and Effect of Mobam on Ruminal Bacteria Mixtures of ruminal bacteria degraded benzo(b)thien-4-yl methylcarbamate (Mobam) to 4-hydroxybenzothiophene, CO2, and polar product(s). The metabolite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrin-hydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3′,6′-diacetate. lite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrinhydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3', 6'-diacetate. Benzo(b)thien-4-yl methylcarbamate (Mobam) is an effective pesticide that exhibits broad spectrum insecticidal activity coupled with an apparent low mammalian toxicity (2,7). Ruminants administered "IC-Mobam as single oral doses excreted 87 to 96% of the 14C dose via the urine in 24 hr (10). Cleavage of the methylcarbamate moiety of Mobam was evident in the production of two primary metabolites, 4-benzothienyl sulfate and 4-benzothienyl sulfate-i-oxide. The purpose of this investigation was to determine whether ruminal bacteria degrade Mobam. The effect of Mobam on ruminal bacteria was also considered. MATERIALS were obtained by stomach tube from calves (>70 days of age, free of ruminal ciliated protozoa) maintained on a pelleted ration of alfalfa, grain, and wheat bran (10:5:1) (13). Bacteria from the ruminal samples were concentrated by centrifuging the contents at 3,000 x g for 8 min and then centrifuging the resulting supernatant fluid at 13,000 x g for 20 min at 5 C. The sedimented bacterial cells were made up to one-fourth the original volume with basal medium (14) and incubated with "4C-Mobam (100,000 to 250,000 dpm/ml of medium with Mobam concentration adjusted to 72 ug/ml). Preparations of cell suspensions of pure cultures were obtained from each culture incubated in RFTY liquid medium for 48 hr at 39 C. The cultures were centrifuged, diluted in basal medium (14), and incubated with "C-Mobam as above but for 18 hr. Cell densities for each culture were diluted so that a 1:10 dilution of 745 the incubating cell preparation had an absorbancy reading of 0.4 at 600 nm in 1.2-cm-diameter cuvettes. Assays. Thin-layer chromatograms were prepared in the laboratory with Silica Gel G (250 ulm thick, Brinkmann) on glass plates (5 by 20 by 0.35 cm) and spotted with "4C-material. The TLC plates were developed ascendingly with hexane-ethyl acetateacetic acid (70:30:2) and scanned for "4C distribution (14). Samples were dissolved in dioxane or toluene (12) scintillation solution. The dioxane formulation consisted of 7.0 g of PPO (2,5-diphenyloxazole), 100 g of naphthalene, 300 mg of POPOP [1,4bis-2-(5-phenyloxazoyl)-benzene], and enough reagent-grade dioxane to bring the volume to 1 liter. Counting of radioactivity was done with a liquid scintillation counter. Counting efficiencies were determined by internal standardization with toluene-1-"4C or by channels ratio standardization. Counting times ranged from 2 to 30 min, depending on the level of radioactivity in the samples. Radioactive "CO2 produced in "4C-Mobam culture experiments was monitored as previously described (12). Gas production of ruminal bacteria in the presence of Mobam (100 to 500 ug/ml) was determined manometrically (14). Bacteria were assayed for carboxylesterase activity by incubating the preparations with 1-naphthyl acetate (NA) for 15 min at 39 C. The reaction was stopped by addition of 10% lauryl sulfate solution, and the 1-naphthol produced was coupled with fast garnet GBC (CI 37210, 4-amino-3, 1'-dimethyl azobenzene) and read photometrically at 560 nm (9). Similarly, fluorescein-3',6'-diacetate (FDA) was used as a substrate for bacterial carboxylesterases, with fluorescein production determined with a spectrofluorometer (4). Protein content of sonically treated bacterial extracts was determined by the method of Lowry et al. (8), with bovine serum albumin (Sigma Chemical Co.) as the standard. Metabolite detection. Bacterial cultures containing "C-Mobam products were extracted with methylene chloride. The extract was evaporated to dryness, taken up in methanol, and applied to a column of Sephadex LH-20 (0.9 by 40 cm) in methanol. The column was eluted with methanol, and the radioactive fractions were analyzed by TLC and gasliquid chromatographic (GLC) techniques. Metabolites were recovered from TLC plates by stripping "IC-containing bands and eluting with methylene chloride. The "C material was taken to dryness under N, and derivatized with 1 gliter of pyridine and 100 Mliter of acetic anhydride. The sample was then analyzed by GLC on an instrument equipped with a "C monitor and effluent splitters to facilitate simultaneous flame ionization detection and trapping with glass capillary tubes (14). A 2% SE-30 Chromasorb W (60/80 mesh) column [6 ft (1.8 m) by 4 mm] programmed from 100 to 200 C at 5 C/min was used. Injection port and detector temperatures were set at 300 C. Helium (55 ml/min) was used as a carrier gas. Metabolites trapped were analyzed by infrared and mass spectrophotometric analysis as previously reported (14). Culture techniques. Effect of Mobam on growth of mixed ruminal bacteria was determined as follows. Gauze-strained ruminal contents (0.1 ml) were inoculated, in duplicate, into 13-mm cuvettes each containing 4.9 ml of RFTY medium under CO, with either 0, 2, 10, 50, 100, or 200 Mg of Mobam/ml of medium. The cuvettes were flushed with CO, sealed with neoprene stoppers, and incubated at 39 C for 24 hr. Absorbancy readings were taken at 600 nm. Readings (at 0, 4, 8, and 24 hr of incubation) were compared with uninoculated medium containing the appropriate concentrations of Mobam. Mixtures of bacteria from ruminal fluid (300 ml) and newly isolated ruminal bacterial strains (see below; no. 29, 53, and 88, each grown 48 hr in 300 ml of RFTY liquid medium) were separately concentrated by centrifugation and suspended in 7.5-ml volumes of basal medium (14). These cells were then sonically treated at maximum intensity (Fisher probe model BP-5; generator model CW-5) for 3 min at 5 C and assayed for NA carboxylesterase activity (9). Agar well-diffusion experiments were run with the sonically treated preparations as follows. A medium of 1.5% agar in 0.2 M sodium phosphate buffer at pH 6.8 was poured into petri dishes containing Mobam dissolved in a minimum volume of ethanol. Final concentration of Mobam was 500 Mg/ml of medium. Fifty to 75 Mliters of the sonically treated cell preparations was added to each 8-mm-diameter well cut in the solidified medium. The preparations were incubated at 39 C. At 0, 4, 8, and 24 hr of incubation, separate preparations were flooded with 10% ferric chloride solution and observed for development of dark blue-black zones due to the presence of 4-hydroxybenzothiophene. These sonically treated cell preparations were compared to sonically treated mixedand pure culture-cell preparations treated to temperatures of near 100 C for 5 min or treated with 15% ethanol. In some instances, FDA (100 Mg/ml) was used as substrate. FDA degradation was indicated by development of a fluorescent zone surrounding the wells when viewed under ultraviolet light. "4C radioactivity in fraction 1, when removed from TLC plates and acetylated, was released from the GLC column when the GLC column oven reached a temperature of 145 C. The retention time of the compound compared favorably with an authentic sample of 4-hydroxybenzothiophene (which was acetylated according to the method used for the metabolite). Infrared and mass spectra of the metabolite were identical to the authentic sample. The metabolite was therefore identified as 4hydroxybenzothiophene. The "4C-compound in fraction 2 was degraded during GLC. It had an infrared spectrum identical with high-purity Mobam. 4C-Mobam degradation. Degradation of "4C-Mobam by mixed-ruminal bacterial preparations is shown in Table 1. Carbonyl-labeled Mobam was progressively degraded with release of 30.7% of the total "4C as "CO2 in 18 hr. Mobam, at RF 0.27 on TLC, accounted for 59.8% of the radioactivity, and 7.2% of the "C remained at RF 0 to 0.1. Total "4C recovered in CO2 and methylene chloride extractions was 97.7%. With ["4C-methyl]Mobam, only a trace of "4CO2 (0.9%) was found at 18 hr. Mobam accounted for 62.0% of the "4C, and the remaining radioactivity (15.1%) remained at the origin. Total "4C recovered was 78.0%. Attempts to account for the loss of the methyl "C were not successful. With "4C-RL-Mobam, no "CO2 was produced, and 33.8% of the "C co-chromatographed with 4-hydroxybenzothiophene at RF 0.49. Remaining "C (65.1%) was at the same RF as Mobam. Total "C recovered was 98.9%. Since no "4C was found at the origin when "C-RL-Mobam was incubated with mixed bacterial preparations but was found when incubated with Mobam labeled in the carbonyl or methyl positions of the methylcarbamate moiety, it indicated that only the methylcarbamate moiety was the source of carbon for production of polar product(s) by the bacteria. These products were not identified. Mobam-degrading bacteria. A number of bacterial isolates from 10-6 and 10-' dilutions of ruminal samples were effective in repeatedly showing zones of hydrolysis in tributyrin emulsion-Mobam medium. Three isolates from this medium were found to degrade Mobam. These obligately anaerobic bacterial strains (no. 29, 53, and 88) are gram-negative motile curved rods that appear similar to isolates of Hobson and Mann (5). These cultures on tributyrin emulsion-Spirit Blue agar medium showed colonies with clearing zones of hydrolysis surrounded by a dark blue coloration. Sonically treated cell preparations of these strains and mixtures of ruminal bacteria (2. 'Remaining 14C in the preparations was methylene chloride extracted and quantitated by liquid scintillation methods. The percentage of 14C recovered was then assigned to the RF values according to thin-layer chromatography (TLC) scanning information for each preparation. WILLIAMS AND STOLZENBERG 159, H-18, and D-32) did not degrade Mobam. Whether these strains could be adapted over a long period of time to degrade Mobam is unknown. With soil bacterial species, adaptation to chlorophenyl-carbamates is necessary before degradation of these substrates occurs (6). Mobam inhibition of bacteria. Manometric experiments with mixtures of ruminal bacteria showed no suppression of endogenous gasses produced in 80 min of incubation in the presence of Mobam concentrations of up to 500 ,ug/ml. Values obtained at the 500-,ug level were 2.86 Aliters of gas produced per min per 38 mg of bacteria (dry weight) from the experimental preparations and 2.65 Mliters of gas produced per min per 38 mg of bacteria (dry weight) from the control preparations (without Mobam). Gas production by the 10 characterized ruminal strains was also not inhibited by Mobam. Growth of mixtures of ruminal bacteria in RFTY liquid medium containing Mobam up to 200 gg/ml suggested that the actively growing members of the mixed population apparently were not suppressed. Evidence that growth could be inhibited by Mobam was demonstrated with a gram-negative, tributyrin-degrading bacillus which was isolated and maintained in Mobam-free medium. This obligately anaerobic ruminal bacterial isolate was inhibited by Mobam and an organophosphate, Phosdrin, as shown in Table 2. Both pesticides at 10-4 M concentrations and above were inhibitory at 72 hr of incubation. This strain (no. 102) was one of only two strains out of ten isolated on Mobam-free medium that was inhibited by Mobam. Strain 53, which was isolated on a medium containing Mobam, as expected, showed a resistance to Mobam at the levels tested in Table 2. Also, strains L-34 and HD-1 showed resistance to these Mobam concentrations. No other strains have been tested at this time. Suppression of ruminal bacterial populations showing zones of tributyrin hydrolysis was demonstrated when numbers of colonies growing in the presence of Mobam (600 ug/ml) were compared to counts growing in the absence of Mobam. At 2 weeks of incubation, 1.2 x 107 colonies/g of ruminal contents showed zones in the presence of Mobam. In comparison, 4.6 x 10' colonies/g of ruminal contents showed zones in the absence of Mobam. Total culturable counts on 0.25% glycerol-2% tributyrin-RFTY-agar medium with and without Mobam, respectively, were 1.4 x 109/g and 1.8 x 109/g of ruminal contents. [On RFTY-agar medium containing glucose, cellobiose, and starch, culturable counts ranged from 7.5 to 14 x 109/g ruminal contents (13)]. When Mobam (500 ug/ml) was incorporated in the agar well preparations with sonically treated preparations of ruminal bacteria, fluorescein production was suppressed. To determine the extent of Mobam inhibition, suspensions of ruminal bacteria were incubated with NA and FDA as shown in Table 3. Mobam, at a concentration of 6.5 x 10-4 M, inhibited 65.5% of the production of 1-naphthol. However, only 5% inhibition was observed with FDA in combination with Mobam at a concentration of 9.7 x 10-2 M. With isolate no. 53, 69.5% inhibition of FDA hydrolysis was observed with Mobam at a concentration of 13.4 X 10-4 M. With isolate no. 29, 35.7% inhibition of FDA hydrolysis with Mobam at 9.7 x 10-2 M was obtained. The levels of Mobam used in these experiments are higher than one might expect in a contaminant of ruminant rations. The rate of Mobam degradation by ruminal bacteria appears to be adequate to detoxify the insecticide. The biological activity of the hydrolysis product (4-hydroxybenzothiophene) is unknown. Data presented on the effect of Mobam on ruminal bacterial endogenous gas production, growth in RFTY liquid medium, and total culturable counts suggested that ruminal bacteria were not inhibited by Mobam. However, colony counts of bacteria showing zones of hydrolysis on tributyrin differential media were reduced in the presence of Mobam, and Mobam inhibited hydrolysis of aromatic ester compounds by pure cultures of ruminal bacteria. The relative importance to rumen metabolism of the bacterial reactions inhibited by Mobam presently cannot be assessed. Further basic knowledge on ruminal bacterial carboxylesterases in relation to water-insoluble aromatic ester compounds and rumen metabolism may be helpful in evaluating Mobam inhibition of microbial reactions.

v3-fos

2019-03-19T13:14:47.361Z

1972-03-01T00:00:00.000Z

82661903

s2

Comparison of Indirect Hemagglutination and Immunodiffusion Tests for Detecting Type II Leukosis (Marek's) Infection in S- and K-Line Chickens 1 The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between S- and K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period. The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between Sand K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period. Type II lymphoid (Marek's) leukosis (8) is common and widespread in poultry. Susceptible (S) and resistant (K) lines of White Leghorn chickens were developed over many years by F. B. Hutt and R. K. Cole of Cornell University by natural exposure to field infection followed by selection on the basis of highest and lowest incidence of leukosis, respectively, in various families (3,6). The response of S-and K-line chickens to artificial challenge with JM virus has been previously established as susceptible and resistant, respectively (3,9). Recently Zacharia and Sevoian (12) reported that birds infected with JM virus developed agglutinins. By sensitizing sheep red blood cells (SRBC) with JM viral antigen or antiserum, they have demonstrated quantitative differences of antigen and antibodies in the circulating blood. The immunodiffusion (ID) technique has been used to detect type II leukosis infections. Kottaridis et al. (7) demonstrated that plasma and bone marrow from infected birds with JM or Connecticut A virus produced double lines against specific rabbit antisera. Antigen from noninfected birds produced a single precipitating line with the same antisera, and the above workers suggested the appearance of an extra precipitin line due to the infection. In other strains of type II leukosis virus (HPRS-B-14 and GA strains), the precipitating an-' Portion of M.S. thesis submitted by Chou C. Hong to the University of Massachusetts, Amherst. tigen in infected kidney tissue culture has also been demonstrated against chicken immune serum by ID technique (2). The present study used the ID and indirect hemagglutination (IHA) tests to determine (i) how soon after exposure each test may be used to detect immunogenic response and viremia in exposure to JM virus, (ii) the comparative relative sensitivity of the two tests in detecting JM infection, (iii) whether there was a corollary between IHA and ID responses to JM virus infections in chickens, and (iv) whether JM antigen and antibody responses were related to host resistance or susceptibility. MATERIALS AND METHODS Experimental design. The general design was experimentally to inoculate with or naturally expose to JM leukosis virus known S and K chickens at 1 day of age and, chronologically to measure in the plasma the sensitivity, rapidity, levels, and duration of antigen and antibody responses by using the ID and IHA tests. One hundred S-line and 100 K-line day-old chicks were each allotted into three JM-infected groups and one control group. Group 1 chicks were infected via air-borne natural exposure, whereas group 2 and 3 chicks were inoculated intraabdominally with 0.25 ml of JM tumor suspension and 0.25 ml of JM-infected duck embryo fibroblasts; [DEF third passage tissue culture, 6 x 104 plaque-forming units (PFU)/ ml], respectively. All chicks were kept in modified Horsfall units for maximal security against crossinfection. Control birds were isolated several miles away at South Deerfield Farm, University of Massa-chusetts. All groups of chicks were identified by wing-band numbers and were fed and managed similarly. Samples of blood from each chicken were drawn via the alar vein into citrated tubes and were centrifuged. Samples were collected weekly from 1 day of age up to the termination of this experiment at 20 weeks postinfection and were tested as fresh plasma. All birds which died during the experimental period were necropsied and recorded up to 20 weeks of age. Preparation of virus inocula. Primary DEF were prepared by conventional trypsinization of decapitated 10-to 12-day-old embryo obtained from a flock of White Pekin ducks in Sterling, Mass. The cell culture (3.3 x 106 cells/ml) was grown in media 199 (Flow Laboratories, Rockville, Md.) supplemented with 10% tryptose phosphate broth, 5% bovine fetal serum, and penicillin, dihydrostreptomycin, and Fungizone with concentrations of 100 units, 100 mg, and 0.25 Ag per ml, respectively. The pH of the medium was adjusted to 7.2 to 7.4 by the addition of sodium bicarbonate. The normal control cultures were incubated at 37 C with a 5% carbon dioxide atmosphere, and the cells were subcultured usually between 3 and 4 days, when monolayer growth became confluent. JM virus (9) inocula were prepared from DEF tissue culture which had been overlaid with JM-infected chicken kidney cells, obtained from a 4-weekold S-line chicken experimentally infected with JM virus via natural air exposure. The same medium was used as described above. The third passage of infected DEF was allowed to grow until the cell sheets peeled and then was harvested in tissue culture fluid to give an approximate 20% cell suspension. The cells were treated with 10% dimethylsulfoxide (DMSO) and stored in sealed glass ampoules in a liquid nitrogen tank. The JM-infected DEF tissue culture was frozen and thawed three times in a dry ice-alcohol solution, and the tissue culture fluid was collected and centrifuged at 1,000 x g for 10 min. The supernatant material was carefully removed, and the sedimented cell debris was discarded. The supernatant material was spun again at 15,000 x g for 1 hr, and the pellet was separated and set aside. The antigens in the infected supernatant material were concentrated by the precipitation method with saturated ammonium sulfate. The fluid was cooled to 4 C and added to an equal volume of saturated ammonium sulfate [(NH4)2SO4 ], and the mixture was homogenized on a magnetic stirrer for 1 hr at 4 C. The precipitate was taken up in the phosphatebuffered saline (PBS) at 4 C overnight to remove the remaining ammonium sulfate. Finally, the dialyzed viral antigen was later combined with pellet, dissolved in PBS (1:1, v/v), and stored in a freezer at -20 C until use. Preparation of JM tumor inocula. The JM tumor suspension inoculum was prepared by using the procedure described by Sevoian et al. (10,11). The virus titer of JM tumor suspension was 104-3 median infective doses (ID,0), as bioassayed in dayold S-line chicks. Preparation of antiserum. JM antiserum was prepared in young adult New Zealand and Dutch Belted rabbits which received a minimum of six injections subcutaneously at weekly intervals. Each dose consisted of 1 ml of JM antigen in DEF mixed with equal volume of incomplete Freund's adjuvant. The rabbits were bled via cardiac puncture for antiserum 10 days after the last injection. Antisera were absorbed with normal DEF and normal chicken tissue powder three times, respectively, and finally with dried fetal bovine sera, and then were centrifuged at 10,000 x g for 1 hr. The absorbed antisera were inactivated at 56 C for 30 min and were kept at -20 C until use. Immunoglobulin was precipitated with 33% saturation of ammonium sulfate. The immunoglobulin precipitate fractions were dialyzed against PBS at 4 C overnight to remove ammonium sulfate. The immunoglobulin fractions were concentrated to 75% of the original volume by dialyzing against Carbowax (Union Carbide and Carbon Co., N.Y.) and stored at -20 C in a freezer. The immunoglobulin was used to sensitize the formalinized tanned sheep erythrocytes for detecting the JM virus levels in the infected chickens. Preparation of formalinized tanned sensitized sheep erythrocytes. The process of formalization was carried out by dialysis method, suspending a dialysis bag filled with 10% sheep erythrocytes in a beaker containing 40% Formalin, pH 6.0. The beaker was gently shaken at 4 C for 2 hr, after which the dialysis bag was released and the Formalin floated over the cells. The mixture was then placed at 4 C for 6 to 8 hr with occasional swirling, after which the fixed cells were washed three times with two volumes of PBS and stored in the freezer at -20 C until use. The fixed-cell suspension (2.5%) was treated with 1/2,ooo tannic acid at 37 C for 15 min and then washed twice in PBS (pH 6.4) solution by lowspeed centrifugation (650 x g). The sediment was resuspended in PBS (pH 6.4) to 2.5% suspension. An equal volume of antigen or antibody was added in the suspension for sensitizing these cells in the water bath at 37 C for 30 min with occasional swirling. After sensitization, the cells were washed twice in four volumes of PBS (pH 6.4), and finally a 0.5% solution of fixed tanned sensitized cell suspension was made up with PBS (pH 6.4). IHA test. Fixed tanned sheep erythrocytes (0.5%) sensitized with either JM virus antigen or rabbit anti-JM serum were used in these trials. The Microtiter system, developed by Cooke Engineering Co., was used. A twofold dilution system was prepared in a Microtiter plate by adding 25 pliters of 1% bovine serum in PBS, pH 6.4, solution as a diluent to the appropriate wells of the plate. The 25gliter microdiluter was placed and rotated in the testing plasma. Then 25 uliters was delivered to the first well, and further dilutions of testing plasma were prepared by transferring 25 juliters from one well to the next down the line. A 25-gliter amount was discarded from the last dilution. Finally, 25 Mliters of 0.5% fixed tanned sheep erythrocytes sensitized with either JM viral antigen or rabbit anti-JM immunoglobulin was added to all wells for detecting 450 APPL. MICROBIOL. on May 5, 2020 by guest http://aem.asm.org/ Downloaded from type II leukosis antibody and antigen response, respectively. The control samples were prepared in the same manner as above. The wells were sealed by transparent tape sealer, incubated in the refrigerator (5 C), and then examined at the end of 12 hr by a test reading mirror. The IHA units were considered the highest dilution of either the antigen or the antibody showing complete agglutination. ED test. The ID test was the petri dish (60 by 15 mm) method performed as described by Crowle (4). Supporting agar was 1.0% Difco agar prepared in 6% sodium chloride that contained 100 units of penicillin and 0.1 mg of streptomycin per ml. Five milliliters of the medium was dispensed into each plate. A central well and six circumferential wells were cut equal distances apart so that the distance from the edge of the center well to that of each surrounding well was 0.5 cm. The positive antiserum or known viral antigen was placed in the central well, and test plasma obtained from infected and control chickens was placed in the circumferential wells. The plates were kept at 37 C in a closed, moist chamber. Plates were observed every 8 hr, and the final readings were made until 72 hr of incubation. RESULTS Leukosis mortality. Mortality resulting from type II leukosis in both K-and S-line chickens during the 20-week experimental period is shown in Table 1. Similar to previous reports (3,11), the K-line had significantly lower mortality (approximately 30%) than the S-line (approximately 80%). In addition, there was a delay of 3 weeks in the initial mortality in the K-line as compared with the S-line. The routes of infection as well as the different inocula used had some influence on the incidence of mortality. Both K-and L-line chickens, when inoculated with the JM-infected DEF, had the lowest total mortality of 20 and 66.6%, respectively. Comparatively, Sline chickens inoculated with tumor suspension had higher mortality (100%) than the same line naturally exposed (82.6%). In contrast, no significant difference in mortality was found between groups of K-line chickens inoculated with tumor suspension or naturally exposed. Detection of antigen and antibody. When JM-infected chicken plasma was tested against rabbit JM antiserum, a precipitin line was observed as early as 8 hr after incubation at 37 C, though more often the precipitin line was observed between 12 and 24 hr of incubation (Fig. 1). However, the final readings were not made until the test plates had been incubated for 72 hr to ensure completion of the reaction. Positive antigen reaction usually consisted of a single precipitin line, but some samples showed double lines when the IHA antigen titers were 6 log2 or above. All attempts to demonstrate antigen in the plasma of control chickens by ID test failed. When the immunodiffusion test was used to determine antibody response in the infected chickens, the precipitin line usually did not appear until after 24 hr of incubation at 37 C. A positive reaction always appeared as one precipitin line. (Fig. 2). No precipitin line was observed in the plasma of the control chickens. Relationship between IHA and ID tests. Table 2 compares the results of IHA and ID tests. Antigen and antibody were detected in ID tests only when IHA titers were 3 to 4 log2 levels or more. Thus the IHA test was the more sensitive of the two tests. Comparison of type II leukosis infection between Sand K-line chickens by ID test. Within the three types of infection, positive antigen reaction was found in 98 to 100% of the S-line and 65 to 80% of the K-line chickens by ID test during the 20-week experimental period. Only 10 to 16% of the S-line and 6 to 7% of the K-line showed the positive antibody reaction during the same period of time. (Table 3). The ID test results showed that the Cornell S-line had higher incidence of antigen or antibody response than the Cornell K-line from 4 to 20 weeks postinfection. DISCUSSION The infected S-line chickens developed higher levels of antigen and antibody titers than did the infected K-line during the early stages of infection from 4 to 8 weeks of age. The line difference became smaller or nonsignificant after that period of time. These observations indicate that the genetically susceptible young S-line chicks may provide better cellular environment for growth or multiplication, or both, of type II leukosis virus as compared with the resistant K-line chicks. The levels of antibody response were proportionally and inversely correlated with antigen titers and the resistance to type II leukosis, respectively. The results of this study suggest that the IHA and ID antibodies do not possess a significant neutralizing or protective ability against type II leukosis. The positive relationship between IHA and immunodiffusion tests for detecting viral antigen and corresponding antibody in infected chickens was observed consistently throughout the experiment. The IHA antigen and antibody titers had reached at least 3 log2 and 4 log2, respectively, before the positive precipitin band could be observed by immunodiffusion test. In this study, IHA test was shown to be more sensitive in detecting infection than was the ID test. This was expected, as Carpenter (1) When IHA antigen titers reached 6 log2 or higher, a double precipitin line could be detected by immunodiffusion test, indicating the presence of at least two distinct antigenic components. One of the bands is undoubtedly of the JM-virus origin, while the other is postulated to be due to virus infection-associated antigen.

v3-fos

2020-12-10T09:04:12.278Z

1972-12-01T00:00:00.000Z

237233622

s2

Klebsiella Biotypes Among Coliforms Isolated from Forest Environments and Farm Produce Samples of water, soil, needles, and bark were collected from three different forest environments and from a pulp and paper mill. In addition, samples of fresh produce were obtained from a local supermarket. All samples were examined for total and fecal coliforms. The counts obtained from the forestrelated samples did not correlate with sample type or location. When 123 isolates were identified biochemically, 71% were Klebsiella, 19% Enterobacter, 8% Citrobacter, and 2% Escherichia. All the Citrobacter, 75% of the Enterobacter, and 65% of the Klebsiella were negative for growth in elevated coliform (EC) broth. All the Escherichia were EC positive. The counts obtained from the fresh produce were generally higher than the forest counts, but the distribution of biotypes was similar. Of the 146 isolates examined 64% were Klebsiella, 14% were Escherichia, 14% were Enterobacter, and 8% were Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of the Klebesiella and 80% of the Escherichia were EC positive. As early as 1929, Edwards (8) noted the similarity in biochemical reactions between Aerobacter aerogenes and Friedlander's bacillus. When Ewing and Edwards (9) proposed a new system of classification for the Enterobacteriaceae, they eliminated the genus Aerobacter and replaced it with Enterobacter and Klebsiella. However, past literature refers to Aerobacter species as being frequent inhabitants of soil and vegetation. Thomas and McQuillin (23) classified at least 73% of the isolates obtained from grazed and ungrazed herbage as aerogenes-cloacae types, and less than 9% as Escherichia coli. Fraser et al. (11) found that the Aerobacter group was present on about 33% of the plants examined, although in their opinion the foliage of field and garden plants, (including trees and shrubs) rarely carried coli-aerogenes bacteria, except when contaminated by animals, insects, or dust. Geldreich et al. (13) examined 152 samples of plants and 40 samples of insects and concluded that typical coliforms of the warm-blooded animal gut contributed a relatively small percentage of the organisms associated with vegetation and insects. They did not attempt to name the nonfecal coliforms they isolated but the indole, methyl red, Voges-Proskauer, citrate (IMViC) reactions listed suggest that more than 50% were Aerobacter types. Papavassiliou et al. (17) examined plant foliage and flowers collected in Greece, found that coli-aerogenes bacteria were seldom isolated from plants grown in uninhabited areas and concluded that they were not native flora. Vegetables and fruits sold in the market in Athens, however, were frequently contaminated with coli-aerogenes bacteria. Geldreich and Bordner (12) have reviewed the subject of fecal contamination of fruits and vegetables destined for market. Nunez and Colmer (16) obtained 384 lactosefermenting isolates from sugar cane and found that 88% of them were IMViC --+ + and would have been classified as Aerobacter aerogenes in the past. By using the classification of Ewing and Edwards (9), they found that 98% of the so-called Aerobacter isolates were Klebsiella and 2% were Enterobacter. Since the major differences between these genera are motility and the presence or absence of ornithine decarboxylase, a similar split might have occurred if these tests had been applied to the isolates of previous workers. Water quality surveys have recently singled out pulp and paper mill wastes (3,6,18,19,22) as major sources of coliform group organisms. In many cases, this is despite the segregation of sanitary waste-treatment facilities. Past studies (14) have identified A. aerogenes as a major component of slime found in pulp and paper 933 mill process streams. However, by using the biochemical classification system proposed by Ewing and Edwards (9), the organisms isolated by Dutka et al. (6) have been identified as Enterobacter, Klebsiella, Escherichia, and Citrobacter, with Klebsiella being the dominant genus. Klebsiella has been associated with upper respiratory tract and hospital-acquired infections, and the epidemiology of Klebsiella pneumoniae has recently been reviewed by Eickhoff (7). Martin et al. (15) have just published their findings from a 3-month study of its epidemiological significance. This investigation was prompted by the fact that a change in nomenclature has resulted in the routine isolation of potentially pathogenic Klebsiella from common environmental systems, which in the past were considered to be the habitat of saprophytic organisms known as Aerobacter. The source or cause of the coliform counts on the fruit and vegetables examined during this study was not the object of this investigation. That subject has been adequately reviewed by Geldreich and Bordner (12). Rather it was the distribution of the coliform group biotypes which was of interest, and the routine exposure of the public to large numbers of such organisms. MATERIALS AND METHODS Source of samples. One series of samples was taken from the Seymour watershed of the Greater Vancouver District, which is a virgin, coastal, evergreen forest, with strictly limited access. The samples of water, soil, bark, and needles were collected in November, 1970, from an area previously remote to humans. A second series of samples was collected from a forested portion of the Nanaimo watershed on Vancouver Island, to which public access has been severely restricted for the last 40 years. Samples of needles and bark were collected in May, 1971, from a public forested area (McMillan Grove Provincial Park on Vancouver Island) and from an integrated pulp and paper mill. Samples of bark were collected from standing trees, from logs hauled by truck from high in the mountains on Vancouver Island, and from logs that had been hauled by truck prior to dumping into the contaminated waters of Alberni Inlet. All samples were collected in an aseptic manner, stored in ice, and returned to the laboratory for testing within 18 hr. Tomatoes and various vegetables were purchased in bulk from a local but nationally represented supermarket early on a Monday morning (August 1971) and returned to the laboratory immediately for processing. The origin, age, and handling procedures prior to purchase are unknown. The samples were homogenized in a Waring blendor at a 1:10 dilution in phosphate buffer (pH 7). Coliform tests. The most-probable numbers (MPN) of total completed and fecal coliforms were determined by the multiple-tube technique (1). Positive tubes of Brilliant Green-bile-lactose broth and elevated coliform (EC) broth were streaked onto either eosin methylene blue (EMB) or MacConkey plates. Identification of isolates. Colonies were picked from either EMB or MacConkey plates and transferred into lactose and EC broths. Lactose-positive cultures were transferred into triple sugar iron agar, sulfide indole motility medium, motility medium, methyl red-Voges-Proskauer medium, Simmon citrate agar, and ornithine decarboxylase medium. Incubation was at 35 C. The isolates were classified by comparing the reactions obtained with those listed by Ewing and Edwards (9) and Fife et al. (10), with emphasis being placed on results from the hydrogen sulfide, ornithine decarboxylase, motility, and citrate tests (HOMoC), as suggested by Closs and Digranes (4). RESULTS Coliform counts. The total completed and fecal coliform counts, expressed as MPN/100 g (wet weight), from the forest samples, are listed in Table 1. All samples of water and soil examined gave a positive coliform count with the highest count recorded being 4,300. However, only one water sample gave a positive fecal coliform count. Positive coliform counts were obtained for all moss, fern, and needle samples taken from the Seymour watershed, whereas hemlock and Douglas fir needle samples taken from the Nanaimo watershed did not have a detectable count. The highest count, 54,000, was obtained from cedar needles taken from the Nanaimo watershed. Positive fecal coliform counts were obtained from the fern sample, one sample of hemlock, and one of spruce needles. However, the counts at 20 to 55 MPN/100 g were low. Table 1 shows that the samples of bark examined gave a highly variable count. Those from the Seymour watershed, regardless of tree type, gave total coliform counts ranging from <20 to >24,000. The samples from the Mc-Millan Grove Provincial Park and the Douglas fir samples from the logging truck had coliform counts below the level of detection. The sample of Grandfir bark taken from the logging truck, gave a count of 130. The samples taken from the log pond, where the logs were floating in sea water, exposed to municipal sewage discharges gave counts ranging from 140 to > 24,000. Fecal coliform counts were confined solely to those bark samples taken from that log pond. The completed coliform counts for the fruits and vegetables, expressed as MPN/100 g (wet weight), are listed in Table 2. They show that all but one of the samples examined gave positive total coliform counts exceeding 2 x 106. To- matoes were the exception with a count of 3,300. The fecal coliform counts listed in Table 2 show that radishes, beets, carrots, and tomatoes gave counts below the minimum number detectable by our procedures, (i.e., less than 200). The leaf lettuce, head lettuce, and whole celery had counts in the vicinity of 400 to 500, whereas the radish tops, beet tops, and carrot tops gave counts between 4,600 and 17,000. The green onions gave a fecal coliform count of 5 x 106 per 100 g (wet weight). Identification of isolates. A total of 149 isolates from the forest environment were picked from EMB plates; 26 were lactose negative and the remainder were classified by their reactions to the IMViC, H2S, ornithine decarboxylase, and motility tests. The probable classification, source, number, and EC reaction of the remaining isolates are listed in Table 3. Numerically, 71% of the isolates were Klebsiella (35% of which were EC positive), 19% were Enterobacter (25% EC positive), 8% were Citrobacter, and 2 were Escherichia, both of which were EC positive. A total of 152 isolates from the fruit and vegetables were picked from MacConkey plates; six failed to ferment lactose, and the remainder were classified by their reactions to the seven biochemical tests used. The probable classification, source, number, and EC reaction of the isolates are listed in Table 4. Numerically, 64% of the isolates were Klebsiella, 14% Escherichia, 14% Enterobacter, and 8% Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of Klebsiella and 80% of the Escherichia were EC positive. DISCUSSION Examination of the data in Table 3 shows that Klebsiella and Enterabacter were obtained from all sources, and Citrobacter from all except soil. Klebsiella and Enterobacter lettuce and the carrots. There was no Citrobacter present on the celery, the carrots, carrot tops, or on the tomatoes. Escherichia was present on the least number of samples. This genus was absent from leaf lettuce, celery, and tomatoes and the underground portions of radishes, beets and carrots, but was present on the tops of radishes, beets, and carrots. Interestingly enough, the underground portions of the vegetables were also free of any EC-positive isolates. The extremely high EC-positive count associated with green onions was primarily due to the presence of K. pneumoniae. Shooter et al. (20) found Klebsiella species on 21 of the 121 salads they examined in their study and E. coli nega-posiisotive tive lates Enterobacter Klebsiella were obtained from all types of bark which gave positive counts, and so no species differentiation is listed in the table. The two Escherichia isolates were both obtained from the stream sample taken in the Seymour watershed. No Escherichia isolates were found on the bark of logs taken from the log pond, which receives settled but unchlorinated sewage. Stewart et al. (21) found that a municipal watershed, closed to public entry since 1917, yielded water with a coliform count four to six times that of an adjacent watershed open to recreational activity. It was suggested that the closed watershed had a large population of wild animals that contributed substantially to the results obtained in their studies. Unfortunately, the authors did not classify their coliform isolates by genus. Examination of the data in Table 4 does not demonstrate any specific relationship between a particular genus and a type of produce. Klebsiella was isolated from all sources, and Enterobacter was isolated from all but the head The data presented here indicate that, when seven biochemical tests are applied to lactosefermenting Enterobacteriaceae, in conjunction with the classification system of Ewing and Edwards (9), approximately 70% of the isolates from the forest and 65% from fresh produce are classified as Klebsiella, and predominantly as K. pneumoniae (Table 5), an organism which is currently considered to be pathogenic. Before the Enterobacteriaceae was reclassified, these organisms probably would have been known as A. aerogenes, a name with nonpathogenic connotations. Cowan et al. (5) recognized this similarity in biotypes and retained the name K. aerogenes for those biotypes which were IMViC --+ +, but HOMoC ---+, a nomenclature which was supported by Bascomb et al. (2) in their numerical classification of the tribe Klebsielleae. Both these authors classify K. pneumoniae as being IMViC -+ -+, and only 10 of the 181 Klebsiella isolated in this study fit that definition. Such organisms would tentatively be classified as K. ozaenae according to Fife et al. (10). In their study of the tribe Klebsielleae, Fife et al. (10) recognized that not all biotypes fit the classic definition, and they listed percentage values to indicate frequency. In our study no one combination of reactions for the seven tests used described the majority of the Enterobacter biotypes. The classic IMViC --+ + reaction described 30 out of the 43 isolates, whereas the HOMoC classification of -+ + + suggested by Closs and Digranes (4) (Table 5). All but 6 of the isolates were HOMoC ---+. The results of this study show that a majority of the coliform group isolates taken from forestrelated samples and from fresh produce were Klebsiella and predominantly K. pneumoniae according to the classification scheme of Ewing and Edwards (9). Previous classification schemes would probably have assigned these organisms to the genus Aerobacter. The Ewing and Edwards classification system evolved through the examination of large numbers of Enterobacteriaceae which were primarily clinical isolates. It remains to be seen if the Klebsiella isolated from the nonmedical environment are of equal pathogenicity to those isolated from clinical sources. [Some data indicate that the mouse pathogenicity of environmental and hospital isolates are indistinguishable, whereas the latter show patterns of multiple antibiotic resistance (J. M. Matsen, personal communication).] Serological typing and antibiotic susceptibility testing of our isolates are planned.

v3-fos

2019-03-19T13:13:03.383Z

1972-12-01T00:00:00.000Z

237230762

s2

Quantitation of the Bioenergetics of a Tuberculosis Infection in Chicks Interaction of an avian tuberculosis infection with a known metabolizable energy yield of dietary corn oil in chicks was used to quantitate total host energy expenditure necessitated by the infectious process. Three trials in which two doses of inoculum were used resulted in mild and severe involvements. Trial 1 (mild) indicated that 6% and trials 2 and 3 (severe) that 96 and 93% of the energy supplied by known quantities of corn oil were utilized by the tuberculosis process. In the birds given the low level of inoculum, the degree of tuberculosis involvement, as measured by increased liver size, was correlated with increased total quantities of hepatic ribonucleic acid, monoglycerides, free fatty acids, free cholesterol, and glucose. All of these effects were observed prior to manifestations of clinical symptoms or failure of the chicks to consume all food offered. A recent comprehensive review (2) on the relationship between lipid metabolism and infectious illness emphasizes that it is not yet possible, from the data available, to define specific changes in the rates of synthesis, mobilization, peripheral utilization, or degradation of lipid moieties during infection. Research in our laboratories related to protein metabolism (9,10) also has indicated the need to assess the role of energy utilization during infectious processes. An avian model for estimating the metabolizable energy of foodstuffs described by Squibb (11) was believed to offer the possibility of casting an infection, avian tuberculosis (TB), into direct interaction with the known metabolizable energy yield of a dietary fat. This combination of experimental models, it was reasoned, would permit the first estimation of total host energy expenditure necessitated by an infectious process. The results of three trials with such a model are reported here, along with a description of biochemical changes in nucleic acids and several lipid moieties in the liver, one of the principal target organs during disseminated avian TB. MATERIALS AND METHODS The procedure for determining the metabolizable energy of foodstuffs with an avian model (11) served as the basis of determining the dietary energy expended during avian TB. A rigidly controlled daily feed restriction schedule was used to restrict chick growth to 40 to 60% of the genetic potential normally achieved with ad libitum feeding. The restricted diet contained adequate quantities of balanced proteins, but was low in total calories. Within the limits used, growth of the chicks exhibited a highly reproducible, direct linear relationship to the availability of calories in the diet. The gain in weight of chicks over a 12-day interval, when compared with an established reference curve (11), was used to provide a readout of total energy expenditure. The model specifically monitors growth response from 9 to 21 days of age (11). Therefore, 1-day-old broiler chicks averaging 40 g in weight were randomly assigned upon arrival from the hatchery to control and TB-infected groups. The chicks in the latter category were injected that day via the left jugular vein with an 0.5-ml suspension of Mycobacterium avium strain Kirchberg. The bacterial suspension used for injection was a 1:10 dilution of a 10to 14-day-old culture in Tween-albumin medium, adjusted to an optical density of 0.45 or 0.60 at 550 nm (7). All birds were kept in separate but similar quarters with constant artificial light and a temperature of 22 2 C. A commercial diet and water were provided ad libitum for the first 8 days after delivery and inoculation of the chicks. The procedure from 9 to 21 days, the end of a trial, was the same as in the published model (11). Quantitation of the amount of energy used was based upon the concept that: (i) by feeding restricted amounts of a balanced 26% protein, low-energy diet to rapidly growing chicks, protein synthesis (one of the principal components of rapid growth) was limited by a single variable, the need for added energy; (ii) any change in energy utilization due to the presence of the infection 924 would alter the growth-related yield of metabolizable energy; and (iii) the percentage change in metabolizable energy values could serve as the basis for quantitating the amount of energy expended because of the infectious process. The 26% protein, low-energy basal ration contained: soybean oil meal (50% protein), 40%; fish meal (60% protein), 5%; alfalfa meal, 4%; ground corn, 35%; complete mineral mix, 4%; vitamin mix, 2%; and, to limit calories, non-nutritive fiber, 10%. To restrain protein synthesis, growth was deliberately held to approximately 55% of that expected under ad libitum feeding by providing each chick in each experimental group with a total of 244 g of diet (11). Since protein intake and body reserves were calculated to be more than adequate, the growth of the controls fed the low-energy basal diet became the point of reference for determining metabolizable energy utilization. Substituting 10% corn oil (known to contain metabolizable energy of 9 kcal/g) for the 10% non-nutritive fiber in the basal diet fueled growth to the extent of the availability of the calories in the corn oil. As previously demonstrated, the resulting synthesis of all components of body mass (weight gain) was linear and, under normal conditions, could be equated to metabolizable energy by reference to the previously published curve (11). At the end of a 12-day trial, by calculation, each chick on the basal ration received a total of 927 calories, and those which received the substitution of 10% corn oil for the inert material in the basal ration received 1,147 calories. A chick given the same basal diet on ad libitum feeding will consume approximately 1,685 calories during this 12-day period and weigh approximately 445 g at 21 days of age. It was assumed that, if the early tuberculous process placed an energy demand on the chick, with the interaction limited to the 220 calories supplied by the corn oil, a comparison of the metabolizable energy yield of corn oil in the control and infected groups would serve as the basis for calculating energy expenditure due to the TB. The assumption required that the infectious process under investigation be regulated so that its energy demands would be less than the 220 calories supplied by the 10% corn oil. An illustration is presented in the Results. Trial 1 consisted of the following groups: basal diet; basal diet plus 10% corn oil, with and without TB (optical density, 0.45); and basal diet plus 15% corn oil, with and without TB (optical density, 0.45). Although not a part of the model, the latter two groups were included to evaluate adequacy of the host reservoir of free amino acids while, at the same time, recording the effect of a higher intake of energy on the TB process. The TB groups had double the number of chicks to allow for possible mortality and to permit classifying biochemical data of the individual chicks in the TB groups according to low or high degrees of severity (12). At the end of the trial, all chicks were weighed and killed, and their livers were weighed and frozen for later analyses. Hepatic nucleic acid and protein content were determined according to modifications of Wannemacher et al. (13); hepatic lipid fractions were separated according to Biezenski et al. (3); and the method of Goodwin (4), which uses the o-toluidine reaction, was used to determine hepatic glucose. Trials 2 and 3 were replicates of trial 1 except that: (i) the chicks were inoculated with a greater corcentration of TB organisms (optical density, 0.60); (ii) the 15% corn oil diet was discontinued; and (iii) no biochemical observations were made. RESULTS In trial 1 (Table 1), as expected, the TB inoculum used resulted in a comparatively mild infection. The efficiency of feed utilization (grams of gain in body weight/total feed consumed, i.e., 244 g) in the 15% corn oil control group was somewhat higher than that in both 10% corn oil groups and significantly higher than that in the 15% corn oil TB group. The following calculations of metabolizable energy utilization in the 10% corn oil groups of trial 1 will serve as examples for trials 2 and 3 also. (i) The final weight of basal controls was 190 g. (ii) The final weight of 10% corn oil controls was 245 g, an increase of corn oil controls over basal controls of 129%. According to the reference curve (11), this 129% increase was equivalent to and confirmed the metabolizable energy yield to be 9 kcal/g, i.e., that of the corn oil. (iii) The final weight of the 10% corn oil TB group was 240 g, an increase of TB over basal control of 127%. According to the reference curve (11), this 127% increase was equivalent to a utilization of metabolizable energy of 8.4 kcal/g of corn oil or 0.6 kcal/g less than the corn oil controls (see above). This difference in growth-related yield of metabolizable energy calculates to be 6.6% (0.6 kcal/g divided by 9 kcal/g) and indicates that 15 kcal of the extra 220 kcal provided were expended in some way as a result of the infectious process, which in this illustration was relatively mild. Liver weights, liver/body weight ratios, and biochemical data of trial 1 ( Table 2) were arrayed according to an index of TB involvement previously reported (12), which uses liver weight of tuberculous chicks as an indication of degree of infection. Within each diet, those with the least increase in liver weight were designated group A and those with the greatest increase, group B. Compared to their respective controls, chicks in the B groups had liver weights and liver/body weight ratios which were significantly higher, indicating that the degree of infection was greater in these groups than in the A groups, i.e., those with a low level of infection. The arrayed data of trial 1 showed that in the 10% corn oil group A, with a low level of TB involvement, there were irregular and nonsignificant disease effects on total quantities of nucleic acids and lipid moieties; chicks with greater TB involvement (group B) showed significantly increased total quantities of liver ribonucleic acid (RNA), monoglycerides, free fatty acids, free cholesterol, and glucose. The increased energy input from the 15% corn oil not only increased the foregoing liver components but also total quantities of protein and the diand triglycerides. A comparison of all TB and diet group interactions (Table 2) with the normal 10% corn oil control group showed that the observed increases in total quantities of various hepatic constituents were also related to severity of the infectious process, with the least effect noted for the mild TB-10% corn oil group and the greatest in the TB chicks fed the excess 15% corn oil. In trials 2 and 3 (Table 1), which used a higher inoculum of TB organisms, body weight gains and feed efficiencies were significantly depressed during the infectious process. The expected 9 kcal of metabolizable energy yield per g of corn oil was reduced in chicks with TB of greater severity to 0.4 kcal/g in trial 2 and to 0.6 kcal/g in trial 3. Calculated as above, these reductions in metabolizable energy values were 96 and 93%, respectively. Thus, energy cost of the infection amounted to 211 and 205 calories of the total 220 calories provided by the 10% corn oil, or to 18.4 and 17.8%, respectively, of the total dietary intake of calories. DISCUSSION Avian TB in the rapidly growing chick is an excellent model for quantitative studies of a chronic infection, even though precise control of the exact number of live bacilli to be injected is not yet available (6). When our standardized method for estimating inoculum dose is used, miliary TB is produced consistently, with the vast majority of tubercles being found in the liver and spleen; regardless of inoculum size, the earliest tissue reaction occurs in the liver during the second week (6). Foci composed of several lymphocytes appear first and become admixed with small epithelioid cells during the third week, the time of termination of the present studies. If the disease process is studied further, weight loss of the chicks may begin; the tubercles increase in size, displacing normal tissue; tubercles eventually develop central necrosis and a peripheral deposition of amyloid; skeletal muscles become severely wasted, and death occurs in 5 to 9 weeks, depending on inoculum size (6). A progressive gain in weight of the liver results from the increase in size and number of tubercles. This initial early reaction, in terms of protein synthesis, was found to be largely anabolic and fueled in the initial stages by nutrients in the diet (12). As the disease progressed beyond the incubation stage, the apparent toxicity of the TB led to depressed diet intake, thus forcing acute catabolism of muscle and other protein stores of the body to continue the reaction. In earlier studies, attempts to aid host resistance by increasing dietary protein (12) and fat (7) above normal requirements resulted in an increased magnitude of the catabolic reactions as well as higher mortality rates. Present data agree with these earlier observations. The recent report of Squibb (11), describing a new model for determining the metabolizable energy content of foodstuffs, served as the basis here for the quantitation of the energy cost of a TB infection. In attempting to quantitate energy utilization in TB-infected chicks, two distinct protein synthetic processes must be considered, namely, those applicable to the ongoing anabolic processes of normal growth, and, second, the superimposed effects on host metabolism associated with, or initiated by, the invading bacteria. When the corn oil reference standard, having 9 kcal/g of metabolizable energy, was substituted for the 10% inert material of the basal diet, growth response (increase in body mass) of the controls ranged from 129 to 131% over the basal groups, within the published experimental error of the model (11). When the tuberculous process was interacted with this known amount of added energy (10% corn oil), the infected birds failed to attain predicted body weight. The magnitude of the growth depression was related to the intensity of the infection; i.e., of the energy provided by corn oil, 94% was utilized for some purpose other than growth during the severe disease (trials 2 and 3), and 6% was similarly used during the mild involvement (trial 1). We recognize that the disease process utilizes energy substrates other than that supplied by corn oil. However, present data relate only to the basic assumption made here: with all other experimental conditions identical, any difference in the known metabolizable energy yield of corn oil can be charged to the stress of the infection; i.e., failure to attain predicted body weight is due to the competition of body growth requirements and the disease process. Although the present report provides, during the period studied, a proximate quantitation of the total energy utilized by the TB-infected host, there is no way to equate such data to the precise amount of overt disease within each bird. Therefore, to relate response to severity, two infecting doses of TB inoculum were used to produce a range of disease effects varying from mild to severe but still remaining within the fiducial limits of the established metabolizable energy reference curve (11). In this regard, the maximal substitution of the reference corn oil for the 10% inert material was further adjusted in one trial to 15% to demonstrate that the regulated intake of the balanced 26% dietary protein had the potential to provide an excess of free amino acids for additional growth. The fact that 4% additional growth was observed in the noninfected 15% corn oil groups confirmed the availability of sufficient free amino acids in the basal ration. Biochemical calculations were obtained from data arrayed according to a proximate index of involvement (Table 1). Artificial infection of groups of animals, even though accomplished with measured concentrations of an inoculum, seldom, if ever, result in an entirely uniform course or severity of disease. Averaging data of widely diverse degrees of infection masks changes in biochemical response, especially when the disease is quite mild (8). The data of the first trial demonstrated that even though the TB inoculum used resulted in a comparatively mild infection during the period of disease studied, liver size and total quantities of RNA, protein and lipid fractions were increased in those chicks with the greatest TB involvement (B groups) compared to those in the mild category (A groups). Serum hyperproteinemia and similar increases in liver size and total quantities of nucleic and free amino acids have been observed in previous studies (12,14). These changes may be associated with increased nitrogen retention in other infections of chicks (5) or humans (1). On the whole, these early increases in nucleic and free amino acids and lipid fractions do correlate with an increase in the rates of synthesis of certain intracellular hepatic and extracellular serum proteins. As stated previously, any such diversion of necessary precursor materials, as well as the molecular machinery required for the synthesis of proteins specifically involved in host responses to the infectious process, are all components of or relate to weight changes, which is the basis of the metabolizable energy model employed here. However, since such newly synthesized proteins contribute to total body weight in the present model, there is the probability of some degree of underestimation of energy costs due to the infections. It is recognized that the restricted feeding regimen required by the Squibb model may in itself affect the progress or intensity of the infection. However, the increases in liver size and total quantities of tissue protein and RNA noted here occurred prior to any voluntary restriction of food intake, confirming observations made under ad libitum feeding (12).

v3-fos

2020-12-10T09:04:12.758Z

1972-11-01T00:00:00.000Z

237231537

s2

Polysaccharide Produced by Anacystis nidulans: Its Ecological Implication An extracellular polysaccharide from Anacystis nidulans was extracted from cell-free medium. Analysis showed that the polysaccharide consisted of glucose, galactose, and mannose in the ratio of 60:14:20. The production of polysaccharide depends on the age of culture, the growth temperature, and the form of nitrogen available. Several freshwater green and blue-green algae are known to excrete varying amounts of organic compounds into their surrounding environments. Polypeptide and free amino acid liberation by a species of Anabaena has been reported by Fogg (9). Organic acid production from species of Chlamydomonas has been studied by Allen (2). Extracellular carbohydrate polymer production has been reported for Anabaena cylindrica, Nostoc muscorum, and Palmella mucosa (4,5,12). Almost all of the common monosaccharide units have been identified in these polymers. Type and amount of polysaccharide production depend on the species employed, and quantitative data are available from papers of Lewin (11) and Moore and Tischer (13). Species of green algae studied by Lewin (11) produced extracellular polymer ranging from 3 to 113 mg per liter, whereas mucilaginous species analyzed by Moore and Tischer (13) produced the polymers in concentrations from 174 to 557 mg per liter of growth medium. This study was undertaken to determine the extent and composition of polysaccharide production by the blue-green alga Anacystis nidulans. Some of the nutritional factors which might influence the polymer production were also examined because of their potential significance during algal blooms in Lake Erie. MATERIALS AND METHODS Culture and media. A bacteria-free, unialgal isolate of A. nidulans was cultured in the medium "c" of Kratz and Myers (10) composed of: MgSO4 .7H2O, 0.25 g; K2UPO4, 1 g; Ca(NO3)2 .4H20, 0.025 g; KNOS, 1 g; NaH5CO7.2H20, 0.165 g; Fe2(SO4J86H20, 0.004 g; and distilled water to make 1 liter (final pH 7.5). Cultures were grown in 3-liter Fernbach flasks in a Psychrotherm shaker incubator (100 rev/min) at 25 or 40 C under the continuous illumination of 130 foot candles from a bank of cool, white fluorescent tubes and with 5% CO2 in air added to the incubator. Isolation of the polysaceharide material and measurement of cell dry weight. Samples were withdrawn aseptically at 2-day intervals. Cells were removed by centrifugation, and total soluble polysaccharide in the supernatant fluid was measured by using the anthrone procedure as described by Ashwell (3). Dry weights were determined after drying the cells in tared aluminum cups overnight at 100 C. At the end of the growth period, the cells were removed by centrifugation, and the cell-free extracts were concentrated to one-tenth volume in a rotatory evaporator at 60 C. After dialysis, the extracellular polysaccharides were precipitated by adding three volumes of cold 95% ethanol. The precipitate was collected by centrifugation, washed with absolute ethanol, dried, and weighed. The harvested cells were killed with 0.5 ml of a 2:1: 1 (v/v/v) mixture of chlorobutane, chlorobenzene, and dichloroethane (11), and capsular polysaccharides were extracted with 50 ml of distilled water at 5 C for 24 hr, precipitated with three volumes of ethanol, and weighed. To extract water-soluble intracellular polysaccharides, the cells were ruptured by ultrasound (Bronson sonifier, model S110) for 30 min. The supernatant fluid was treated with 10% trichloroacetic acid to precipitate protein, and the polysaccharides were precipitated with ethanol from the proteinfree solution. Polysaccharide material was also extracted by the alkali extraction method of Bishop et al. (4). The culture of Anacystis was made alkaline by adding sodium hydroxide (4: 1, w/v). The mixture was boiled for 6 hr under reflux, filtered through sintered glass, adjusted to pH 4 with HCl, and dialyzed for 3 days at 4 C. The nondialyzable material was isolated by freeze-drying. Hydrolysis and chromatography. Polysaccharide samples (50 mg) were hydrolyzed in 5 ml of 0.7% HSO4 in sealed ampoules for 6 hr at 100 C. Barium carbonate was added to neutralize the excess acid and BaS04 was removed by filtration. Supernatant fluids POLYSACCHARIDE PRODUCED BY A. NIDULANS were concentrated under reduced pressure at 60 C, and monosaccharides were separated by descending paper chromatography on Whatman no. 1 sheets with three different solvent systems: butanol-acetic acidwater (4: 1:5, v/v/v), ethyl acetate-pyridine-water (8:2: 1, v/v/v), and isopropanol-water (4: 1, v/v). Sugar spots were located on the dried, developed chromatograms by a dip in alkaline silver nitrate (14). Known sugars were included with each run. Molar ratios of the monosaccharides were determined by gas-liquid chromatography of the hydrolysates by the procedure of Albersheim et al. (1). The polysaccharide material (20 mg) was hydrolyzed with 2 N trifluoroacetic acid for 2 hr at 121 C. After evaporating the acid, the hydrolysate was reduced with sodium borohydride in 1 N ammonia and acetylated with acetic anhydride, and a 10-uliter amount was injected into a Varian model 200 gas-liquid chromatography apparatus. To determine the effect of nitrogen sources, the alga was grown on various nitrogen sources supplied at a level of 1 mg of culture medium per ml. The response was measured both as dry weight of cells and as extracellular polysaccharide production on the dialyzed cell-free supernatant fluids. RESULTS AND DISCUSSION Analysis of spent growth medium and capsular and alkali-extracted polysaccharides showed the presence of glucose, galactose, and mannose as components. The average molar ratio for glucose-galactose-mannose was 66: 14: 20. Chromatographic and colorimetric analysis did not reveal hexuronic acids or ninhydrin-positive components. The composition of polysaccharide was similar to the sugar moiety of the cell wall of Anacystis as reported by Drews and Gollwitzer (7), although these authors also tentatively identified fucose as a cell wall constituent. No fucose could be identified in the present study. The amount of extracellular polysaccharide, determined gravimetrically after ethanol precipitation, was 366 mg per liter of growth medium after 21 days at 25 C. Capsular and water-soluble intracellular polysaccharide accounted for 10 and 15 mg per liter, respectively. Subsequent experiments gave an average of 414 mg (range 360 to 446 mg) of extracellular polysaccharide per liter and 12.5 mg (range 8 to 16 mg) of capsular polysaccharide per liter. Intracellular polysaccharide was not determined. The extensive accumulation of extracellular polysaccharide as compared with capsular polysaccharide may be attributed to agitation during growth and has been reported with Anabaena flos-aquae (13). After extraction and removal of ethanol-soluble as well as ethanolprecipitable material, 0.0826 g of cell residue per liter of medium remained. Production of extracellular polysaccharide, as determined with the anthrone reagent, in comparison with cell growth at 25 and 40 C over a period of 14 days is presented in Table 1. The total anthrone-positive sugar values are considerably less than expected on the basis of the gravimetric determination of total alcoholprecipitable polysaccharide. Although incubation time (21 versus 14 days) might account for some of the difference, it is assumed that some polysaccharide is lost during our analytical procedure. A comparison of the values shown in Table 1 indicates that cells grow much more rapidly at 40 C than at 25 C. Total cell growth is nearly the same after 12 days at both temperatures and could be attributable to depletion of a limiting nutrient in the growth medium. Production of total extracellular polysaccharide during the growth period was higher at 25 C than at 40 C. Also, the calculated rate of polysaccharide production per unit of cell synthesis ranged from twoto threefold greater at 25 C than at 40 C. It appears that CO2 is fixed and directed toward polysaccharide synthesis preferentially at the lower temperature. The 14-day values may be misleading because the low total cell weight at 40 C probably reflects cell lysis, and this would bias the calculated amount of polysaccharide produced per gram of cells. Anacystis is one of the blue-green algae responsible for algal blooms in fresh water (8), and most fresh water bodies have a temperature lower than 30 C. It is therefore likely that soluble polysaccharides are produced which diffuse into the water and cause a greater organic enrichment than can be accounted for by many procedures used to measure primary productivity. The report of Moore and Tischer (13) on polysaccharide production by several green and blue-green algae in life support systems also supports the present conclusion. The significance of bacterial extracellular polysaccharides in aquatic ecosystems has been reported (8), and the algal polysaccharides undoubtedly have a similar influence. The influence of several sources of nitrogen on cell yield and polysaccharide production after 10 days at 25 C is presented in Table 2. Although all media contained less available nitrogen, and therefore supported less cell growth, than the Kratz medium used to produce the data in Table 1, relative influences can be compared. Nitrate supported more growth and polysaccharide production than either ammonium or urea, which indicates that nitrate is more metabolizable than ammonium or urea as a nitrogen source. However, the amount of polysaccharide produced per gram of cells is significantly higher in the presence of the less metabolizable ammonium or urea nitrogen than in the presence of nitrate. This suggests that the available carbon source (CO2) is directed toward extracellular polysaccharide production rather than toward cell material when either total available nitrogen is a limiting nutrient or when ammonium or urea are the nitrogen sources. Therefore, nitrate concentration has more relevance than total available nitrogen in determining the fate of metabolically fixed CO2. As an ecological consideration, we would predict rapid growth of Anacystis nidulans (potential algal bloom) only after the process of nitrification has converted reduced nitrogen (e.g., from organic pollutants) to nitrate.

v3-fos

2016-05-04T20:20:58.661Z

1971-07-01T00:00:00.000Z

6389559

s2

Nutrition of the dwarf layer SUMMARY Feeding levels of dietary protein varying from 1 6 to 23 p. ioo and metabolizable energy levels varying from 2 .8 7 to 3. 31 kcal per gram failed to significantly influence egg production or egg size of dwarf layers. An increase in weight of dwarf birds at 6 weeks of age was obtained by feeding thyroprotein, thus suggesting that the dwarf layer may be a hypothyrotic bird. Body temperatures were r/2 0 C lower for dwarf as compared to normal birds, however, feeding thyroprotein brought the body temperature of the dwarf up to that of the normals. The dwarf bird contained approximately 5 p. roo more carcass fat on a dry weight basis than did normal birds. Feeding thyroprotein resulted in a decrease in carcass fat for the dwarf bird while thiouracil feeding increased carcass fat. The very opposite effect was noted for carcass fat of the normal birds. There was no significant difference in packed cell volume, hemoglobin, red blood cell count, mean corpuscular hemoglobin, total cholesterol, serum protein and serum albumin of dwarf and normal birds. Of the plasma amino acids methionine was significantly lower for the dwarf while all other essentials were similar for the dwarf and normal birds. Although there has been a lot of discussion about the possibility of using dwarf layers in the commercial egg industry, results to-date with regard to egg production and egg size have not been good enough to make the dwarf a serious contender as a replacement for our present day commercial layer. In table i, is shown the average weight and average weekly feed consumption of egg production type dwarfs reared to 8 weeks of age on a normal 20 p. ioo protein starter ration. Body weight and feed consumption are approximately i/ 3 lower than that for most of to-day's modern layers. The pullets were randomized into floor and cage pens at 8 weeks of age and fed ad ti bitum high and low energy growing diets (2 gq.6 vs. 2 77 6 kcal ME/kg) containing 14 p. 100 protein to 20 weeks of age. Birds reared in cages were slightly heavier than those reared on the floor (table 2 ). Weight gain was not affected by the energy level of the ration, however, less food was consumed on the lower energy diet. At twenty weeks of age the pullets were placed in 8 inch laying cages (one bird per cage) and placed on diets containing 15 , 17 and I g p. 100 protein. After 3 , 2 8 day periods, when it was evident that there was no difference in response to the diets the levels of protein were increased 4 p. 100 to I g, 21 and 23 p. 100 . At the end of 5, 2 8 day periods, the level of energy was increased in the diets, since it was felt that perhaps energy rather than protein may have been a limiting factor in these diets. The two extremes in diets are shown in table 3 . Since there was no significant difference in performance between any of the treatments, the results have been averaged to show the overall performance of the birds from 23 to 53 weeks of age (table 4 ). Feed consumption was very low, however, for the rate of production and egg size obtained, efficiency of protein utilization was extremely good. Several small tests were conducted using higher additions of DL-methionine and also higher levels of vitamins and trace minerals. All these treatments were without effect in enhancing performance. In order to try and find some answers as to why the dwarf bird did not respond to increased levels of nutrients work was initiated to try and determine whether the dwarf was a hypothyrotic bird. Normal and dwarf chicks were fed a regular chick starter ration containing protamone ( 1 ) or thiouracil. Weight gain and feed consumption values to 6 weeks of age are shown in table 5. A marked improvement in weight gain was observed for the dwarf bird when fed protamone while little or no difference was noted for the normal birds. Thiouracil resulted in a marked depression in performance for both dwarf and normal birds. A similar pattern was noted for shank length (table 6). Body temperature was recorded for dwarf and normal birds at 8 weeks and 7 months of age. It can be noted in table 7 , that the dwarf birds had a lower body temperature than the normal. Feeding thyroprotein raised the body temperature of the dwarf up to that of the normal bird. In table 8 is shown carcass composition of normal and dwarf birds at 6 weeks of age. The dwarfs had significantly more body fat than the normals. Thyroprotein feeding reduced the fat content of the dwarf bird but increased body fat of the normal. The opposite effect was noted with the feeding of thiouracil. These results suggest a marked difference in metabolism between the dwarf and normal bird. It was decided to grow dwarf pullets to 20 weeks of age and attempt to enhance their feed consumption and hence egg production and egg size by the feeding of thyroprotein. Various levels of thyroprotein were fed with little or no success. However, in the latest test (table 9 ) there appeared to be some indication that thyroprotein supplementation may be of benefit to the dwarf bird. In an additional experiment with dwarf layers, housed and fed individually and again testing various levels of protein and energy, no response to dietary treatment was noted. However out 4 in table io is shown the number of hens laying at different ( 1 ) Trade name of thyroprotein contains p. ioo thyroxine activity. rates of production for the above test. Even though there were large differences in composition of the diets employed there was no consistant effect on performance. Number of birds in various clutch sizes was summarized for the dietary treatments (table 11 ). Again there is no difference between the treatments, but the large number of clutch sizes of 3 days or less, indicates the main problem with the dwarf bird. Clutch size must be improved before any major improvement in production can be obtained. A further study was undertaken to compare various blood constituents of the dwarf with those of normal White Leghorn hens. There was no significant difference in packed cell volume, hemoglobin, red blood cell count, or mean corpuscular hemoglobin concentration (table r2). No difference in total cholesterol, serum protein or serum albumen was also noted for the dwarf and normal birds (table r 3 ). Plasma amino acids were determined for dwarf and normal chicks 8 hours after feeding. Most amino acids were similar, however, there was a marked decrease in the level of methionine for the dwarf as compared to the normal bird. Further work is underway to verify this finding and expand this area of investigation. Re!u pour publication en septemb y e 1971.

v3-fos

2019-03-19T13:13:58.502Z

1972-01-01T00:00:00.000Z

236892339

s2

Procedure for Isolation and Enumeration of Vibrio parahaemolyticus, An evaluation of criteria used in the identification of Vibrio parahaemolyticus showed that cultural responses varied with respect to growth in broth with 10% NaCl, type of hemolysis, reactions in triple sugar-iron-agar, and serological reactions. With few or no exceptions, cultures were positive for cytochrome oxidase, utilized glucose fermentatively, were sensitive to pteridine (0/129) and novobiocin, and failed to grow in Trypticase soy broth (TSB) without NaCl. A procedure employing a direct plating technique, with or without prior enrichment, was designed for the isolation and enumeration of V. parahaemolyticus. The plating medium consisted of 2.0% peptone, 0.2% yeast extract, 1.0% corn starch, 7% NaCl, and 1.5% agar, with the pH adjusted to 8.0. The enrichment broth was TSB with 7% NaCl. Dilutions of food homogenates were either spread directly on the plates or inoculated into enrichment broth. TSB enrichments were incubated at 42 C for 18 hr. A loopful of the TSB tubes then was streaked onto the direct plating medium. Incubation of plates was at 42 C for 24 to 48 hr. Smooth, white to creamy, circular, amylase-positive colonies were then picked as suspect V. parahaemolyticus. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by a fluorescent-antibody technique. With this procedure, a satisfactory quantitative recovery of known V. parahaemolyticus from inoculated seafoods was made possible. V. parahaemolyticus was nto isolated from other salted foods. Information on the isolation, identification, and public health significance of Vibrio parahaemolyticus is presented in a recent review (R. Nickelson and C. Vanderzant, J. Milk Food Technol., in press). Much of the published information concerning this organism originates from Japan where V. parahaemolyticus is responsible for the majority of foodborne gastroenteritis. Although several isolations have been reported from marine environments and seafoods in the United States, little is known about its presence in other salted foods and about its significance in foodborne illnesses in this country. Present isolation and identification procedures are lengthy and require large quantities of various media. Confirmation of suspect V. parahaemolyticus involves numerous biochemical tests and serological typing. The objectives of this investiga- ' Published with the approval of the Texas Agricultural Experiment Station, College Station. 2 A preliminary account of this work was presented at the 71st Annual Meeting of the American Society for Microbiology at Minneapolis, Minn., 2-7 May 1971. tion were (i) to evaluate criteria now used for presumptive identification of V. parahaemolyticus and to determine the fewest and most reliable characteristics for identification and (ii) to develop a rapid and reliable procedure for the isolation and enumeration of V. parahaemolyticus. MATERIALS AND METHODS Cultures. The sources of the V. parahaemolyticus cultures and other Vibrio species are shown in Table 1. Other organisms mentioned in this study were purchased from the American Type Culture Collection or were isolated from seafoods. All cultures were maintained at 25 C on Trypticase soy agar (TSA; BBL) with 3% NaCl. Morphological and biochemical characteristics. Gram stains of 24-hr agar slant cultures were made by Hucker's modification (13). For flagella stains, cultures were incubated at 25 C for 18 to 24 hr in a flagella broth (pH 7.0) consisting of 1% Tryptose (Difco), 0.1% K2HPO4, and 3.0% NaCl. After centrifugation (3,500 x g for 10 min), packed cells were resuspended in distilled water and poured over a process clean slide (Corning no. 2948). Air-dried 26 slides were stained for 6 to 18 min with flagella stain (Difco). Motility was determined by microscopic examination of the broth by the hanging-drop method (13). Growth from TSA slants was used to determine catalase production in 3% H202 and cytochrome oxidase on filter paper moistened with 1% aqueous tetramethyl-p-phenylenediamine. Utilization of glucose was tested in Hugh-Leifson medium at 25, 35, and 42 C (5). Sensitivity to pteridine 0/129 and novobiocin was determined on TSA plates by the paper disc method. Reactions on triple sugariron-agar (TSI; Difco) were recorded in 24 hr. Salt tolerance was determined in Trypticase soy broth (TSB) with 0, 3, 7, and 10% NaCl. Hemolysis was determined on sheep, human, and rabbit blood with 0.5, 5.0, and 7.0% NaCl. Hemolysis was also tested on the medium proposed by Wagatsuma (7) Serological identification. Serological identification was accomplished by slide agglutination with K antisera (Nichimen Co., New York, N.Y.). K pool and corresponding monovalent reactions were determined by the scheme of Sakazaki et al. (10). A suspension of a 24-hr TSA culture and diluent (3% NaCl) was mixed with an equal volume of polyvalent or monovalent antiserum. After mixing for 30 to 60 sec, agglutination was rated (1+ to 4+) by visual observation. Cultures were considered nontypable with any of the following: no reaction with K pool, reaction with more than one K pool, reaction with K pool and absence of any monovalent reaction, and reaction with K pool plus reaction with more than one monovalent antiserum. Isolation of antigenic substance fromV. parahaemolyticus. The procedure was a modification of that reported by Miwatani et al. (6) for the isolation of A substance. Thirty grams of wet-packed cells of V. parahaemolyticus (ATCC 17802) was used. The procedure was only carried out to the elution of the partially purified A (PPA) substance, and diethylaminoethyl (DEAE) Sephadex A-50 was used in place of DEAE cellulose. Preparation of antiserum for PPA. A 40-ml sample of fraction PPA containing 3.44 mg of protein was reduced to 2.5 ml with Lyphogel (Gelman Instrument Co.). This was mixed with 2.5 ml of Freund's complete adjuvant (Difco). The mixture was injected subcutaneously into five sites (1 ml per site) of a rabbit's back. Titer was determined by slide agglutination with live cells of culture 17802. After 17 days the rabbit was exsanguinated, yielding 120 ml of whole blood. After clotting, the serum (49 ml) was removed and the globulin fraction was separated by dialysis (overnight at 4 C) against 50% saturated (NH4)2SO4. The precipitate was removed by centrifugation (16,000 x g for 20 min) and was washed three to four times with 50% saturated (NH4)2SO4 until most of the red pigment was removed. The precipitate was suspended in 0.85% NaCl and desalted on a G-25 Sephadex column. Conjugation of rabbit anti-PPA globulin with fluorescein isothiocyanate. Labeling procedures were those described by Goldman (4). The globulin solution was reduced to 16 ml with Lyphogel. Protein concentration by the method of Lowry et al. was 17.5 mg/ml. To this solution in an ice bath was added 10% (v/v) 0.5 M carbonate buffer (pH 9.5) in saline. Fluorescein isothiocyanate (FITC) was added at the rate of 15 Ag of dye per mg of protein. The labeling reaction was allowed to proceed overnight at 4 C with gentle stirring. Unreacted dye was removed by passing the solution through a Sephadex G-25 column. The column was eluted with 0.0175 M phosphate buffer at pH 6.3. Highest working dilution of conjugate was found to be 1:8; hence a 1:4 dilution with FA buffer (Difco) was made yielding a total of 84 ml. Fluorescent-antibody examination of V. parahaemolyticus and other organisms. Cultures of Proteus, Salmonella, Aeromonas, Escherichia coli, Bacillus, Flavobacterium, Providence group, Staphylococcus, Alcaligenes, V. parahaemolyticus, and other Vibrio species were streaked on TSA and incubated at 35 C for 24 hr. A loopful of each culture was spread in 3% NaCl on a FA slide (Clay Adams, Parsippany, N.J.) and allowed to dry in the air. They were then lightly heat-fixed before staining. A few drops of diluted labeled antibody were placed on each smear and incubated in a humidifier at 25 C for 30 min. Excess conjugate was removed by rinsing with FA buffer (Difco). Slides were then soaked for 10 min in the same buffer with at least one change of buffer in that time period. After air-drying, the slides were either mounted by using FA mounting fluid (Difco) and a cover slip or were examined directly. Controls were run by exposing antigen to normal (no antibody to antigen) conjugate and exposing antigen to unlabeled and then to labeled antibody. Indirect stains were made in a similar manner with minor exceptions. Smears were first reacted with unlabeled anti-PPA serum for 30 min and then were stained with fluorescein-conjugated goat antirabbit globulin (Nutritional Biochemicals, Cleveland, Ohio). Intensity of fluorescence was rated from 0 to 4+. Reactions of greater than 2+ were considered positive. RESULTS Morphological, biochemical, and serological characteristics. All V. parahaemolyticus cells were short, gram-negative rods exhibiting pleomorphism. Curved, straight, coccoid, and swollen forms were observed. There was also a strong tendency towards bipolar staining. All cultures were motile, had a single polar flagellum, and hydrolyzed starch. With few or no exceptions, cultures of V. parahaemolyticus (i) showed a weak catalase reaction; (ii) were posi-tive for cytochrome oxidase; (iii) utilized glucose fermentatively at at least two of the three incubation temperatures; (iv) were sensitive to pteridine (0/129) and novobiocin; (v) did not grow in TSB without NaCl; (vi) grew well in TSB with 3 or 7% NaCl; and (vii) produced an alkaline slant, acid butt, and no H2S or gas in TSI. Growth in 10% NaCl varied. Other Vibrio species were sensitive to pteridine 0/129 and novobiocin. Hemolytic activity of the test strains varied greatly, depending on the type of blood and concentration of NaCl (Table 2). Table 3 shows the serological reactions observed in this laboratory and those reported by others. Only eight cultures were typable. The other 20 cultures were nontypable for the following reasons: 9 cultures failed to react with any K pool, 3 reacted with more than one K pool, 7 reacted with a K pool and failed to react with the monovalent antisera in that pool, and 1 reacted with more than one monovalent antiserum. Reactions on 13 cultures had been reported previously. Only 7 of the 13 cultures showed similar reactions in the present study. V. anguillarum (14181S) showed a 2+ reaction with the group III K pool and a slight agglutination with monovalent antisera 4 and 29. V. alginolyticus (17749) showed no reaction. Isolation and identification procedure. The various plating media tested were as follows: Staphylococcus 110 medium with 5 IU of penicillin per ml; Twedt medium (Bacteriol. Proc., p. 6, 1970); Brain Heart Infusion agar with 5% NaCl; Brain Heart Infusion agar with tetramethyl-p-phenylenediamine (0.1%); bismuth-sulfite-agar; Brilliant Green-sulfadiazine-agar; tellurite-polymyxin-egg yolk-agar; tellurite-polymyxin-egg yolk-agar without polymyxin; Baross and Liston medium (1); Twedt medium with 5% NaCl and 0.2% bile salts; Twedt medium with crystal violet (0.0001%); Twedt medium with 1% corn starch, 7% NaCl, and 10 IU of penicillin per ml; and Twedt medium with 1% corn starch, 7% NaCl, and no penicillin. The enrichment broths were as follows: Trypticase soy broth with 5% NaCl at pH values from 8 to 11, Trypticase soy broth with 7% NaCl and 10 IU penicillin per ml, Trypticase soy broth with 7% NaCl. Plating media were inoculated by spreading 0.1 ml of appropriate dilutions of TSB cultures on the surface of the plates. In addition, seafoods with a natural microbial flora were inoculated with V. parahaemolyticus cultures and plated in a similar manner. All plating and enrichment media were incubated at 35 and 42 C. With few exceptions, a modification of a medium NaCl. After 18 hr at 42 C, the tubes were streaked onto MT agar plates with a wire loop. All MT agar plates were incubated aerobically at 42 C for 24 to 48 hr. White to creamy, circular, smooth, amylase-positive colonies were picked as suspect V. parahaemolyticus. These isolates were then tested for Gram reaction, morphology, glucose utilization, presence of cytochrome oxidase, and sensitivity to pteridine 0/129. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by fluorescent-antibody technique. Recovery efficiency of plating and enrichment media. Seafoods (50 g) were blended with 450 ml of sterile 7% NaCl and inoculated with V. parahaemolyticus. The population level of the inoculum (TSB culture) was determined by plating on MT medium and on TSA with 3% NaCl. The number of cells recovered was determined by direct plating on MT medium. Recovery of V. parahaemolyticus from seafoods inoculated with various isolates was generally acceptable ( Table 4). The average recovery of V. parahaemolyticus from all seafoods was 85%. All enrichment broths which contained seafoods inoculated with V. parahae- Roman numerals refer to polyvalent antisera, and arabic ones refer to monovalent antisera; NT = did not react with polyvalent and/or monovalent antisera; + to + + + + refers to degree of reaction. FDA, Food and Drug Administration. parahaemolyticus from these samples. Immunofluorescent reaction. When tested with the conjugated globulin, all V. parahaemolyticus cultures (except 7BW, 8C, A4280, A6540, and A7606) were positive. These five cultures differed in one or more characteristics from the majority of V. parahaemolyticus cultures. Some enteric pathogens such as Providence group, Proteus, and Salmonella showed positive reactions. Of the V. anguillarum cultures, only one (14181S) exhibited a positive reaction. Other species of Vibrio (including V. alginolyticus) exhibited slight or no fluorescence. The indirect staining procedure increased the intensity of fluorescence in only one case (Providence group). Fluorescence remained the same or diminished with the other test organisms. Comparison of isolation procedures. Eighteen commercial seafoods were examined for V. parahaemolyticus by the present procedure and that described in the Bacteriological Analytical Manual (15). Seafoods included were oysters, clams, shrimp, squid, and crab meat. These samples were either fresh from the Gulf of Mexico or were purchased in local restaurants. Both procedures recovered V. parahaemolyticus from Galveston Bay clams and oysters from the Gulf of Mexico. With the new procedure, V. parahaemolyticus was recovered from two additional samples, oysters and crab from Galveston Bay. V. parahaemolyticus cells detected with both procedures were in low numbers and were recovered from 1:10 and 1:100 dilutions placed in enrichment broths. Organisms which mimicked V. parahaemolyticus on MT medium (gram-negative, oxidase-positive, and amylase-positive rods) were usually V. alginolyticus. Organisms picked as suspect V. parahaemolyticus from thiosulfate citrate bile salts sucrose agar (TCBS) were screened by the oxidase reaction and reactions in TSI. Many of these produced H2S, gas, or were oxidase-negative. The number of colonies picked as suspect V. parahaemolyticus from the MT medium for further confirmatory tests was lower than that from TCBS medium. Incidence of V. parahaemolyticus in other salted food products. Sixteen commercial foods including ham, salt pork, corned beef, stuffed crab, olives, pickles, and some canned seafoods (oysters, clams, sardines, tuna) were checked for the presence of V. parahaemolyticus. No V. parahaemolyticus was isolated from the salted foods. Amylase-positive colonies were noted on plates which contained corned beef. These were gram-negative rods which failed to utilize glucose fermentatively. With the enrichment procedure, amylase-positive colonies were recovered from three samples, but none could be confirmed as V. parahaemolyticus. The predominant organisms on MT plates usually were gram-positive cocci. DISCUSSION Numerous morphological and biochemical characteristics have been reported for V. parahaemolyticus. One of the objectives of this study was to determine a set of characteristics which would best identify V. parahaemolyticus. Strains of V. parahaemolyticus used in this study were morphologically similar to those reported by Sakazaki et al. (9) and Twedt et al. (14). They were short, gram-negative pleomorphic rods, with a single polar flagellum. Sakazaki (8) recommended growth in 10% NaCl as one of the methods of separating V. alginolyticus (+) from V. parahaemolyticus ( -). Results of the present study and those reported by Twedt et al. (14) showed that growth of V. parahaemolyticus in media with 10% NaCl was variable and that this characteristic should not be used as a key test in identification. Aeromonas can be separated from Vibrio because of its resistance to pteridine 0/129. This substance is a known vibriostatic compound as described by Collier et al. (2), Shewan et al. (12), and Sakazaki et al. (9). Reactions in TSI agar as a preliminary screening test (Bacteriological Analytical Manual) are not considered useful, since many other organisms, for example anaerogenic Aeromonas and some Enterobacteriaceae, produce reactions similar to those of V. parahaemolyticus. Hemolysis has been related to pathogenicity by Miyamoto et al. (7) and Sakazaki et al. (11). It is also used as a preliminary screening criterion by Baross Another objective of this study was to develop an isolation procedure that would require less time than those currently employed. Baross and Liston (1) used anaerobic hydrolysis of starch and hemolysis of human blood in 1% NaCl as criteria for selecting suspect colonies for further identification of V. parahaemolyticus. The lack of dependability of hemolysis as a criterion for identification has been discussed. Although all strains of V. parahaemolyticus tested in this study hydrolyzed starch, results on salt water-starch-agar (Baross-Liston) were not clear. Growth of V. parahaemolyticus was not good, and in mixed cultures amylase-positive colonies were difficult to recognize. Twedt et al. (Bacteriol. Proc., p. 6, 1970) eliminated the tedious process of anaerobic incubation; however, many other marine organisms are capable of growth on their starch medium. Starch hydrolysis was used as a means of picking suspect V. parahaemolyticus. The procedure described in the Bacteriological Analytical Manual (15) is a combination of Baross and Liston's and Japanese methods. The procedure employs two plating and two enrichment media. Suspect colonies are screened on TSI reaction with 34 additional biochemical tests that could require up to 9 days for the final identification of V. parahaemolyticus. The procedure reported in this study requires 3 to 4 days for the identification of V. parahaemolyticus. Gram-positive bacilli and cocci are frequently encountered but present no problem because of distinct morphological differences of colonies and cells. Confirmation of suspect colonies is based on the previously mentioned morphological and biochemical characteristics and fluorescentantibody reactions. Although V. alginolyticus mimicked V. parahaemolyticus on MT plates, separation was easy on the basis of morphology and fluorescent-antibody reaction. Recovery of V. parahaemolyticus from inoculated seafoods by this new procedure was good. The method is comparable or more effective than those currently used for the isolation of V. parahaemolyticus from fresh seafoods. Although V. parahaemolyticus was not isolated in this study from other salted foods, the possible presence of this halophilic organism in other salted foods should not be discounted. Although serological identification of V. parahaemolyticus is at the present time questionable, the A substance described by Miwatani et al. (6) seems a very specific antigen. The isolation of this substance is quite complex and requires large numbers of wet-packed cells. With the modifications in procedure recommended in this study, the isolation of fraction PPA (assumed to be partially purified A substance) is not difficult. Fluorescein conjugated anti-PPA rabbit globulin was specific for V. parahaemolyticus when tested with other species of the genus Vibrio. Some cross-reactions were observed with enteric pathogens (Enterobacteriaceae), but it is doubtful that these organisms would be encountered on MT medium containing 7% NaCl.

v3-fos

2020-12-10T09:04:12.620Z

1972-12-01T00:00:00.000Z

237230100

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Bacteriological Survey of the Blue Crab Industry During sanitation inspections of 46 crabmeat processing plants on the Atlantic and Gulf Coasts, 487 samples of whole crabs immediately after cooking, cooked crabs after cooling, backed or washed (or both) crab bodies and whole crab claws, as well as 1,506 retail units of finished product were collected and analyzed bacteriologically. The 1,506 retail units (1-lb [373.24-g] cans) included 518 cans of regular (special) meat, 487 cans of claw meat, and 501 cans of lump meat. Statistical analyses showed that crabmeat from plants in Mississippi, Louisiana, and Texas had higher counts in 19 of 24 cases for the four bacteriological indices than crabmeat from plants located along the Atlantic Coast and the Gulf Coast of Florida. Aerobic plate counts of retail units collected from a previous day's production were significantly higher than those collected on the day of inspection. Regular crabmeat had consistently higher aerobic plate counts than claw or lump meat. When the product was handled expeditiously under good sanitary conditions, the bacteriological results were significantly better than the results from plants operating under poor sanitary conditions. Crabmeat produced in plants operating under good sanitary conditions had the following bacteriological content: (i) coliform organisms average most-probable-number values (geometric) of less than 20 per g; (ii) no Escherichia coli; (iii) coagulase-positive staphylococci average most-probable-number values (geometric) of less than 30 per g in 93% of the plants; (iv) aerobic plate count average values (geometric) of less than 100,000 per g in 93% of the plants, with the counts from 85% of these plants below 50,000 per g. bacteriologically. The 1,506 retail units (1-lb [373.24-g ] cans) included 518 cans of regular (special) meat, 487 cans of claw meat, and 501 cans of lump meat. Statistical analyses showed that crabmeat from plants in Mississippi, Louisiana, and Texas had higher counts in 19 of 24 cases for the four bacteriological indices than crabmeat from plants located along the Atlantic Coast and the Gulf Coast of Florida. Aerobic plate counts of retail units collected from a previous day's production were significantly higher than those collected on the day of inspection. Regular crabmeat had consistently higher aerobic plate counts than claw or lump meat. When the product was handled expeditiously under good sanitary conditions, the bacteriological results were significantly better than the results from plants operating under poor sanitary conditions. Crabmeat produced in plants operating under good sanitary conditions had the following bacteriological content: (i) coliform organisms average most-probable-number values (geometric) of less than 20 per g; (ii) no Escherichia coli; (iii) coagulasepositive staphylococci average most-probable-number values (geometric) of less than 30 per g in 93% of the plants; (iv) aerobic plate count average values (geometric) of less than 100,000 per g in 93% of the plants, with the counts from 85% of these plants below 50,000 per g. An outbreak of food poisoning in Chicago in September, 1926, from crabmeat containing Salmonella suipestifer led to an investigation by the Food and Drug Administration (FDA) of the crabmeat industry in Maryland and Virginia (5). It was determined that crabmeat was being produced under grossly insanitary conditions, which were reflected by the high incidence of coliform organisms in the finished product. In 1932, additional outbreaks of food poisoning in Washington, D.C., Baltimore, Md., and Philadelphia, Pa., were traced to microbial contamination in crabmeat (5). During the past 4 decades, the FDA has been taking regulatory action against firms producing crabmeat under insanitary conditions when the observed insanitary conditions were substantiated by the presence of indicator organisms such as E. coli and coagulase-positive staphylococci, and by high coliform organisms and aerobic plate counts (APC). During the last decade, the FDA has been conducting bacteriological surveys to determine the numbers and types of certain microorganisms in food prod-ucts and relating their presence to sanitary conditions observed in the plants (6)(7)(8)(9). This paper reports the results of such a survey of the blue crab processing industry along the Atlantic and Gulf Coasts in 1968 and 1969. The primary purpose of this survey was to determine the relationship of industrial sanitary practices to the microbiological quality of freshly picked blue crabmeat that was neither frozen nor pasteurized. The blue crab (Callinectes sapidus Rathbun) is fished commercially from Maryland to south of Galveston, Texas. The greatest concentration of the industry is in the Chesapeake Bay area. Crabs are brought to the processing plant alive and are generally cooked immediately either in boiling water or in a steam retort. The latter method is most common, with cooking times ranging from 7 to 23 min at approximately 121 C (approximately 15 psi). Cooked crabs are usually cooled at ambient temperatures for 3 to 16 hr. Where the firms are equipped with refrigeration facilities, the crabs are placed in a cooler for storage until the beginning of opera-958 tions. Cooler temperatures are usually between 4.4 and 6.7 C, although some were as high as 15.6 C at the time of inspection. Two different processes are used to prepare the crabs for removing the meat (picking): a wet process and a dry process. Forty-four percent of the firms inspected used the wet process in which the crabs are backed (carapace removed) and declawed. The crab bodies are then washed by hand or machine and the meat is removed immediately, or the bodies may be refrigerated overnight. The claws are picked separately. The dry process, most commonly used in the Chesapeake Bay area, does not include the washing step. Each employee backs, declaws, and removes all the meat from each crab. Sometimes the claws are picked separately as in the wet process. There are three general types of crabmeat: (i) regular or special, consisting of white body meat; (ii) lump or backfin, consisting of the large pieces of body meat taken from the muscles which control the back swimming legs; and (iii) claw, consisting of the darker meat taken from the claws. The crabmeat is packed into 1-lb metal or plastic cans with snap-on lids or packed into 5-to 6-lb (1866.2 to 2239.4-g) plastic bags. The finished product is placed in wet ice, stored in the cooler, and shipped in wet ice, usually within 3 days. The crabmeat in the plastic bags is sold for further processing into crabcakes, deviled crabs, etc.; crabmeat in the 1-lb cans is intended for the retail market. MATERIALS AND METHODS Collection of samples. A total of 53 inspections of 46 processing plants were made by inspector-microbiologist teams during May to October in 1968 and 1969. The inspected plants were located in three geographical areas: Baltimore (Maryland, Virginia, and North Carolina); Atlanta (South Carolina, Georgia, and Florida); New Orleans-Dallas (Mississippi, Louisiana, and Texas). The majority of the inspected plants were located on the Gulf and southern Atlantic Coasts. During each inspection, samples were collected aseptically at various stages of processing. These samples were limited to whole crabs immediately after cooking, whole cooked crabs after cooling overnight, backed or washed (or both) crab bodies, and whole crab claws. Retail units (1-lb cans) of each type of crabmeat were collected from that produced on the day of inspection. When available, retail units of at least one type of crabmeat produced on a date prior to the inspection were collected. Immediately after collection, the samples were placed in wet ice in the firm's cooler. After the inspection was completed, samples were packed in an ice chest with wet ice and transported to the laboratory. The analyses, in which four FDA district laboratories participated, were started within 48 hr after collection. A total of 1,506 retail units were collected including 518 cans of regular meat, 487 cans of claw meat and 501 cans of lump meat. Also, 487 samples were collected of whole crabs immediately after cooking, cooked crabs after cooling, backed or washed (or both) crab bodies, and whole crab claws. Analytical procedures. Aerobic plate counts, coliform, E. coli, and coagulase-positive staphylococcus counts were determined on samples by the official first action method for the examination of frozen, chilled, precooked, or prepared foods (41.013-41.018) of the Association of Official Analytical Chemists (1). Correlation of bacterial findings with inspectional evidence. Establishment inspection reports prepared by the inspectors were evaluated, and the firms were classified as good or poor based on the degree of the insanitary conditions observed. Plants which received good ratings were not necessarily operating under ideal sanitary conditions, but their operations were visibly cleaner than those of poor plants. The bacteriological results were statistically analyzed to determine if significant differences occurred (i) between plants rated good versus those rated poor; (ii) among the three types of crabmeat examined; (iii) among the three geographical areas; and (iv) between production lots. Statistical procedures. Tests of significance were performed by using logarithms of counts per gram and percentage of positive units. The logarithms of the counts were assumed to be normally distributed, and tests of significance and confidence limits were computed by using normal theory (Ostle [3D. The differences between good and poor plants and between geographical areas were examined by using the within-lot variation as the estimate of random error. Since a large number of observations were reported as <3, frequency distributions and percentage of positive units were found to be more useful in evaluating the differences for coliform organisms, E. coli, and coagulase-positive staphylococci. RESULTS Plants operating under poor sanitary conditions displayed many poor employee practices, building and equipment defects, and operational inadequacies which contributed to contamination of the finished product. Flies and other insects entered through various windows and door openings. Equipment such as carts and dollies used to transport cooked crabs in wire baskets were rusted and pitted and were very seldom cleaned or sanitized. Cooked crabs came into contact with unsanitized objects such as retort hoist chain, cooler walls, employees' clothing, and rusted storage racks, and were usually air-cooled in areas subject to dust and flies. Cooked crabs frequently were not rotated on the picking tables, so that some crabs remained at room temperature for several hours. In addition, some picked crabmeat was left at room temperature for several hours before 959 VOL 24,1972 being iced. Other practices observed in poor plants included failure of employees to wash and sanitize their hands after touching unsanitized objects, reuse of paper towels and rags, and chlorine dip stations for hands and equipment with less than an effective amount of residual chlorine. Floor filth was introduced to cooked crabs by splatter from water during clean-up operations or by contact of baskets of crabs directly with the floor. Crab claws were left at room temperature most of the day and picked last. In contrast, the plants operating under good conditions of sanitation had fewer insanitary infractions by employees, handled the product quickly, and maintained buildings and equipment in reasonably good condition. However, some of the plants classified as good had very few employees present on the day of inspection, and other plants operated only part of a day. Thus, the opportunity for bacterial buildup was minimized. The cooking times and temperatures, with the exception of the processing plants that boiled crabs, were usually difficult to establish due to either the lack or the malfunctioning, or both, of temperature and pressure gauges. In most cases, the cooking times for the crabs were not sufficient to sterilize them. Sample results of whole crabs collected immediately after cooking showed that 83% of the 104 samples collected had APC values below 10,000/g. Three samples were over 100,0001g. Table 1 shows a comparison of bacteriological results of samples collected at various stages of processing for plants observing good and poor sanitary practices. In the plant operating under good sanitary practices, crabs were cooked for 23 min at approximately 121 C, which was sufficient to reduce the population density to less than 300/g. The plant operating under poor sanitary conditions cooked the crabs for 10 min at the same temperature, and the geometric average APC of two samples was 1,800/g; one sample had an APC of 310,000/g. All samples of cooked crabs were free of coliform organisms, E. coli, and coagulase-positive staphylococci. The results for the poor plant show an increase in counts for all four bacteriological indices after backing, washing, and cooling overnight. The results for the good plant showed an increase in APC after overnight cooling and the appearance of low numbers of coliform organisms, but the samples contained no E. coli and only low numbers of coagulase-positive staphylococci after handling by the employees removing the meat. The low level of counts in the good plant was probably due to the expeditious handling of the crabs. The whole claws were grossly mishandled in the poor plant (Table 1). They were left at room temperature for several hours and allowed to touch unsanitized or rusty equipment, or both. Since claws are more difficult to pick than are the bodies, they remained on the picking tables at room temperature for longer periods of time. APC (geometric means) and 99% confidence limits for three geographical areas and three types of meat produced in good and poor plants are shown in Table 2. Significant differences between the New Orleans-Dallas area and the other two areas were observed for regular and lump meat in both good and poor plants by Duncan's (2) test. This difference was probably due to the higher mean temperature and humidity in the New Orleans-Dallas area, which afforded better incubation conditions. The claw meat did not show significant differences among the New Orleans-Dallas, Baltimore, and Atlanta areas for good plants, but did show significant differences between good and poor plants. This was probably due to the differences in operating procedures in the various areas. Some poor plants in the Baltimore area allowed the claws to remain in wooden baskets all day at room temperature and were picked last, whereas in the other areas the claws were picked simultaneously with the crab bodies. When the claws were handled expeditiously and kept cool, the counts remained at a low level all through the process. As further evidence of the differences between good and poor plants, the overall APC geometric mean per gram was: for regular meat, 18,000 for good plants and 190 tained coliform organisms and coagulase-positive staphylococci, but the geometric averages were very low because most of the retail units were negative (Table 3). The APC geometric averages were below 50,000/g with the exception of plant no. 4. The higher APC average in this sample could not be explained, since the claw and lump meat collected the same day had much lower counts. None of the retail units from the good plants contained E. coli. Table 5 shows the percentage of good and poor plants with aerobic plate counts in seven count ranges for the three types of crabmeat. Regular meat from good plants had APC values (geometric means) below 100,000/g 93% of the time; all samples of claw and lump meat were below this level. This compares with 41, 34, and 51% for regular claw and lump meat (poor plants), respectively, which were below 100,000/g. The regular meat APC values (geometric means) from poor plants were above 1,100,000/g 23% of the time, with the claw and lump meat each above this level 5% of the time. Table 6 summarizes the results for coliform organisms, E. coli, and coagulase-positive staphylococci by the percentage and number of plants having geometric averages in four count ranges. All coliform counts (geometric means) for all three types of crabmeat from good plants were below 20/g (most-probable number [MPN D. Coliform counts (geometric means) for regular, claw, and lump meat were below 10/g (MPN) in 39, 42, and 49% of the poor plants, respectively, while the counts were above 50/g (MPN) in 36, 21, and 23% of the poor plants. All values (geometric means) for E. coli, good, and poor plants, were below 10/g (MPN). Coagulase-positive staphylococci values (geometric means ) for regular, claw, and lump meat were below 10/g in 79, 69, and 77% of the good plants, respectively. The counts were below 10/g (MPN) in 54, 55, and 62% of the poor plants, and above 50/g (MPN) in 21, 24, and 15% of the poor plants. Since coagulase-positive staphylococci are normally found on the human skin and crabmeat is picked by hand, it was expected that these organisms would be present in the finished product. In some cases, coagulase-positive staphylococci counts from finished product units within a lot varied greatly, even when collected from good plants. This could be due, in part, to the difference in the microflora of each employee's hands, since most retail units of finished product represented crabmeat picked by one employee. This difference was demonstrated by Puncochar and Pottinger (4). However, where the product was handled expeditiously and under good sanitary conditions, the finished product counts were usually low. Tables 7 and 8 show the percentage of retail units positive for coliform organisms, E. coli, and coagulase-positive staphylococci for good and poor plants, respectively, and compare products from the three geographical areas. In all three analytical categories and all types of crabmeat, except the coliform results of claw meat from the Atlanta area poor plants, the percentage of units positive was significantly higher in the New Orleans-Dallas area than in the Baltimore and Atlanta areas, regardless of whether the retail units were from good or poor plants. There was very little difference among the types of meat for all geographical areas and all analytical categories. Some differences were noted between good and poor plants in the percentage of units positive for coliform organisms. The percentage of units positive for E. coli in the Baltimore and Atlanta area poor plants was relatively low compared with the New Orleans-Dallas area, and, as stated before, the products of the good plants had no E. coli. Coagulase-positive staphylococci results did not vary significantly, although the poor plants had a slightly higher percentage of units positive for most of the types of meat in the Baltimore and Atlanta areas. The retail units from the New Orleans-Dallas area good plants, however, were higher than the units from the poor plants. This could be due to the fact that fewer units were examined from good plants, since coagulase-positive staphylococci results were shown to vary considerably between lots, regardless of whether the samples were from good or poor plants. Tests were performed on geometric means observed on retail units packed the day of the inspection and units packed on a date prior to the inspection. The APC results in Table 9 (7) 2.1 (3) 120.Oe (10) 450.Oe (10) 12.Oe (7) 90.0 (10) 38.0 (10) 20.0 (9) 140.Oe (10) 61.0 (10) 320.Oe (10) 500.0 (5) 1.7 (3) 11.0 (6) 33.0 (10) 83.0 (10) 89.1 (10) 74.7 (10) 98.6e (9) 26.8 (9) 390.8e (10 210,000-400,000 0 Although the possibility exists that the higher mean counts of the previous day's production could have been due to the additional 24-hr incubation period, even though the product was stored in ice, the probability of significant bacterial growth during this period is not likely. DISCUSSION Tobin and McCleskey (10) showed that the steaming process destroyed all coliform organisms and greatly reduced the numbers of other bacteria in the whole crabs. The results from the few samples of cooked crabs analyzed in our survey confirmed their findings. Furthermore, our survey detected no coagulase-positive staphylococci after the crabs were cooked either by steam retort or boiling. Thus, the presence of large numbers of either coliform organisms or coagulase-positive staphylococci in the finished product indicates insanitary conditions during processing. As a result of a study of the sanitary requirements for crabmeat processing plants, Puncochar and Pottinger (4) reviewed the state of the industry and also made suggestions for technological improvements. The cooking times they reported ranged from 15 to 22 min at pressures of 12 to 18 psi, and were sufficient to kill all bacteria except for a few heat-resistant sporeformers. However, our survey indicated that the industry is generally cooking for 8 to 12 min at approximately 15 psi and, therefore, is not getting a sterile cook. Their study also showed a marked increase in total counts on cooked crabs cooled overnight. Industry practices have 964 PHILLIPS AND PEELER APPL. MICROBIOL. BACTERIOLOGICAL SURVEY OF BLUE CRAB INDUSTRY changed in that most firms air-cool for only a few hours and then refrigerate, rather than air-cool all night. However, the coolers used now are not of a construction design to insure sanitary conditions, and the crabs contact rusty racks, cooler walls, and sometimes raw fish used as crab bait. Since the crabs are not generally sterile when placed in the coolers, which are usually above 4.4 C, there is the possibility of bacterial growth during the overnight cooldown cycle. This may explain why the samples of cooked, cooled crabs in our survey showed a marked increase in counts over the cooked crabs taken out of the retort. Punochar and Pottinger (4) also studied the microflora of the employee's hands and found that 44% were carriers of E. coli. Washing with soap and water followed by sanitizing in a chlorine solution greatly reduced the microflora of the hands, if done periodically throughout the day. Many of the plants inspected had inadequate toilet facilities. Some had no hand washing facilities in or near the toilets, which meant the employees had to enter the processing area to wash their hands. Doors, faucets, and other objects handled before washing could then become a source of contamination with E. coli and other bacteria. The employees also

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Effect of Extracts of the Bark of Saccoglottis gabonensis on the Microflora of Palm Wine Direct addition of the bark of Saccoglottis gabonensis to fresh palm juice followed by microbial enumeration every 6 hr showed that the bark inhibited bacterial growth appreciably. The effect of various extracts of the bark on two bacterial species isolated from palm wine (Leuconostoc mesenteroides and Lactobacillus plantarum) was studied by using the paper disc method. It was observed that three of the five constituents of the bark showed inhibitory effect on the bacteria. Direct addition of the bark of Saccoglottis gabonensis to fresh palm juice followed by microbial enumeration every 6 hr showed that the bark inhibited bacterial growth appreciably. The effect of various extracts of the bark on two bacterial species isolated from palm wine (Leuconostoc mesenteroides and Lactobacillus plantarum) was studied by using the paper disc method. It was observed that three of the five constituents of the bark showed inhibitory effect on the bacteria. Palm wine is a fermented palm juice which is consumed mainly by the peasants in the Southern region of Nigeria. Palm wine tappers in Eastern states of Nigeria usually add the bark of Saccoglottis gabonensis Urban (family Humiriaceae) to palm wine. The bark is sold in the local markets as dry sheets. When added to palm wine this bark imparts amber color to the wine and gives it a bitter taste. The tappers also claim that the bark retards the tendency of the wine to become sour. Microfloral analyses of palm wine have shown that the initial inhabitants of palm wine are predominantly Saccharomyces cerevisiae and, to a small extent, Schizosaccharomyces pombe and a Pichia species. The bacteria isolated during this period of palm wine fermentation have been mainly Leuconostoc mesenteroides and Lactobacillus plantarum, and at times Micrococcus sp. and Bacillus sp. (2, 5; Faparusi, Ph.D. thesis, Univ. of Ibadan, Nigeria, 1966). M. 0. Eze and A. U. Ogan (Department of Biochemistry, University of Nigeria, Nsukka, Nigeria) have reported (in a private communication) that water extracts of the bark lowered titratable acidity levels in palm wine. The aim of this study was to investigate the possible inhibition of growth of microflora of palm wine by various solvent extracts of the bark. MATERIALS AND METHODS Test organisms. For the antibiotic test experiments, S. cerevisiae yeast cells and the bacteria L. mesenteroides and L. plantarum were used. S. cerevisiae cells were isolated from fresh palm wine on malt extract at pH 3.5 (12). L. mesenteroides and L. plantarum were grown on Rogosa, Mitchell, and Wiseman medium (12) excepting the addition of 1.3 ml of acetic acid; drops of the acid were used to bring the pH of the medium to 5.8 (3). This medium is subsequently referred to as modified Rogosa, Mitchell, and Wiseman medium. Extraction of active principle(s). To isolate the active principle(s) the dry bark of the tree, which was bought from a local market, was ground in a Waring blender. Thirty grams of the ground bark was extracted (by direct immersion of the bark in the solvent) with about 70 to 80 ml of solvent at a time, until a total volume of about 300 ml was used. Each extract was concentrated in vacuo to 20 ml. The solvents used were light petroleum (40-60 C), ethanol, ethyl acetate, and methanol. Chromatography of extracts. The extracts were analyzed by paper chromatographic methods. We found from the results of using various techniques and resolution solvents that one-way descending method with two solvents-isopropanol-formic acid-water (2:5:5, v/v) and ethyl acetate-formic acid-water (10:2:3 v:v)-gave good separations. These solvents were used in all subsequent separations. The chromatograms were viewed under an ultraviolet (UV) lamp. The spots were located by using ferric chlorideferricyanide reagent (7). Test for antimicrobial activity. In the preliminary experiment 10 g of the bark was added to 100 ml of fresh palm wine (pH 7.0). Changes in the yeast (S. cereviszae) and bacteria (L. mesenteroides and L. plantarum) population in the palm wine and control (palm wine without the bark) were followed by culturing these organisms on appropriate media every 6 hr. Test for action of the extracts. The inoculum was prepared by growing the above bacteria in modified Rogosa, Mitchell, and Wiseman medium (pH 5.8) for 18 hr at 30 C. The cells were washed and resuspended in sterile saline, and the concentration 853 was adjusted to 104/ml. This concentration was chosen because it was found in the preliminary experiments that inhibition zones were rarely obtained from a concentration greater than 106/ml (see Table 3). Twenty milliliters of agar, inoculated with 1 ml of the suspension of the bacterial cells in saline (104/ml), was placed in sterile petri dishes. Sterilized paper discs (10 mm diameter), which were soaked in the bark extracts and dried in hot petri dishes, were placed directly on the surface of the agar. Sterilized paper discs in the solvent blanks were similarly treated. The plates (both tests and blanks) were incubated at 37 C for a sufficient period to permit accurate measurement of the inhibition zones produced. Test for antibacterial action of eluates from the chromatograms. A total of 5 ml of each of the extracts tested above was spotted on chromatography papers (Whatman no. 20) and run in ethyl acetate-formic acid-water solvent. The spots on the chromatograms were eluted with the particular solvent used in the extraction. Eluates of each compound were pooled together and concentrated in vacuo to 5 ml. Paper disc assay of these eluates was performed on the bacteria as described above. Paper discs, treated with only solvents, were run as controls. RESULTS From the preliminary assessment of inhibition of growth of the microflora of palm wine, it was observed, as shown in Table 1, that there was a considerable inhibition of bacterial growth but the yeast population was not significantly affected. These results therefore show that the reduction in the rate of souring of palm wine is due to inhibition of the rate of growth of the acid-producing bacteria. Table 2 shows the zones of inhibition of bacterial growth when different solvent extracts of the bark were used. These zones of inhibition were measured after the plates were incubated at 37 C for a period of 24 to 36 hr. It was observed earlier that these zones disappeared after incubating the plates for a period exceeding 48 to 60 hr. The methanol extract produced the largest zones of inhibition, whereas there was no inhibition with the ether extract. The effect of inoculum size on inhibitory capability of methanol extract showed that from a concentration of 108/ml there was no inhibition zone formed (Table 3). In a similar experiment, ethyl acetate did not produce any zone of inhibition with a concentration greater than 104 organisms/ml. Ether extract failed to show any zone of inhibition at all concentrations of the extract tested. Assay of eluates from chromatographic separation of extracts of the bark for bacterial inhibition was recorded in Table 4. Ability to inhibit growth of the two bacteria (L. mesenteroides and L. plantarum) was based on formation of zones of inhibition after an incubation period of 24 to 36 hr. When a concentrate of methanol extract was left at room temperature (22-25 C) for about 2 weeks, snow-white pyramidal crystals separated. Upon recrystallization from ethanol a white, crystalline powder was obtained. The RF value of the compound in isopropanol-formic acid-water (2:5:5, v/v) was 0.45. This shows that this compound was the same as the compound on spot c in Table 4. The melting point of the compound was 238 C, and the molecular weight by mass spectrometery was 328. Results of infrared (IR), UV, and nuclear magnetic resonance spectra and action of alkali which gave bathochromic shift in the UV spectrum (UV maximum at 275 nm) showed that the compound was bergenin. The compound has been reported by Ogan (10). The compound failed to inhibit bacterial growth. Spots a and b, which were brown on the chromatogram, were very potent in inhibiting the bacterial growth. The IR functional peaks and the UV absorption peaks at 237, 280, 330, and 395 nm indicated that the compound on spot a is a substituted 1, 2-naphthoquinone. Acid hydrolysates of the compound gave two sugars (D-digitalose and D-fucose) and an aglycone. The aglycone melted at 316 C, and the molecular weight by mass spectrometery was 334. Catalytic reduction of the aglycone reduced two double bonds and removed one hydroxyl group. The aglycone has Vmax at 1,755 and 1,700 cm-'. These values are consistent with the presence of two lactone rings, one of which suffers a bathochromic shift. When heated with aqueous alkali, the aglycone lost CO2, and the product was the monolactone ( Vmax at 1,724 cm-'). This new compound failed to give a green reaction of the aglycone with the ferric chloride reagent. All of these tests showed that the compound on spot a is similar to chartreusin which was reported to be a product of Streptococcus chartreusis by Leach et al. (8). The compound on spot b had a molecular weight of 198, and it melted at 148 to 150 C. When hydrolyzed with NaOH it gave a white, crystalline compound from ethanol, and the ethyl ester peak disappeared. The melting point of this crystalline compound was 253 C. The IR and UV spectra of the compound were superimposable with those of gallic acid. Thus, the compound was identified as the ethyl ester of gallic acid. The remaining two compounds (spots d and e) gave blue fluorescence under the UV light. Tests for antimicrobial activity of eluates of these two spots showed that spot e (RF 0.85) failed to inhibit growth of the bacteria, whereas spot d (RF 0.62) gave a positive inhibition result. The chemical nature of these compounds was not determined. DISCUSSION Palm wine consumers rarely drink palm wine which is more than 1 day old. The reduction of the rate of bacterial growth by the bark explains the lowering of titratable acidity levels in palm wine by extracts of the bark as reported by Ogan in a private communication. The result of analyses of the active principles showed that they were essentially phenolic compounds, and methanol is known to be one of the best solvents in extracting plant phenolic compounds (11). Therefore, the size of the zone of inhibition attributed to this solvent could be related to its efficiency in extracting the active principles, because all other solvents except ether extracted the same number of compounds. It is likely that this also explains why ether extract failed to produce a zone of inhibition. Analyses of the ether extract produced only oils which did not give any zone of bacterial inhibition. Ogan (10) has also reported the absence of alkaloids and phenolic compounds in ether extract of the bark. Gallic acid and derivatives are usually obtained from soluble tannins. In many cases gallic acid occurs as its dimeric condensation product, ellagic acid. Turbovsky et al. (13) and Negre (9) considered tannin to be of some value in resistance of wine to bacterial spoilage. Thus, the antibacterial action of the compound on spot b (ethyl ester of gallic acid) is not surprising. Also, this compound would be one of the factors responsible for the bitter taste the bark imparts on palm wine. Amerine et al. (1) have asserted that ethyl ester of gallic acid gives a bitter taste to wine. Bergenin (spot c) has been obtained from Bergenia crassifolia and a plant Humiria balsamifera of the family Humiriaceae (4). Eze and Ogan (personal communication) reported that bergenin accelerated alcohol production in palm wine at the same time it inhibited acid production. The compound was shown not to inhibit bacterial growth. It could be that the compound altered metabolic pathways leading to acid formation in favor of alcohol production. Experiments on the effect of the compound on enzymes of the pathways of sugar conversion to acid could supply an answer to this suggestion. Chartreusin, which was shown to be similar to the compound on spot a, has been reported to be an antibiotic substance (8). Geissman (6) reported that many of the naphthalene derivatives in plants have been identified as products of mold metabolism. Thus, the possibility of the compound on spot a originating from metabolic activities of molds could not be ruled out. Before the bark is bought as sheets in the market, molds could grow on it. The high relative humidity of the rain forest regions from where the bark is harvested would favor rapid colonization of molds. However, analysis of a fresh bark would give an insight into this problem. An active principle could be bacteriocidal or bacteriostatic in action, but the fact that the zones of inhibition usually disappeared on incubating the plates for a period longer than 48 hr shows that the active principles in this case are bacteriostatic. The bark would be effective in reducing the rate of souring of palm wine provided the degree of infection was not high (not more than 104 to 106/ml.) The population of bacteria in palm wine is about 102 to 104 organisms per ml when most consumers drink it. At this stage, addition of the bark to palm wine will produce a useful effect.

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Bacteriological Survey of Fresh Pork Sausage Produced at Establishments Under Federal Inspection At the time of manufacture, 75% of 67 sets of finished fresh pork sausage collected in 44 plants had aerobic plate counts in the range of 500,000 or fewer/g; 88% contained 100 or fewer E. coli/g; and 75% contained 100 or fewer S. aureus/g (geometric means of 10 samples). Salmonellae were isolated from 28% of 529 samples of pork trimmings used for sausage, and from 28% of 560 finished sausage samples. Semiquantitative analysis revealed that salmonellae were at low levels; more than 80% of the salmonellae-positive samples were positive only in 25-g portions (negative in 1.0- and 0.1-g portions). A survey was conducted to determine the bacterial levels in fresh pork sausage during preparation and as packaged for shipment from representative establishments under Federal inspection in the United States. To produce fresh pork sausage, chilled pork trimmings are first ground to permit easy mixing with spices. The ground pork is placed in a mixer with spices where water may be added. In some establishments, a chopper (cutter) is used for grinding-mixing. To maintain the chilled condition of the mixture for proper extrusion through stuffers, some operators add water in the form of wet ice and some operators add dry ice "snow." The total added water may not exceed 3%. The mixture is transferred to a sausage-stuffer through which it is extruded into casings (natural or artificial) held over the stuffing horn of the sausagestuffer. The strands of stuffed casings are fed into a mechanical linker which twists the casing at regular intervals into links. Skinless links are produced by means of a dispenser attached to the outlet of the stuffer which forms and deposits six links automatically on paper. Whole-hog sausage is processed similarly, using the warm meat and fat from sows conveyed directly from the slaughter-eviscerating lines. Most whole-hog sausage is prepared as I A preliminary account of this work was presented at the 1971 Annual Meeting of the American Society for Microbiology, Minneapolis, Minn., 2-7 May 1971. rolls, by stuffing into 2-inch-diameter polyethylene casing. MATERIALS AND METHODS Sampling. From September 1968 to June 1969, 67 sets of samples were collected from 44 federally inspected plants producing fresh pork sausage; some operations were sampled more than once. Eight of the plants were located in the Northeast, eight in mid-Atlantic states, nine, in the South and Southeast, 13 in the West and Midwest, and six on the West Coast. Twenty-seven of the plants produced sausage from trimmings of chilled carcasses of market hogs slaughtered and eviscerated on the premises. Ten of the plants utilized pork trimmings which arrived chilled from local off-premises sources or frozen from more distant sources. Six of the plants produced whole-hog sausage, which consists of the meat and attached fat from eviscerated sow carcasses. One plant produced both whole-hog sausage and sausage from the trimmings of market hogs. Production line samples totalling 1,152 and finished sausage samples totalling 710 were collected and analyzed. Sets of samples were collected aseptically at the following sites when possible: (i) skin from an eviscerated carcass, (ii) interior tissue from an eviscerated carcass, (iii) pork trimmings or cuts utilized for the sausage, (iv) meat at discharge of grinders, (v) meat at discharge of mixer (some plants used a chopper for grinding-mixing), (vi) meat at discharge of stuffer, (vii) sausage at discharge linkformer, (viii) spices, (ix) natural casings (if used), and (x) finished product. In most cases a set of samples included 10 samples of pork trimmings or cuts and 10 samples of the finished product. Each set of samples was placed promptly into a freezer or under dry ice and shipped frozen to the laboratory for analysis. Analysis was begun 2 to 4 weeks from the dates of collection. Laboratory methods. Methods used for aerobic plate count (APC), Escherichia coli, and Staphylococcus aureus have been fully described (9). Most samples were examined semiquantitatively for salmonellae. From the blended 1:10 dilution prepared for the other determinations, 250 g (equivalent to 25 g of the sample) was weighed into a sterile jar containing 2.5 ml of sterile Tergitol and 26 ml of sterile lOx lactose broth. The jar and contents were shaken thoroughly, after which 11 ml (equivalent to 1.0 g of the sample) and 1.1 ml (equivalent to 0.1 g) were transferred to sterile tubes. The jar and tubes were incubated for 24 hr at 35 C (lactose pre-enrichment). After incubation, 0.5 ml from each portion was transferred to 10 ml of tetrathionate (TT) broth of Hajna and Damon (5) and incubated for 18 to 24 hours at 35 C. Loopfuls of the TT broth were streaked onto Brilliant Green sulfa agar (BGS) and xylose-lysine-deoxycholate-agar (XLD) plates and incubated for 24 hours at 35 C. Characteristic colonies from the selective agars were transferred onto triple-sugar-iron (TSI) and lysine-iron-agar (LIA) slants and incubated for a minimum of 24 hours at 35 C. Isolates with characteristic reactions on TSI and LIA slants that grouped with Salmonella somatic "O" antisera and demonstrated flagellar antigens with Salmonella polyvalent "H" antisera were recorded as Salmonella species. Isolates not meeting these criteria were examined further in accordance with the procedures of Edwards and Ewing (3) until either identified or eliminated as Salmonella species. Commercially dehydrated media were used and were prepared in the manner suggested by the suppliers. RESULTS The bacterial content of the pork sausage is shown in Fig. 1, 2, and 3. The APC values, plotted in Fig. 1, show that the bacterial counts of the finished fresh pork sausage were primarily dependent on the bacteriological condition of the pork trimmings. Sausage made from trimmings with APC values of 100,000/g or below contained fewer than 200,000 75% of the time, and fewer than 500,000 96.4% of the time. Sausage made from trimmings with APC values over 100,000 exceeded 200,000 87% of the time and exceeded 500,000 49% of the time. The processing of fresh pork sausage includes no step that will kill bacteria. If the APC values and S. aureus content of the sausage were equal to that of the trimmings, all points in Fig. 1 and 2 would fall on the solid diagonal line. Normal sample-to-sample variation would result in these points falling in a ,scattered pattern above and below the line. However, in Fig. 1 only 13 of the 67 points are below the line, and in Fig. 2 only 19 of the points are below the line. This indicates that there was some contamination during the processing of trimmings into sausage in about 60% of the sample sets. One would expect some contamination in even the very best operations. Conversely, 44 of the 67 points in Fig. 3 are below the solid diagonal line, indicating a reduction of E. coli in the pork sausage as compared to the pork trimmings. The addition of spices, including about 2% NaCl and 1% sugar, to ground pork lowered the water activity to 0.97. The apparent loss of E. coli may have been due to a deleterious effect of the depressed water activity during the 2-to 4-week pre-examination frozen storage of the sausage. Some salmonellae and, of course, staphylococci are more resistant to depressions of water activity. Most sample sets fell within 0.75 logarithm of the solid diagonal lines in Fig. 1, 2, and 3. Those sample sets above the broken diagonal lines represented unusually high contamination which, in most cases, appeared to result from inadequately cleaned and sanitized food contact surfaces. These conditions have since been corrected in the plants concerned. However, the set marked "A" in Fig. 1 was high because the black pepper used in the spice mixture contained 40,000,000 bacteria per g. The incorporation of approximately 4% of high-count spice mixes to low-count pork also increased the APC significantly in four other sets of samples (sets "B", Fig. 1). Additional data from plant Y samples are shown in Table 1. In sets 2 and 3 of Table 1, the high count in the spice mix raised the count in the final packaged product because the bacterial level in the pork cuts was low. On the other hand, in set 1, high levels of bacteria in the trimmings masked the adverse effect of the high-count spice. Most fresh pork sausage processors purchased premixed spices for the product. In most cases, the spices did not add appreciably to the bacterial count of sausage. Analysis of 59 premixed spice samples collected at the plants revealed that all were negative for salmonellae, E. coli, and S. aureus; and 35 had APC values below 100,000 per g. However, 10 had APC values greater than 1,000,000 per g. These would add measurably to the bacterial level of low-count meat. Six firms prepared their own mixes from individual spices. Among 28 samples of these The set marked "C" in Fig. 1 was high because of the poor bacteriological quality of the casings. This, and a companion set collected in the same plant on a different date, are shown in Table 2. The trimmings had very low APC values. If the plant had used trimmings with APC values greater than 100,000 per g, the effect of the bacterial load of the casings could not have been measured. Figure 1 shows that there was no discernible difference in the range of APC values on pork trimmings from carcasses cut on premises and from those cut off premises. Initially, the APC values of pork trimmings and finished sausage were determined at both 35 C (2 days of incubation) and 25 C (4 days of incubation). The APC values at these temperatures were consistently similar, indicating that the bacteria were predominantly mesophiles and that the pork trimmings had not been in prolonged chill storage. It was noted during plant visits that pork sausage processors recognize fresh-ness to be essential for an acceptable product with a reasonable shelf-life. APC values at 25 C were discontinued during the latter stages of this survey. Figure 1 also shows that the range of bacterial counts of whole-hog sausage was similar to sausage processed from trimmings, though most whole-hog sausage was processed from freshly slaughtered, warm carcasses. Apparently the flushing of processing equipment by the surge of tissue, and prompt chilling of the finished product make whole-hog sausage neither more nor less susceptible to bacterial contamination and growth. Also, samples collected at intervals during the day in whole-hog sausage plants did not vary significantly in bacterial counts. Table 1 shows that in plant Y the samples of set 3 had counts similar to those of set 2, which had been collected 4 hr earlier. Salmonellae were isolated from 150 (28%) of 529 pork trimming samples and from 158 (28%) of 560 sausage samples. Semiquantitative analysis revealed that salmonellae, when present, were at low levels. Of the 150 salmo- Thus, on the average, the processing of trimmings into sausage did not lead to increased salmonellae contamination. Samples of skin from each of 36 eviscerated carcasses in 29 plants were examined. Thin strips of the skin were cut aseptically from the shoulder of carcasses on rails. In most cases, the animals had been slaughtered and eviscerated the previous day; in every case, the carcasses were to be cut and trimmed on the day of sampling. Salmonellae were recovered from 5 of the samples; low levels of E. coli and S. aureus were isolated from 11 and 15 samples, respectively. Three of the samples had APC values of more than 106/g, but the geometric mean of the APC values of the 36 skin samples was 46,000/g. This figure is in close agreement with the bacterial levels found by Dockerty et al. (2) on pork carcasses at postevisceration, prechill, and postchill steps. Samples of tissue below the skin were collected from the same carcasses and, as expected, almost all were sterile by the test methods employed. Almost invariably, pork trimmings had much higher bacterial loads than eviscerated carcasses. Thus, the bacterial counts on pork for sausage increased during cutting, trimming, and boning of the carcasses. At the time of manufacture, 75% of the sets of fresh pork sausage samples had APC values of less than 500,000/g; 88% contained 100 or fewer E. coli/g; and 75% contained 100 or fewer S. aureusig (geometric means of 10 samples). Such bacterial counts are not excessive for a raw, ground-meat product which is cooked thoroughly by the consumer. Figure 1 shows that the APC (geometric mean of 10 samples) of only 20% of the sets of sausage samples was below 100,000/g. The plants consistently producing low-count sau-sage not only used low-count trimmings, but also maintained excellent control of sanitary conditions, particularly in that all equipment and contact surfaces were cleaned thoroughly and treated with a sanitizing agent daily. Brooks (M.S. thesis, Univ. of Tennessee, Knoxville, 1968) demonstrated that, in a fresh pork sausage plant, thorough cleaning and pro- DISCUSSION After manufacture, chilled storage permits bacterial growth in fresh pork sausage during distribution and retailing. Elliott and Michener (4) described the factors affecting the growth of phychrophilic bacteria in chilled foods. Miller (8) found that 7 of 11 sets of pork sausage samples representing 10 brands collected at retail outlets had initial median counts of more than 106I/g and that some of the sausage had counts above 108/g. Miller also noted that the sausage with higher counts developed abnormal odors after 3 to 7 days of additional storage at chill temperatures. Freezing would, of course, prevent bacterial growth and extend shelf-life. Butler (1) and Hall, et al. (6) reported that seasoned pork sausage remained stable at frozen storage as long as unseasoned ground pork because the antioxidant properties of sage suppresses the pro-oxidant effect of sodium chloride. However, freezing cannot be a substitute for good sanitation in maintaining consumer protection or attaining a quality product. Hendrickson (7) reported that rapid chilling, proper management, and clean condiments were essential in retaining the quality of pork sausage during frozen storage; that large numbers of organisms were found to be conducive to off-flavors and rancidity development; and that palatability scores of the quality were found to correlate closely with the quantity of bacteria in the sausage.

v3-fos

2018-04-03T04:43:55.079Z

1972-06-01T00:00:00.000Z

22233785

s2

Dry heat inactivation of Bacillus subtilis var. niger spores as a function of relative humidity. Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Dry heat is the principal process being considered for spacecraft sterilization by the National Aeronautics and Space Administration. It is known that, at a given temperature, Dvalue (time to sterilize 90% of a population) varies with air moisture content (3). However, there is a paucity of data available for setting spacecraft sterilization cycles as a function of relative humidity. This is a report on dry heat sterilization of Bacillus subtilis var. niger spores at relative humidities (RH) from 0.0033 to 1.67% at oven temperature. 105 C D-values were determined for 20 RH values in this range. These values were used to set parameters in a kinetic model which incorporates relative humidity as an environmental parameter. The model is then used to predict D-values at 115 C and 125 C and compare them to experiments at these temperatures. MATERIALS AND METHODS Humidity determination. The standard formula for RH is (5): % RH = (e/e,.t) x 100 (1) where e is the pressure of water vapor present and esat is the pressure of saturated water vapor at the same temperature. Since, for air of a given moisture content, saturation temperature and dewpoint are the same, the RH inside an oven during dry heat sterilization (RH,) is given by % RH. = (ed/eo) x 100 (2) where ed is the saturated vapor pressure at dewpoint and e. is the saturated vapor pressure at oven temperature. The saturated vapor pressures are found in handbook tables (4). If the RH is known at a given temperature (T), the relative humidity of the same air at oven temperature is given by % RH, = (% RHTeT)/eo (3) where RHT is the relative humidity at T and eT is the pressure of saturated water vapor at T. Accuracy of RH determinations. RHO was determined either by use of equation 2 and direct dewpoint readings or by use of equation 3 and RHT readings from LiCl sensor-strip chart recordings. It was assumed that the direct dewpoint readings were accurate to 40.5 C. If ed is the saturated vapor pressure at dewpoint, ed+0.0 -ed > ed -edO0.5. For this reason we took the accuracy range for dewpoint RH0 determinations to be (ed/e0) + I(ed+0.Ied)/eol x 100. The LiCl sensor-strip recorder units were calibrated as a system by the Primary Standards Laboratory of Sandia Laboratories and gave % RH + 1% at ambient temperature. The temperature at which these readings were taken was 26 C. It can be seen from equation 3 that the error in RHO which results from a 1% error in RHT is the ratio eT/eo. The accuracy range for LiCl RH0 determinations were calculated as % RHO 4 (eT/eG). Humidity control systems. The humidity control used for a given experiment depended on the desired air moisture content. This determined the dewpoint for the oven input air. The initial step in the humidity control system assured an excess air moisture content. The ambient RH at our location is frequently as low as 10%. Thus for dewpoints above -10 C the input air was first passed through fritted glass tubes submerged in deionized water at 26 C. For dewpoints below -10 C, the moisture content of the incoming air was adequate. After assurance of a sufficient moisture content, the air was then either cooled to the desired dewpoint and the excess moisture was removed by condensation, or, if the dewpoint was below 2 C, the air was cooled to 2 C and excess moisture was removed by condensation under pressure. Condensation under pressure enabled us to attain dewpoints as low as -18 C. For dewpoints below -18 C, air having this dewpoint was passed through a desiccant bed having a bypass arrangement for dewpoint regulations. For dewpoints above 2 C, control was maintained by regulation of condensation temperature. Condensation occurred in cooling coils submerged in a tem-perature bath controlled to ± 1 C. For dewpoints in the range -18 to 2 C, dewpoint was controlled by regulation of the pressure under which condensation took place. If condensation takes place under the pressure P. absolute, RH % = f[e(P./P.)I/e..dt x 100 (4) where P. is ambient pressure absolute. Thus the dewpoints covered by this system configuration were easily controlled by pressure regulation. This pressurized air was expanded to ambient pressure prior to entry to the subsequent phase. A dewpoint of -52 C was attainable by passage of -18 C dewpoint air through a desiccant bed. Control was achieved through a bypass arrangement which allowed the mixing of the desiccated and -18 C dewpoint air. Air having the proper moisture content was warmed to 26 C, the RH determination was made either by dewpoint or LiCl sensor measurement, and the air was then fed into the oven. Air flow was metered at a level which maintained an oven overpressure of 0.03 ± 0.001 inch H20 and provided a 1-min replacement of oven air. The equipment configuration shown in Fig. 1 is for a test using input air with a dewpoint above -18 C. The desiccant bed, the only item not shown, was constructed from a sealable cylinder of approximately 3 ft3 volume. A 6-inch layer of the desiccant, CaSO4, was supported at the center of the chamber by a porous mesh. Air entered at the bottom and exited at the top. Spores. B. subtilis var. niger spores acquired from Fort Detrick were cleaned of vegetative material by multiple centrifugation and suspended in 95% ethanol at a concentration of approximately 107 spores/ml. The suspension was stored at 4 C. Sample preparation. The spore suspension was insonated for 2 min to distribute the spores uniformly. For each sample, 0.1 ml of the insonated spore suspension was pipetted onto the surface of an aluminum disc 1.25 inches in diameter. These discs were cut from 0.0015-inch biological grade aluminum foil. After the ethanol had evaporated, four of the inoculated discs were placed on an aluminum strip 1.5 by 7 by 0.020 inch. A single clean foil disc was placed over each inoculated disc, and the entire unit was covered by another aluminum strip. The assembly was held together by wire clamps. One such assembly was prepared for each sampling period. The sample strips were then placed in an evacuated, 23-inch Hg vacuum, dessicator over CaSO4 for approximately 16 hr before exposure to the dry heat environment. Exposure method. The spores were exposed to the RH-controlled dry heat environment in the temperature chamber shown in Fig. 1. The strips were placed on a perforated metal cage within the chamber. The chamber door was modified by the addition of small plugs at its center. Thus, individual strip assemblies could be inserted or removed The slight overpressure, 0.03 inch H2O, in the temperature chamber was necessary for the maintenance of RH stability. This overpressure prevented the diffusion of outside air into the temperature chamber. Recovery methods. Each sample strip of -four inoculated discs represented a single sampling period. After exposure, the spores were removed from the foil discs by 2-min insonation at an energy level of 11 watts/inch2 in 10 ml of sterile 0.1% Tween 80water. Tenfold serial dilutions were made as required and plated out in duplicate on Trypticase Soy Agar. Plate counts were made after 72 hr of incubation at 35 C. All inoculation, assembly, and recovery operations were carried out in a class 100 vertical laminar airflow clean room. Data analysis. Each experiment covered from four to six sampling periods. Data for each sample time came from eight plate counts for each of the serial dilutions. All data for a given experiment were input to a computer program which (i) computed the coefficient of variation for the eight plate counts for each dilution; (ii) selected from the data for a given sampling time that dilution which had the smallest coefficient of variation; (iii) computed a best linear least squares fit in the semilog plane to those data points from the dilution values with smallest coefficient of variation; (iv) computed the D-value from this least squares fit; (v) computed a 95% confidence interval about the D-value as follows: 0.95 CI = D 4 1.96 DE/S where D is the D-value, E is the standard error in the estimate of the slope of the least squares linear fit, and S is the slope of that line; and (vi) computed the average coefficient of variation for the data used for the least squares fit. The coefficient of variation provides a measure of the tightness of the data for a given dilution. The 95% confidence would be 0 if the standard error in the estimate of the slope were 0. This provides a measure of the linearity of the data and indicates the extremes of the D-value range for that experiment. Since there are inherent errors in both the D-value and RH determination, the data were smoothed by taking a moving average of each three consecutive points in the D-value-RH plane, consecutive in the sense of increasing RH. RESULTS The test series consisted of 27 experiments. Twenty-two were carried out over an RH range of 0.0033 to 1.6% at a sterilization temperature of 105 C. Twenty of these experiments were for determining 105 C D-values as a function of RH and two were designed for checking possible diffusion effects resulting from our use of covered discs. The environmental conditions, D-values, average coefficients of variation, and 95% confidence intervals on the D-values for the 20 D-value determination experiments are shown in Table 1. Figure 2 shows the D-values and 95% confidence intervals as a function of RH at sterilizing temperature. The moving averages discussed above are also shown in Fig. 2. Figure 3 shows a comparison of survivors from covered foil disc assemblies and open planchetts. The solid line is the least squares fit to the covered disc data, and the dotted line is that for the planchetts. The simultaneous exposure to 105 C of the covered discs and planchetts was at an RH of 0.0033 ± 0.00014%. To set sterilization parameters, it was necessary to have D-values for a fixed RH at two temperatures. D-values other than 105 C were desired for predictive checks. To this end, three experiments were carried out at 125 C and two at 115 C. The results of these experiments are shown in Table 2. Kinetic analysis. A model for dry heat sterilization which gives D-value as a function of temperature and relative humidity is useful for spacecraft sterilization applications. It was suggested in (1) that if AH# were constant over the RH range of interest, D-value could be modeled by expressing AS# as a function of RH. Here AHI and AS$ are the thermody- The assumption that sterilization can be expressed in terms of D-values is equivalent to the assumption that sterilization is logarithmic. Analogously, first-order kinetics is logarithmic. We will assume that both temperature and RH can be modeled as environmental sterilization parameters by first-order kinetics. The relationship between D-value and the reaction rate constant of equation 5 is given by r = (loge 10)/D (7) where D is D-value. From equations 5 and 6 we see that TAS# -AHf = RT loge (rh/kt). namic parameters of Eyring kinetics in which reaction rate (r) is expressed by (2) r = (kT/h) exp ( -AF/RT) (5) where k is Boltzmann's constant, T is the temperature in degrees Kelvin, h is Planck's constant, R is the gas constant, and AF# is the energy of activation. Table 3 shows the moving averages of Fig. 2 and AS as a function of RH under the assumption that AH# = 25 kcal/mole. Figure 4 shows these AS# values as a function of RH. Finally, the utility of a modeling technique depends on its predictive qualities. Figure 5 shows D-values with 95% confidence intervals for the 115 and 125 C~sterilization experiments given in Table 2 DISCUSSION The technique of holding AH# constant and expressing ALSa as a function of RH appears to be a useful supplement to the kinetic modeling of dry heat sterilization. This allows for the inclusion of both RH and temperature as envi- ronmental parameters. Equation 3 shows how one can convert the RH scale of Fig. 4 to a temperature other than 105 C and equation 10 shows how to construct a AS4 curve for a different AH# base. Considering the number of experiments and lengths of confidence intervals, the drop in Dvalue through the oven temperature RH range of 0.02 to 0.2% must be real. An analysis which predicted this drop was presented in (1).

v3-fos

2014-10-01T00:00:00.000Z

1972-02-01T00:00:00.000Z

335900

s2

Further characterization of bovine keratohyalin. Extraction of serial sections of cattle hoof epidermis with solutions of calcium chloride, magnesium chloride, potassium chloride, sodium chloride, guanidine hydrochloride, ammonium sulfate, and potassium phosphate buffer (pH 7.0) at varying salt concentrations demonstrates that keratohyalin (KH) is extracted by these salts at certain molarities. Under given conditions of time and temperature, each salt has a specific extraction pattern, and similar salts have similar extraction patterns. Dialysis of the salt extracts of hoof epidermis against distilled water results in the macroaggregition of KH, as assayed by histochemical methods. Although the various macroaggregates appear identical at the histochemical level, they display different ultrastructural characteristics. Polyacrylamide gel electrophoresis of the sodium decyl sulfate-solubilized macroaggregates results in the fractionation of a 20 (or more) member homologous series of oligomers. Isolation of the various oligomeric species of bovine keratohyalin and re-electrophoresis indicate that the various KH species can undergo depolymerization. Amino acid analyses of the unfractionated bovine macroaggregates and the various molecular weight species of bovine KH are similar, further demonstrating homology of the oligomers. The molecular weight of the subunit (monomer) of bovine KH is 14,955, estimated from the amino acid analyses. . Physicochemical analysis of the resultant aggregates (termed "macroaggregates") (25), has indicated that they are com?osed of a 13 (or more) member homologous series of oligomers with a subunit (monomer) molecular weight of 16,900 (26) . In agreement with histochemical (12,15,18,21,25) and radioautographical (6,7,9) studies of in situ KH granules, the protein species contain significant amounts of serine, arginine, glycine, and histidine, and appear THE JOURNAL OF CELL BIOLOGY . VOLUME 52, 1972 . pages 4 5 3 -464 to be complexed to ribonucleotides (25) . Further evidence for homology and identity of the isolated material as KH is the production of specific antibody to bovine KH after immunization of rabbits with the isolated bovine macroaggregates or specific molecular weight species of bovine KH . 1 The present study was directed at defining the relationship between extraction of bovine KH and salt concentration, and further demonstrating the homology of the isolated KH by physicochemical methods and amino acid analyses of the various oligomeric states . Serial Extractions of Bovine Hoof Epidermis was obtained from the posterior aspect of cattle hooves (27) . Specimens were frozen in liquid nitrogen and sectioned with a cryostat set at 6 p . Other specimens were fixed in 80% methanol, 10% buffered formaldehyde, or 6% buffered glutaraldehyde (25), embedded in paraffin under vacuum at 40°C, and serially sectioned at 6 z. A single section of each preparation of cattle hoof epidermis (cryostat, methanol-fixed, formaldehyde-fixed, glutaraldehyde-fixed) was extracted in a solution of specified molarity for 15 min at 37°C . Solutions, varying at 10th molar intervals, were as follows : (a) sodium chloride (0 . Only potassium phosphate solutions were buffered ; in all other cases the extractions were performed at the pH of the specified solution . All chemicals were reagent grade . Guanidine hydrochloride and urea were obtained from Mann Research Labs . Inc ., New York . After extraction, sections were rinsed in distilled water for 2 min, then stained with either Harris' hematoxylin, Congo red, or diazotized sulfanilic acid (18,25) . Preparation of Macroaggregates from Bovine Hoof Epidermis Cattle hoof was dissected, and the entire epidermis located beneath the stratum corneum was retained (26,27) . Dissected tissue was minced with scissors, then extracted with 1 .0 M potassium phosphate buffer (pH 7 .0) (24, 25) . Additional samples of cattle hoof epidermis were extracted with 1 .0 M solutions of the reagents described above (sodium chloride, potassium chloride, guanidine hydrochloride, magnesium chloride, calcium chloride, ammonium sulfate), or with 8 M urea . Cleared extracts were dialyzed against 32 volumes of distilled water, and onset of visible aggregation was recorded . Macroaggregates were harvested by centrifugation (24, 25) . Additional samples of minced cattle hoof epidermis were fixed in 80% methanol for 12 hr at 4°C, rinsed in distilled water, then extracted with 1 .0 M potassium phosphate buffer (pH 7.0) as described above. Extracts were dialyzed against distilled water, and macroaggregates were harvested as described above . (25) . Macroaggregates (on the surface of Millipore filters) and specimens of cattle hoof epidermis were fixed in 80% methanol or Carnoy's solution (25), then stained with Harris' hematoxylin, Congo red, diazotized sulfanilic acid, sodium alizarin sulfonate, toluidine blue, methyl green-pyronin, and acridine orange (18,22,25) . Additional specimens were stained with fast green (1) . Electron Microscopy Macroaggregates (on the surface of Millipore filters) were fixed in phosphate-buffered glutaraldehyde, thin sectioned, then stained with uranyl acetate and lead citrate (24, 25) . Specimens were examined in a Siemens electron microscope at 80 kv . Polyacrylamide Gel Electrophoresis of Solubilized Macroaggregates Macroaggregates were solubilized in sodium decyl sulfate (NaDS) (Eastman Organic Chemicals, Rochester, N. Y.) (25) . Polyacrylamide gel electrophoresis of solubilized macroaggregates was performed on 9°,ßc separation gels (3% cross-linked) as previously described (19,25,26), except that the time of electrophoresis was extended until the glycinate-decyl sulfate boundary (26) approached gel bottom (in order to further separate the various oligomers) . Protein was determined by the method of Lowry et al., using bovine serum albumin (Armour Pharmaceutical Co ., Chicago, Ill .) as a standard (16) . Preparation of Bovine Keratohyalin Fractions 300-500 µg samples of solubilized bovine KH were fractionated on 9 670 polyacrylamide gels as described above . A sample gel was placed in 7.5% acetic acid to rapidly precipitate the fractionated species (25) . Visualization was aided by passing a beam of light through the gel which resulted in the scattering of light at the sites of precipitation . Areas corresponding to the monomer were excised and extracted (25) . Oligomeric species 2, 3, 4, and 5 were excised together and extracted as above, and are referred to as "fraction II", and oligomeric species 6 through 20 and nonhomologous species were similarly isolated and are referred to as "fraction III" . Additional samples of solubilized bovine macroaggregates were fractionated on 9% polyacrylamide gels, and the area corresponding to the nonhomologous species and the largest oligomers (approximately species 18, 19, and 20) were also excised and extracted . These species are referred to as "fraction IV ." Purified oligomers were prepared by selective unstacking of nonhomologous species on 3% polyacrylamide gels (3% cross-linked) (25) . Gel slices were homogenized with a high-speed shearing device (Polytron, Brinkman Instruments Inc., Westbury, N . Y .) in the presence of an equal volume of 0.02 M NaDS, and extracted for 48 hr at room temperature . Extracts were centrifuged at 75,000 g to remove gel particles (25) . 100 to 200-µg samples of the above preparations were subjected to electrophoresis on 9% polyacrylamide gels, then stained with amido black as described above . Additional samples of the KH monomer, fractions II, III, IV, and purified oligomers were precipitated from the extract by the addition of an equal volume of 10% trichloracetic acid . The resultant turbid suspensions were stored at 4°C for 24 hr, then collected by centrifugation at 75,000 g for 30 min . Pellets were washed in 90% methanol, lyophilized, and stored at -90°C . Amino Acid Analyses Samples of bovine monomer, fractions II and III were prepared by dissociating fraction IV as described above. These fractions and additional samples of fraction IV, purified oligomers, and unfractionated bovine macroaggregates were hydrolyzed in 6 N hydrochloric acid under nitrogen for 24 hr at 110°C . Amino acid analyses were performed by an automated amino acid analyzer (Beckman, Model 117) . All values are corrected for losses of threonine, serine, and tyrosine by extrapolation to zero hydrolysis time, as determined by hydrolysis of macroaggregates for 24, 48, and 72 hr . Tryptophan was estimated colorimetrically by the method of Gaitonde and Dovey (10) . Extraction of Bovine Keratohyalin from Tissue Sections In all cases, extraction was similar with either cryostat or methanol-fixed, vacuum-embedded sections (see below) . Formaldehyde or glutaraldehyde fixation of tissue, however, prevented extraction by all salts at any molarity . Magnesium and calcium chloride completely extracted bovine KH at the lowest salt concentrations of the salts studied (both between 0 .05 M and 0 .10 M) . As noted with guanidine hydrochloride, sodium chloride, and potassium chloride, extraction of bovine KH also occurred at all higher molarities of magnesium and calcium chloride (Fig. 2) . Ammonium sulfate completely extracted bovine KH at a salt concentration of 0 .5 M and continued to extract KH up to a salt concentration of 2 .9 M . Above 2 .9 M ammonium sulfate, extraction of bovine KH was greatly reduced (Fig . 3) . Potassium phosphate buffer (pH 7 .0) completely extracted bovine KH at a salt concentration of 0 .5 M and continued to extract KH up to 1 .3 M . Above a salt concentration of 1 .3 M, extraction was greatly reduced, although KH granules appeared swollen and in certain areas appeared to have coalesced (Fig. 4) . In Vitro Aggregation of the Salt-Solubilized Keratohyalin In all cases, dialysis of the high salt extracts of cattle hoof epidermis resulted in the aggregation of protein(s) (see below) . Grossly visible aggregation occurred in the sequence : (guanidine hydrochloride, sodium chloride, potassium chloride), (potassium phosphate buffer [pH 7.0]), (ammonium sulfate), (magnesium chloride, calcium chloride) . Dialysis of 8 M urea extracts of cattle hoof epidermis against 32 volumes of distilled water resulted in the formation of a gel which maintained the configuration of the dialysis bag . Light Microscopy Bovine macroaggregates formed from the various salt extracts were similar at the histochemical level to those formed from potassium phosphate buffer (pH 7 .0) extracts (25), staining with Harris' hematoxylin, Congo red, diazotized sulfanilic acid, sodium alizarin sulfonate, toluidine blue, and pyronin . Fast green stained in situ KH granules and macroaggregates, but diffusely stained other epidermal structures . Electron Microscopy Although bovine macroaggregates formed from the various salt solutions appeared identical at the histochemical level, finite differences were noted at the ultrastructural level (Figs . 6, 7, 8) . The most homogeneous-appearing macroaggregate was that formed from the potassium phosphate buffer (pH 7.0) extract (Fig . 6) . Macroaggregates formed from potassium and magnesium chloride extracts appeared to be composed of tightly packed smaller aggregates, somewhat similar in appearance to the small particles located at the margins of in situ KH granules (Fig . 7) . Macroaggregates formed from sodium chloride, guanidine hydrochloride, calcium chloride, or ammonium sulfate were more disordered in substructure, and had areas which appeared vacuolar (Fig . 8) . These latter macroaggregates also varied considerably in size and shape . Polyacrylamide Gel Electrophoresis of Solubilized Macroaggregates As previously described (25,26), electrophoresis of solubilized bovine macroaggregates formed from 1 .0 M potassium phosphate buffer (pH 7 .0) extracts resulted in the fractionation of a 13 (or more) member homologous series of oligomers (which migrate as doublets), and two nonhomologous species . By extending the time of electrophoresis (as described in Methods), a total of 20 darkly staining oligomers were observed (Fig . 9) . Electrophoresis of solubilized macroaggregates formed from the other salt solutions resulted in similar fractionation patterns . The homologous oligomers and the conformational isomers and nonhomologous species 21 and 22 (previously designated 14 and 15) (25) were stained by all the histochemical reagents, as has been previously described (25) . Identical fractionation patterns were obtained with KH which had been extracted from methanolfixed cattle hoof epidermis . Electrophoresis of the solubilized gel formed after dialyzing the 8 M urea extract against distilled water resulted in the fractionation of numerous bands which were heterogeneous and not a homologous series . Isolation of Bovine Keratohyalin Fractions Figs. 10, 11, 12, and 13 demonstrate the fractionation patterns of the monomer, fractions II, III, and IV obtained as described in Methods . In 45 8 THE JOURNAL OF CELL BIOLOGY . VOLUME 52,1972 all cases, the species originally excised from the gels are present in large amounts, but lesser amounts of the smaller oligomers and relatively larger amounts of the monomer are also present . Amino Acid Analyses The amino acid analyses of the bovine KH monomer, fractions II, III, IV, purified oligomers, and unfractionated bovine macroaggregates are shown in Table I . All values are corrected for losses of threonine, serine, and tyrosine as described in Methods . Solubility of Keratohyalin The isolation of bovine KH was predicated on extraction experiments performed on serial cryostat sections of hoof epidermis (23) . These experiments demonstrated that bovine KH could be extracted from hoof epidermis by potassium phosphate buffer (pH 7.0) in the range of 0 .5 M-1 .3 M, and suggested that the solubilized KH would be precipitated by lowering the salt concentration of the high salt extract of hoof epidermis below 0 .5 M . The present studies demonstrate that bovine KH is extractable by numerous salt solutions . Similar extraction as a function of salt concentration has been noted with protein-polysaccharide complex of bovine nasal cartilage (including decreased extraction at high salt concentrations) (20), but a precise explanation for the decreased extraction at high salt concentration is not known . Urea solutions (up to 8 M) failed to solubilize KH (at least as monitored histologically), presumably because the reagent is nonionic . Again, similar results have been noted with protein-polysaccharide complex, although urea did act synergistically with other salts (20) . Preliminary studies, however, FIGURE 6 Isolated macroaggregate which has been formed by dialysis of 1 .0 M potassium phosphate buffer (pH 7 .0) extract of cattle hoof epidermis against 32 volumes of distilled water . The core (2) appears electron-opaque and homogeneous, and is surrounded by a shell of less densely staining material (1) . Uranyl acetate and lead citrate . X 46,000 . FIGURE 7 Isolated macroaggregate formed by dialysis of 1 .0 M magnesium chloride extract of cattle hoof epidermis against 32 volumes of distilled water . The macroaggregate appears to be composed of closely grouped smaller aggregates which appear somewhat like the shell surrounding the macroaggregate in Fig. 6, and like the particles which are seen at the margins of in situ KH granules . Similar macroaggregates are formed by dialyzing potassium chloride extracts of cattle hoof epidermis against distilled water. Uranyl acetate and lead citrate . X 46,000 . FIGURE 8 Isolated macroaggregate formed by dialysis of 1 .0 M guanidine hydrochloride extract of cattle hoof epidermis against 32 volumes of distilled water . The macroaggregate has some characteristics of the particle described in Fig . 6 in that a dense central core (2) and a less dense shell (1) are apparent, but differs in that the central core has vacuolar areas (3) . Similar macroaggregates are noted when sodium chloride, calcium chloride, or ammonium sulfate solutions are dialyzed against distilled water . Uranyl acetate and lead citrate. X 46,000 . indicate that isolated bovine KH is also soluble in 8 M urea, provided sufficient time is allowed for solubilization . The marked difference in the solubility of KH noted with small changes in salt concentration (at least as small as 0 .05 M for magnesium or calcium chloride), as revealed by the extraction patterns, strongly suggests that macroaggregation is due to ionic or hydrogen bonding between the various KH oligomers . The formation of macroaggregates by in vitro aggregation as a function of salt concentration further supports this interpretation . could have been employed . The difference in appearance of the various macroaggregates at the ultrastructural level might indicate that salt-KH complexes are important in determining the macromolecular structure of the native KH granules and the isolated macroaggregates . The resemblance to in situ KH granules was most obvious with macroaggregates formed from potassium phosphate extracts, and may indicate that potassium and phosphate ions are complexed to the in situ KH granule . Isolation of Bovine Keratohyalin Fractions All oligomer fractions isolated from gels produced monomer and smaller oligomers on reelectrophoresis . Apparently, in the presence of NaDS, the oligomers break down by releasing monomeric units, and not by splitting . However, the relative stability of the oligomers as compared to the macroaggregates in the presence of NaDS suggests that oligomer formation and macroaggregation probably occur by different mechanisms, or that the stability of bovine KH oligomers in the presence of anionic detergent decreases as the size of the oligomer increases . Presumably, the macroaggregate or the in situ KH granule, which may represent the largest polymeric state, is the most sensitive to salt or detergent concentration . Because the solubility of the macroaggregate in NaDS and the continued breakdown of the oligomers in NaDS, it is possible that the entire mechanism of polymerization or depolymerization is noncovalent . Amino Acid Analyses The amino acid analyses of the bovine macroaggregates, monomer, fractions II, III, IV, and purified oligomers were similar (Table I) . These results further suggest the homology of the species and also suggest that the nonhomologous species 21 and 22 are either size or charge isomers of KH, as has been previously indicated (25) . The similar analyses also indicate that the entire macroaggregate is composed of a homogeneous group of proteins . The analyses resemble those reported for rat and human "histidine-protein" (2, 3, 4, 13, 28), but are markedly different from the analyses of rat KH as reported by Matoltsy and Matoltsy (17) . The molecular weight of bovine KH is 14,955 (after rounding off to nearest whole number) . Adding 8% (due to the presence of ribonucleotides) (25), the molecular weight is 16,150, which is in fair agreement with the subunit (monomer) molecular weight of 16,900 as determined on 9% polyacrylamide gels by the linear regression of the logarithm of molecular weight on mobility (26) . Definition of Keratohyalin Explicit in any definition of KH is the assumption that the particular substance(s) being defined composes in toto the in situ KH granule . In defining the homologous series as bovine KH, therefore, it is necessary to assume that the in situ KH granule is composed entirely of these homologous oligomers, i .e ., that the in situ KH granule is homogeneous . At present, there are no definitive data to indicate whether the in situ KH granule is homogeneous or heterogeneous . However, certain data favor a homogeneous nature for in situ KH granules . At the histochemical level, KH stains homogeneously with all stains employed (25) . Ultrastructurally, most KH granules appear homogeneous (5,24,25), although some granules appear heterogeneous (8,14) . However, as previously demonstrated, macroaggregates of bovine KH may appear very heterogeneous at the ultrastructural level, but are homogeneous at the molecular level (25 Tryptophan~( 0 protein species has large amounts of serine, glycine, arginine, and histidine (25) . Finally, immunization of rabbits with bovine macroaggregates produces antibody to the entire in situ KH granule .' The previous statement that there is no FIGURES 9-13 Electrophoresis of bovine KH fractions on 9% polyacrylamide gels stained with amido black . Fig. 9, electrophoresis of solubilized macroaggregates, demonstrating 20 bands distributed in a geometric series . Nonhomologous species XXI and XXII are not sufficiently separated to be visualized . Fig. 10, electrophoresis of bovine monomer revealing a single prominently stained band . Fig . 11, electrophoresis of fraction II, demonstrating the presence of oligomers 2 through 5, as well as the monomer. Fig. 12, electrophoresis of fraction III, revealing relatively greater amounts of oligomers 6 through 20 and the nonhomologous species, and lesser amounts of the KH monomer and the smaller oligomers . Fig . 13, electrophoresis of fraction IV, demonstrating the presence of relatively greater amounts of oligomers 18 through 20 and the nonhomologous species, but lesser amounts of the other species are also present. absolute method of identifying KH (at the molecular level), despite the present assumption of homogeneity, may still be true, depending on whether the definition is limited to a particular species (25) . In the case of bovine KH, however, it is possible to speculate that KH exists in situ as a biopolymer, probably stabilized by salt or hydrogen bonds . Relatively weak salt or hydrogen bonding seems to be involved in macroaggregation whereas stronger noncovalent linkages may be involved in formation of the molecular species which range in size from the monomer to the 20-mer . The subunit (monomer) molecular weight is 16,000-17,000, and is composed of a complex consisting of 87, RNA and 9270 protein, the protein being basic and containing large amounts of serine, glycine, arginine, and histidine . The function of KH remains unknown .

v3-fos

2018-04-03T02:13:09.457Z

1972-12-01T00:00:00.000Z

32701801

s2

Associative Growth Studies in Three-Strain Mixtures of Lactic Streptococcil A recently developed differential agar medium was used to study associative growth patterns in 17 different heterologous, three-strain mixtures of Streptococcus lactis, S. cremoris, and S. diacetilactis grown in milk. Mixtures were made by combining equal volumes of 18-hr milk cultures of the three species. Relative populations of component species were followed through three succes- sive transfers in milk after the initial mixed propagation. Direct evidence for strain dominance and compatibility was obtained. A procedure also was developed to estimate the extent of suppression of S. lactis and S. diacetilactis in a mixture containing a dominant S. cremoris strain. The technique described could be successfully applied in quality-control work in the dairy-starter manufacturing industry. Several investigators have examined associative growth relationships among the lactic group streptococci widely used as starters in the dairy industry. Hammer and Babel (4) and Knudsen (8) were the first to point out that, when lactic acid and flavor microorganisms are grown together in butter cultures, each type undoubtedly has an influence on the other. In 1954, Czulak and Hammond (3) determined "compatibility" among strains of lactic streptococci in triple-species, starter mixtures containing Streptococcus lactis, S. cremoris, and S. diacetilactis by applying phage tracer techniques. Their data indicated that mixtures of S. lactis, S. cremoris, and S. diacetilactis retained their near-original composition longer than did starters made exclusively of different S. cremoris strains. Later, Henning et al. (5) and Vedamuthu et al. (13) studied associative growth patterns in mixed cultures containing the three Streptococcus species also by using phage tracer techniques. They observed that S. diacetilactis invariably became dominant after even a few successive subcultures in milk. In our study, we attempted to obtain direct evidence for domination or compatibility by application of differential enumeration techniques (M. S. Reddy and 31-2) were included in this investigation. These strains were obtained from the culture collection of the Department of Food Technology, Iowa State University. The S. lactis and S. cremoris strains were selected on the basis of satisfactory acid-producing activity in the Horral and Elliker test (6). Cultures were maintained by transferring three times a week into reconstituted, pretested, nonfat dry milk (11% solids) and incubating at 21 C for 18 hr. Between transfers, the cultures were stored at 5 C. Counting medium and plating technique. The differential agar medium and the specific plating technique described by Reddy and his associates (M. S. Reddy, M. S. thesis, Iowa State University, Ames, 1971; 12) were used. Associative growth relationships in threestrain mixtures. The experimental design shown in Fig. 1 was followed. All platings for counts represented in this figure were made at culture dilutions of 10-7 and 10-8. These dilutions provided the best differential counting efficiency. Wherever an S. cremoris strain entirely dominated in a given mixture (as determined by counts at the 10-7 dilution), the extent of suppression of the corresponding S. lactis and S. diacetilactis strains was determined by inoculating lower dilutions (<10-f) of the culture mixtures into Niven broth (9) and sterile, reconstituted, nonfat dried milk. After sufficient incubation, the broth cultures were tested for NH3 (9), and the milk cultures were tested for diacetyl-acetoin (7 (7) provided the most probable numbers of the S. lactis culture in the mixture; in a parallel series, a positive test for both arginine hydrolysis and diacetyl-acetoin production revealed the most probable numbers of the S. diacetilactis strain in the mixture. Graphical representation of this procedure is shown in Fig. 2. RESULTS AND DISCUSSION Associative growth patterns in four representative S. lactis-S. cremoris-S. diacetilactis combinations from a total of 17 different mixtures are shown in Tables 1 and 2. All counts reported in these tables represent the numbers of colonies developing on the agar at the dilution of the mixed culture. Statistical analyses of the data were not made because of the obvious differences in the counts of the component strains for several of these mixtures. Associative growth responses shown in Tables 1 and 2 represent possibly four different types of growth relationships. In the combination consisting of S. cremoris ML,, S. lactis 7963, and S. diacetilactis 18-16, S. cremoris abruptly and totally inhibited S. Iactis and S. diacetilactis. In such instances, dominance could be directly attributed to specific antibiotic production as suggested by Collins (2). We reported earlier on similar observations with two-strain mixtures (11). The second type of dominance involving a progressive suppression of component strains is represented by the mixture comprising S. cre-moris DR7, S. lactis C2, and S. diacetilactis 18-16. In this instance also, the dominant component was a S. cremoris strain. Staggered inhibition observed in this mixture probably results from differences in competitive growth abilities and relative acid tolerances (1,3). An instance where dominance by a component culture other than a S. cremoris strain was encountered when S. cremoris ML., S. lactis C2, and S. diacetilactis 26-2 were grown together. Here, the S. diacetilactis strain entirely dominated the mixture after the second successive propagation. Other workers attributed the dominance exhibited by S. diacetilactis strains in starter mixtures to greater metabolic efficiency and versatility in utilizing the lactose and citrate of milk as carbon sources; another factor proposed was their relatively greater acid tolerance (13). In this mixture, S. cremoris ML, does not seem to produce antibiotic(s) against S. diacetilactis 26-2, although this S. cremoris strain effectively inhibited S. lactis C2 and 7963 as well as S. diacetilactis 18-16. Compatibility among component strains in a mixture is exemplified by the combination composed of S. cremoris HP, S. lactis 7963, and S. diacetilactis 31-2. From a practical standpoint, mixed cultures with similar associative growth behavior would be desirable for commercial application in the dairy industry (14). These experiments were repeated twice, and similar associative growth patterns were ob- Fig. 3. In the three-strain mixture containing S. cremoris DR,, S. lactis C2, and S. diacetilactis 18-16, both S. lactis and S. diacetilactis were suppressed progressively until the second transfer. S. diacetilactis 18-16 totally disappeared from the mixture at the third transfer. On the other hand, when S. cremoris ML, was combined with S. lactis 7963 and S. diacetilactis 18-16, the heterofermentative strain was not detectable even after the first transfer. These results also were confirmed by parallel inoculations of the various dilutions into the differential broth described by Reddy et al. (10). These observations again emphasize the need for discarding the practice of day-to-day transfer or "carrying" of mixed lactic cultures used in the dairy industry unless the mixtures in question have been pretested for compatibility and maintenance of the near-original proportions of the component strains through several transfers. In the combination involving S. cremoris ML,, S. lactis C2, and S. diacetilactis 26-2, the extent of inhibition of S. cremoris by the S. diacetilactis culture could not be ascertained with this technique. Also, it is not feasible to determine the degree of suppression of S. lactis (if it were to be inhibited below the detectable level at the 10-7 dilution) in a mixture where the dominant strain happens to be a strain of S. diacetilactis. Such problems could be solved if this technique is used in conjunction with phage tracer methods. Combinations of these two techniques also would facilitate examination of associative growth patterns in mixtures containing multiple strains of each of the three lactic group Streptococcus species.

v3-fos

2020-12-10T09:04:12.944Z

1972-12-01T00:00:00.000Z

237232784

s2

Effects of Irradiation on the Survival of Virus in West Coast Oysters Gamma irradiation was evaluated as a means of inactivating poliovirus in shucked and whole shellfish. Results indicated that there was a significant survival of virus at all levels of radiation tested. In recent years the use of ionizing radiation has been proposed as a potential means of eliminating possible food spoilage or pathogenic microorganisms from a variety of foods, such as shellfish (2,3,5). However, among these pathogens are viruses which are known to possess a certain degree of resistance to the inactivating effects of gamma radiation (4,6,7). Therefore, an investigation was conducted in our laboratories to determine the ability of ionizing radiation to inactivate viruses in shellfish. This report presents our preliminary findings. Two separate series of experiments were conducted. In the first study, 2-year-old Pacific oysters (Crassostrea gigas) and 3-year-old Olympia oysters (Ostrea lurida), obtained from a Shelton, Washington, oyster grower, were placed in 19-liter stainless-steel aquaria to which was added 3,500 ml of filtered, sterile seawater contaminated with poliovirus 1 (strain Lsc-2ab). Virus titer was approximately 1.0 x 104 plaque-forming units (PFU)/ml. The oysters were allowed to contaminate for 24 hr after which time they were dipped in a 1% hypochlorite solution to inactivate any virus adhering to the shell surfaces, rinsed in distilled water, and dried. The contaminated shellfish were divided into two equal lots. Those to be used in the first experiment were sealed, whole' in polymylar pouches (eight per pouch) and irradiated. The source of the gamma radiation was a Mark II food irradiator having a cobalt 60 source of 40,000 Ci, with a dose rate of 400 krads/hr. All samples were irradiated at an ambient temperature of 20 C. Doses represented 50, 100, 150, 200, 300, and 400 krads of radiation. The samples were allowed to remain at ambient temperature for 1 hr to provide for latent effects of irradiation, and then were assayed for virus content. Control samples consisted of contaminated oysters sealed in polymylar pouches, but not irradiated. In these studies, 100% virus survival was considered to be the virus titer existing in the shellfish after 24 hr of contamination. Samples were readied for assay by preparing 10% (w/v) homogenates of tissue using nutrient broth as a diluent. All homogenates were blended for 2 min at 6,500 rev/min in a Lourdes homogenizer. After clarification, serial decimal dilutions were prepared in nutrient broth, and the samples were assayed for virus by plaquing in duplicate monolayer bottles. The assay technique was that of Davis and Dulbecco as modified by Hsiung and Melnick (1). Primary African green monkey kidney tissue was used to prepare monolayers which were grown in 3 oz. (ca. 90 ml) prescription bottles. In the second study samples of contaminated shellfish were shucked, as aseptically as possible, and then the oysters and fluid were sealed in polymylar pouches (eight per pouch). Samples were then irradiated and assayed as described above. The results of studies with whole, irradiated oyster samples are presented in Table 1. Although there was a reduction in the total number of viable virus present from all samples, the greatest reduction in virus titer occurred in samples subjected to a dose of 400 krads. However, even after this dose of irradiation 88 virus PFU/g were recovered from C. gigas and 100 PFU/g from 0. lurida or, on a per unit weight basis, approximately 13% of the original virus contained per gram of oyster tissues. The results of irradiation studies with shucked oysters are shown in Table 2. As in the previous experiments, increasing doses of ir- The results reported are preliminary observations. However, they do indicate that viruses in at least one food product, oysters, are able to survive the inactivating effects of gamma radiation. This rate of survival, under experimental conditions, varied from 87 to 7.3%, depending upon the dose of radiation and the nature of the sample. However, due to the mode of pathogenicity of viruses, even a low percent survival of these pathogens cannot be considered satisfactory. In addition, taste testing of noncontaminated oysters subjected to the dose required to inactivate 90% or more of the viruses (400 krad) revealed that they had undergone organoleptic changes which rendered them unpalatable. Thus, it appears that further research is required to determine the ability of viruses in various foods to survive the inactivating effects of irradiation before this method of preservation can be safely applied.

v3-fos

2020-12-10T09:04:16.581Z

1972-06-01T00:00:00.000Z

237232027

s2

Microbial Utilization of Crude Oil The utilization of two crude oil samples of different quality at 4 and 30 C has been studied by using pure and mixed bacterial cultures obtained by enrichment procedures. Growth, emulsification, and utilization occurred readily at both temperatures. The crude oil residue is increased in specific gravity and readily sediments out of solution. A comparison of the chemical analysis of the oils by liquid and gas-liquid chromatographic procedures before and after growth showed that the n-saturate fraction had been preferentially used. Some utilization of the aromatic fraction also occurred. Enrichments obtained with a high-quality crude oil were not as effective in utilizing a lower quality crude oil as sole carbon source as a population enriched on the low-quality crude oil. The utilization of two crude oil samples of different quality at 4 and 30 C has been studied by using pure and mixed bacterial cultures obtained by enrichment procedures. Growth, emulsification, and utilization occurred readily at both temperatures. The crude oil residue is increased in specific gravity and readily sediments out of solution. A comparison of the chemical analysis of the oils by liquid and gas-liquid chromatographic procedures before and after growth showed that the n-saturate fraction had been preferentially used. Some utilization of the aromatic fraction also occurred. Enrichments obtained with a high-quality crude oil were not as effective in utilizing a lower quality crude oil as sole carbon source as a population enriched on the low-quality crude oil. In the last 15 years, there has appeared a large number of publications concerning the use of hydrocarbons as substrates for microbial growth (1,4,11,12; J. A. Williams and J. C. Williams. Microbial alteration of crude oil in the reservoir, Amer. Chem. Soc. Annu. Meet. N.Y., 7-12 September 1969). Such studies, however, usually involved the use of purified substrates, such as alkanes of specified chain lengths. Bacteria have been implicated as agents of whole oil catabolism both in oilbearing formations (in situ) and in artificial fermentation systems by Davis (2) and Williams and Winters (Microbial alteration of crude oil in the reservoir, Amer. Chem. Soc. Annu. Meet. N.Y., 1969). It is also apparent that whole oils spread artificially, or accidently, on soil are degraded within months or years of application depending on the quality and frequency of the oil spilled and upon the prevailing environmental conditions. A study of the relationship between crude oil quality and its biodegradability was carried out by comparing the effect of growth of pure and mixed bacterial cultures obtained by soil enrichment on the composition of crude oils of different quality. The effect of temperature on this relationship also was studied, as psychrophilic environmental conditions prevail during the greater part of the year in northern areas which could affect the rate of biodegradation of crude oils. MATERIALS AND METHODS Microbiological methods. All cultures used in these studies were obtained by enrichment procedures from soils which had been contaminated with crude oil over a period of time. Soil samples were obtained from a producing well site in the Judy Creek area of north-central Alberta, from a diesel fuel spill near Salmon Arm, British Columbia, and from a green house soil which had been saturated with crude oil 2 years prior to sampling. Enrichments were carried out in 2-liter Erlenmeyer flasks containing 1 liter of basal salts medium consisting of K2HPO4, 0.5 g; NH4Cl, 1.0 g; Na2SO4, 2.0 g; KNO., 2.0 g; CaCl2 6H2O, 0.001 g; MgSO4-7H2O, 1.0 g; and a trace of FeSO4 in 1,000 ml of distilled water. Fifty milliliters of a 10% (w/v) suspension of soil plus 1.0 ml of crude oil was added to each flask which was incubated at 4 or 30 C on a rotary shaker (300 rev/min, 1 inch eccentricity). Transfers were made into the same medium at appropriate intervals with 50 ml of culture as inoculum. Growth was monitored by using the plate count technique and a medium consisting of the above salts solution (minus oil), to which was added yeast extract (Difco), 1.0 g/liter; sodium lactate (60% solution), 2.5 ml/liter; and 15.0 g of agar (Difco) per liter. This medium also was used for the isolation of pure cultures. An incubation temperature of 4 C was used for the psychrophilic studies and 30 C for the mesophilic experiments. Different levels of aeration were obtained by varying the amount of medium in the 2-liter Erlenmeyer flasks and keeping the agitation constant, i.e., 300 rev/min and a 1 inch eccentricity. The sulfite oxidation method (3) was used to determine the rate of oxygen transfer into solution. Analytical methods. Residual crude oils were extracted from cultures by using n-pentane as a solvent. Cultures were divided into two equal fractions and extracted three times with a volume of solvent equal to 30% of the original divided culture volume. The aqueous phase was removed each time by using separatory funnels and draining to the interfacial area which contained the salts, asphaltenes, and sol-1082 vent. This interfacial material was removed after the last solvent extraction, pooled with the n-pentane washes, and evaporated to dryness in a fume hood. The residual petroleum was recovered from the beakers by using 10 to 20 ml of benzene. The walls of the beaker were rigorously washed with this benzene prior to its removal and storage for analysis. Liquid chromatographic fractionation. Samples containing 0.05 to 0.4 g of residual or crude petroleum were placed in a tared beaker and "topped" by exposing them to forced draft conditions for 19 hr at either 32 or 36.5 C. This treatment removed volatile materials, e.g., light aromatics, napthenes, and nalkanes up to and including C,, chain length, leaving a weight referred to as the "topped weight of oil." The asphaltenic component of the "topped oil" was precipitated by addition of n-pentane. The pentane solubles and precipitated asphaltenes were then applied to a 1 by 15 cm bed of Hyflo Super-Cel (Fisher Scientific Co.) suspended in n-pentane. The column was sequentially developed with 50 ml of npentane and 40 ml of benzene to elute the deasphaltened oil and benzene-soluble asphaltenes. The weight of benzene-insoluble asphaltenes remaining on the column was calculated by difference. The deasphaltened oil (n-pentane-soluble) was fractionated by adsorption chromatography by using a dualphase column (1 by 40 cm) containing in the bottom half 10 4 0.2 g of activated 28-200 mesh silica gel (Matheson) and in the upper half 10 + 0.2 g of activated F-20 alumina gel (Matheson). Both phases were suspended in n-pentane. The deasphaltened oil was layered on the top of the column and then eluted sequentially with 65 ml of n-pentane, 100 ml of benzene, and 100 ml of a 1:1 mixture of benzenemethanol. This procedure ( Fig. 1) eluted the saturate, aromatic, and soluble NSO components in that order. (NSO component-fraction recovered by elution with a 1:1 benzene-methanol mixture. This fraction should contain more polar compounds than those eluted with benzene which yields the aromatic fraction of crude oil.) The sum of these three weights subtracted from the deasphaltened weight originally applied to the dual-phase column yielded the insoluble NSO component weight. This procedure is similar to that used by Imperial Oil Research Laboratories, Calgary, Alberta. Gas-liquid chromatographic analysis of saturate hydrocarbons. The separation and determination of n-saturate alkanes was achieved by using a Varion Aerograph Chromatograph model 1740-1 equipped with a flame ionization detector and a 10 foot (N6 inch inner diameter) stainless-steel column containing (100 to 200 mesh) chromosorb P precoated with 3% OV-1. Columns were conditioned at 325 C for 72 hr prior to use, and all carrier gases were purified by passage through Hydro-Purge Molecular Sieve 5A filters prior to passage through column or detector. The instrument was programmed as follows: linear temperature program 50 to 325 C; rate of programming, 10 C per minute; injection block temperature, 300 C; nitrogen flow rate, 12 Forty micrograms of saturates could be readily resolved by using these conditions. Benzene was used as solvent as it passed through the OV-1 column well in advance of all other components and is less volatile than n-pentane. Chemicals. The solvents used in the fractionation of the crude oil were of spectral quality and obtained from Fisher Scientific Co. The two crude oil samples used in these studies were obtained through Imperial Oil Research Laboratories, Calgary, and originated in the Weyburn area of south-east Saskatchewan. The sample from the North Cantal field represents a high-grade crude oil having more saturates and less sulfur than the sample from the Lost Horse Hill field which represents an inferior grade of crude oil. Both samples are classified as having the same origin and their analyses are presented in Table 1. RESULTS Microbial utilization of a high-grade crude oil. The utilization of crude oil, i.e., North Cantal sample, at mesophilic temperatures, i.e., 30 C, is shown by the increase in number of viable cells as a function of time (Fig. 2). The most striking effect of bacterial action on the crude oil was a change in its specific gravity. Repurified, residual crude North Cantal oil showed a specific gravity of 1.046 after 21 days of incubation as compared to its original specific gravity of 0.827 (as determined by pycnometric measurement). This resulted in a residual material which gradually settled to the bottom of the flasks. However, even before this specific gravity change had occurred, emulsification of the petroleum was observed. Similar growth patterns were obtained through seven successive transfers at which time the population consisted of at least three distinct morphological types. These were identified as being a nonpigmented Pseudomonas species, a Flavobacterium species, and an Achromobacter species. A fourth colonial type appeared, which was classified as a Bacillus'. species, but its incidence suggested that it could have arisen as a contaminant rather than being part of the original enrichment mixture. Since their initial enrichment, this mixture has been maintained for over a year by semiweekly transferring on salts medium plus 0.1% crude oil without any detectable changes in the proportion of colonial types comprising the mixed population. Liquid chromatographic analysis of the crude oil residue after the growth of the mesophilic mixture is presented in Table 2. The saturate fraction is the primary source of carbon and energy for growth as there has been about a 30% reduction in weight of this fraction during the 21-day incubation period. However, a calculation of residual weights which would be expected if only the saturates were used shows a decrease in weight of the aromatic fraction. This suggests that the aromatic fraction was also subjected to microbial attack. The utilization of the components of the saturate fraction was followed by gas-liquid chromatography and the results are presented in Fig. 3. The short-chain saturates present, i.e., C16 to C25, are used before the longer chain ones although they all disappear within 14 days of incubation at 30 C. The isoprenoids pristane and phytane are more resistant to microbial attack, requiring at least 2 weeks of incubation before being utilized. Similar results were obtained at 30 C with a pure culture (tentatively identified as a Micrococcus species) except that there was no preferential earlier digestion of the shorter chain saturates. Phytane and pristane were also metabolized at a faster rate by this Micrococcus than by the mixed culture (Fig. 4). Enrichment at psychrophilic temperatures, i.e., 4 C, resulted, after several subcultures, in the establishment of a population consisting of a small gram-negative pseudomonad, a small, thick, unidentified gram-negative rod, and a gram-positive rod tentatively identified as a Bacillus species. Similar mixtures were observed in enrichments from both the Judy Creek and Salmon Arm soil samples. These were considered to be nonobligate psychrophiles as they grew as well at 30 C as at 4 C. The growth of this mixed population on whole crude oil is shown in Fig. 5. Liquid chromatographic analyses of the crude oil residue after growth of the Judy Creek mixture shows that the saturate fraction is preferentially used (Table 3). However, calculation of expected weights of various fractions if only the saturates were degraded indicates that, in contrast to mesophilic growth, under psychrophilic conditions very little of the aromatic fraction is used. Gas chromatographic analysis of the saturate fraction as a function of psychrophilic growth is presented in Fig. 6. The utilization pattern is more similar to that of the mixed mesophilic population than is the Micrococcus species pattern. The isoprenoids phytane and pristane again show a greater resistance to microbiological degradation than the n-alkanes of the saturate fraction. Effect of aeration on utilization of crude oil. The effect of aeration on increase in viable counts is presented in Fig. 7. Sulfite oxidation values (millimoles of 02 per liter per hr) of 27.5, 19.0, and 12.5 millimoles of 02 per liter per hr were obtained with liquid volumes of 250, 500, and 750 ml/2-liter flask, respectively. Increasing the liquid volume to 1,000 ml did not further reduce the sulfite oxidation value. It would appear that the rate of growth and total yield of cells are not affected by aeration levels under these conditions. However, analyses of the crude oil by liquid chromatography (Table 4) and by gas-liquid chromatography (Fig. 8) indicate a differential utilization of crude oil components. The results of both analytical techniques show a more rapid utilization of the saturate fraction under conditions of maximum aeration. Utilization of purified oil components by mixed cultures at mesophilic temperatures. The utilization of the individual components of the North Cantal crude oil sample as growth substrates is shown in Fig. 9. In the purified state, only the saturate fraction sustained the growth of the mixed population which had been derived by enrichment on whole crude oil Table 1, footnote a. at mesophilic temperatures. In contrast to the suggested concomitant utilization of the aromatic fraction when the whole crude oil was subjected to microbial digestion, very little growth was produced with the aromatic fraction as the sole carbon source. Utilization of an inferior grade crude oil. Liquid chromatographic analyses of the residues of Lost Horse Hill crude oil samples after exposure to the mesophilic soil mixture enriched on a high-quality North Cantal oil sample and to the fresh enrichment obtained on the Lost Horse Hill sample are shown in Table 5. The population obtained from enrichment on the high-quality crude oil had very little effect on the composition of the lower quality Lost Horse Hill crude oil sample. However, an enrichment using the same soil as for the North Cantal oil sample brought up a population which readily brought about changes in this lower quality crude oil similar to those obtained with the high-quality North Cantal crude oil sample. Gas-liquid chromatographic analyses of these samples (Fig. 10) confirm the above observations. DISCUSSION The first evidence of bacterial activity on a highor low-quality crude oil is a quick and extensive emulsification of the oil followed by an increase in its specific gravity to a level greater than that of the medium used in these experiments. Sequential chemical analyses of the oil residues as microbial growth proceeds indicate that the n-alkane components of the n-saturate fraction were preferentially utilized as carbon and energy sources. The residue which forms the envelope in the gas-liquid chromatogram profile was comprised of iso-and cycloparaffins. The pattern of n-alkane utilization does vary depending on the population present, the longer chain components tending to be more resistant to microbial attack. For example, the psychrophilic mixture preferentially utilized nalkanes of chain length up to Cal whereas the mesophilic mixture used up to C25 prior to metabolism of the rest of this fraction. This suggests that a factor or factors other than solubility determines the utilization of n-alkanes by microorganisms. This preference had previously been noted (12). The isoprenoid compounds, phytane and pristane, are more resistant to microbiological degradation. However, the fact that they were degraded indicates that their use as biological markers has to be interpreted with care. The mechanism by which they were utilized was not investigated but may be similar to that outlined by McKenna and Kallio (8). The utilization of the remaining components, with the exception of the aromatic fraction, appears to take place slowly, if at all, under these experimental conditions. The apparent increased utilization of the aromatics in the presence of whole crude oil which contains the readily utilized saturates as opposed to the lack of growth when aromatics were the sole carbon source suggest that cooxidation is involved in their metabolism. They also would appear to be more readily utilized at mesophilic than at psychrophilic temperatures. Similar studies in this laboratory (unpublished observations) concerning the metabolism of this aromatic fraction of crude oil confirm this hypothesis. The works of other investigators (7,9) also confirm the requirement for the presence of an assimilable substrate in order to obtain utilization of certain model aromatic compounds. The insoluble NSO components in the metabolized oils have been increased beyond that value predicted from the theoretical enrichment calculations. This suggests that some modifications of the crude oil, presumably brought about by microbial action, have taken place yielding an increase in the amount of polar N-, S-, and 0-containing materials. Aeration, although not having a marked effect on either the rate of growth or the total amount of cells produced, did affect the utilization of the n-saturate fraction. The highest level of aeration used resulted in a 15 weight % reduction in the saturates present in the residual oil, whereas lower levels of aeration resulted in a reduction of only approximately 5 weight %. The constant yield of cells, however, suggests a more efficient conversion of n-saturates to cell material under conditions of reduced aeration. The quality of the crude oil used in enrichment studies has a marked effect on the capabilities of the populations to utilize crude oils of lower quality. A mixed population derived from enrichment procedures by using the highquality crude, i.e., North Cantal, had only a limited capability of utilizing the lower quality crude, i.e., Lost Horse Hill. However, populations derived by using a low-quality crude oil during enrichment procedures can readily metabolize a high-quality crude oil (unpublished observations). It is possible that the increased asphaltenic content of the low-quality crude oil or the altered aromatic character inhibits its utilization by a population derived when these components are present in lower concentrations. This result suggests that the successful use of "bacterial cocktails" to deal with accidental oil spills will be dependent, in part, on the mixture being composed of microorganisms capable of utilizing oils of the lowest quality comprising the spill. So far it has been impossible to degrade a crude oil completely under laboratory conditions. Populations are readily derived which can utilize a significant proportion of the saturate fractions and possibly some of the aromatic fraction. These chemical changes are accompanied by a change in the specific gravity of the oil from lighter to heavier than water and results in a product which forms an emulsion with the medium. This phenomenon has been well documented in the literature (6) and is attributed to a microbial product of hydrocarbon metabolism. There is, however, a substantial portion of the original crude oil material still found within the emulsified material. We suggest that this residual material is analogous to the humic matter remaining after the readily digestible material of organic matter has been metabolized by microorganisms and thus could be regarded as humic material. ACKNOWLEDGMENTS We thank N. Bailey and A. Rogers, Imperial Oil Research Laboratories, Calgary, Alberta, for their advice and technical support. In addition, we thank Colleen Dmytriw and Jack Kinnear for their assistance in photography and gas chromatography. The work was supported by the National Research Council of Canada, operating grant N.R.C. A-3687, and by the Imperial Oil Co. of Canada.

v3-fos

2018-04-03T05:33:45.265Z

1972-04-01T00:00:00.000Z

22842323

s2

Ammonium, nitrate, and total nitrogen in the soil water of feedlot and field soil profiles. A level feedlot, located in an area consisting of Wann silt loam changing with depth to sand, appears to contribute no more NO(3) nitrogen, NH(4) nitrogen, and total nitrogen to the shallow water table beneath it than an adjacent cropped field. Soil water samples collected at 46, 76, and 107 cm beneath the feedlot surface generally showed NO(3) nitrogen concentrations of less than 1 mug/ml. During the summer months, soil water NO(3) nitrogen increased at the 15-cm depth, indicating that nitrification took place at the feedlot surface. However, the low soil water NO(3) nitrogen values below 15 cm indicate that denitrification takes place beneath the surface. The possible movement of nitrogen-containing compounds from feedlot surfaces to groundwater is of serious concern to scientists and laymen alike. This concern is justified when one considers that in Nebraska alone about 3.5 million beef animals are fed annually with about 1.2 million on feed at a given time (8). These numbers are significant, inasmuch as a steer will excrete 0.18 kg of N per day (11), which amounts to approximately 216,000 kg of N deposited on Nebraska feedlots each day. Many of these feedlots are located on permeable soils over shallow water tables. The large quantities of N excreted on feedlots could provide a groundwater pollution hazard. However, to date, the effect of feedlots on groundwater quality is controversial. Several studies have been conducted on the quality of groundwater beneath land subjected to diversified uses. In Colorado, Stewart et al. (10) found high NO3nitrogen levels beneath some corrals and heavily fertilized, irrigated crops. However, they found almost no NO3nitrogen beneath other corrals. They felt the low NO3nitrogen values were due to denitrification beneath these corrals. In another study, Smith (9) attributed high NO3nitrogen levels in some Missouri aquifers to livestock feedlots. In contrast, Mielke et al. (7) found a level feedlot contributed limited NO3nitrogen to the groundwater beneath it. This feedlot was located on a coarse-textured soil 'Published as paper number 3248, Journal Series, Nebraska Agricultural Experiment Station. with a high water table. These studies seem to show that feedlots may or may not allow NO3nitrogen to move downward into the groundwater. Investigations are required to determine why some feedlots may contaminate the groundwater while others do not. If feedlot management schemes are available or can be devised to prevent contamination, research needs to delineate them. This study was conducted to measure NH,+ nitrogen, NO3nitrogen, and total N compounds present in the soil water from the surface to groundwater in a feedlot and a cropped field. MATERIALS AND METHODS The level, beef feedlot studied by Mielke et al. (7) was used as the research site. The feedlot has a permeable soil, in its natural state, over an aquifer that fluctuates between 76 and 305 cm beneath the surface during the year. The water table is lowest at the end of the irrigation season and highest in the spring. The feedlot soil profile consists of a Wann silt loam becoming sandy at 91 cm and changing to sand and gravel at lower levels. The feedlot is stocked at a normal rate of about 1 animal/37 m2, and no manure has been removed for the past 15 years. The manure has been mounded the last 2 years. Mounding is practiced as a method of on-site manure disposal, animal comfort, and a feedlot drainage aid. When the feedlot was scraped for mounding, care was exercised to leave a shallow layer of manure on the surface. Mielke et al. (7) found the infiltration rate was slow in the feedlot as a result of a dense layer that formed at the feedlot VOL. 23, 1972 AMMONIUM, NITRATE, AND NITROGEN IN SOIL WATER soil surface-manure interface. The thin manure cover was left to preserve the layer. Two caissons were installed in the feedlot, and one was placed in an adjacent cropped field (Fig. 1). The caissons served to protect soil water samplers (in duplicate in caisson) installed in the respective soil profiles at increments of depth (4). The soil water samplers consisted of porous ceramic cups in the soil profile to which vacuum was applied to obtain liquid samples. Samples were collected at 2-week intervals, or as weather permitted, between April 1970 and April 1971. A Technicon AutoAnalyzer was used to analyze for NO3,nitrogen by the hydrazine reduction method of Kamphake et al. (5); NH4+ nitrogen was determined by the indophenol method of Bolleter et al. (1). Nitrogen was measured by the microkjeldahl method described by Bremner (2). RESULTS Average monthly soil water NO3nitrogen levels were low in the feedlot except for the 15cm samples in late summer and fall ( Table 1). The 15-cm soil water NO3nitrogen increased from July through November. Probably this change can be attributed to nitrification near the feedlot surface. Soil water samples were not obtained at the 15-cm level in December and January because the feedlot surface was frozen. The other missing monthly samples were due to the soil being too dry for a suction sample to be obtained. Soil water NO3nitrogen values in the field varied, but usually were highest in May and June following fertilization and then declined rapidly. Because the field soil was quite dry at the 122-and 152-cm depths during and immediately following the growing crop, only scattered samples were obtained in this period, and these data were not felt to be representative. The feedlot NH4+ nitrogen was high in the soil water samples obtained at the 15-cm depth but declined markedly to low levels at 76 and 107 cm ( Table 2). The declining trends found with NH4+ nitrogen were similar to those found for NO3nitrogen. Ammonia values were generally very low in the field soil water samples. The total N in the feedlot soil water samples (Table 3) was high at the 15-cm depth, but at 46 cm and lower appeared comparable to, or less than, the field values. The highest value obtained from samples taken at 107 cm was 14 jug of total N/ml. Obviously, limited amounts of N-containing compounds were present in the feedlot soil profile. The field total-N samples followed the trends obtained for NO3nitrogen and NH4+ nitrogen. Table 4 shows a comparison of average yearly concentrations of the nitrogen compounds beneath a feedlot and a cropped field. The feedlot NO3nitrogen values at 46 cm and below appeared lower than those obtained from the cropped fields. The NH4+ nitrogen samples from the feedlot and field were similar at 76 cm and below. At these depths, total-N values seem to be higher in the field than in the feedlot. However, the total-N values include the NO3nitrogen values, which were higher in the cropped field. If the NO3nitrogen values are subtracted, the total-N values from 76 cm and below in the feedlot and the cropped field would be comparable. DISCUSSION Soil water samples indicate this feedlot contributes low amounts of NO3nitrogen, NH,+ nitrogen, and soluble N-containing compounds to the groundwater. Samples from the feedlot obtained at the 15-cm depth were high in these compounds; however, at the 76-cm depth the levels appeared as low as, or lower than, comparable field samples. There was evidence that nitrification took place in the feedlot because NO3nitrogen at the 15-cm depth increased during the summer with no increase at lower depths at any time. In another study, Elliott and McCalla (3) found that reducing conditions existed beneath this feedlot. That report, coupled with the data of Mielke et al. (7) and the information in this paper showing that NOnitrogen declined rapidly beneath the 45-cm depth, indicates denitrification probably takes place in this feedlot. The presence of CH4 (3) and soluble-N compounds indicates that organic matter, which is a requirement for denitrification, is present in the soil profile. Denitrification would account for the fact NO3-nitrogen was generally low in the feedlot. The infiltration rate in this feedlot is low because a dense layer forms at the soil-manure interface (7). However, the samplers at 15 cm are just beneath this dense layer, and these samples did show elevated levels of NO3nitrogen, NH4+ nitrogen, and total N. Therefore, it is assumed some materials pass through the dense layer. Because soil water NO3nitrogen increased during the summer, it must be assumed nitrification took place in the feedlot and NO3nitrogen did get below the dense layer. In view of the fact low NO3nitrogen was found below this depth, it would seem reasonable to postulate denitrification occurred. It may be argued that soil water NO3nitrogen content will fluctuate widely depending on soil moisture. This is true to a point; however, the sampling method will collect a sample only when suction is 0.7 bar, or less. Consequently, this effect would not be great enough to affect seriously the trend of the results. The literature shows some feedlots may contaminate groundwater, and this particular feedlot would seem a likely candidate. The feedlot has not been cleaned for 15 years and is situated on an originally permeable soil, and the water table fluctuates between 76 and 305 cm from the feedlot surface (7). However, the data presented here and elsewhere (7) show this feedlot does not contaminate the groundwater. Possibly, the answer lies in management. This feedlot is used continuously throughout the year. The surface of the feedlot, when the manure pack is intact, has a low infiltration rate (7). Therefore, it seems that if a feedlot is kept well stocked and the manure pack-soil interface is not disturbed, only limited organic matter and NO3nitrogen will reach the underground water supply (6). Also, the feedlot soil profile should remain anaerobic.

v3-fos

2020-12-10T09:04:16.712Z

1972-04-01T00:00:00.000Z

237231010

s2

Bruised Poultry Tissue as a Possible Source of Staphylococcal Infection Bacteriological analyses were made on 45 swab samples secured from hands of poultry workers on processing line, on 31 bruised and 15 normal poultry tissue samples, and on 15 swabs obtained from infected lacerations and exudates of abcesses on hands, arms, chest, and abdomen of poultry workers. A total of 170 Staphylococcus cultures were isolated from samples examined. These cultures were characterized morphologically and biochemically and then grouped into six distinct patterns. S. aureus was found in 86.6% of swab samples obtained from infected workers, in 40% of swabs from hands of workers who handle bruised birds, and in 38.7% of bruised tissues, and was absent from all samples obtained from hands of workers who do not handle bruised birds. All the coagulase-positive staphylococcal isolates were bacteriophage-typed, and the results showed that the same phage-type S. aureus was found in many poultry bruises and in infected lesions of poultry workers as well as on hands of workers who handle bruised birds. These results indicate that poultry bruises are a source of staphylococcal infection encountered among poultry workers. Microbiological examination of poultry bruises by McCarthy et al. (10) revealed that these tissues harbor large numbers of various types of bacteria, particularly Staphylococcus aureus. It was also reported that bruised tissue supported and stimulated the growth of Escherichia coli and S. aureus in vivo (5,6). The latter organism was able to persist in injured tissues for 18 days, even in the absence of noticeable infection (5). Although higher incidence of staphylococcal infections occurred in the wounds of poultry workers in the Clarke County areas as compared to the population at large (5), no epidemiological studies had been conducted to ascertain a definite source. These infections appeared among the employees of the poultry processing plants who came in contact with live chickens and carcasses during the initial stages of dressing. To substantiate the existence of a health hazard to these workers, the log book kept by the company nurse at a large poultry processing plant in Clarke County was examined. Analyses of 252 complaints recorded over a 4-day working period revealed that requests for treatment of staphylococcal infections occurred 35 times during this interval (Table 1). The majority of the infections were characterized as "oozing sores" resulting from cuts and scratches that became infected and failed to heal properly. These areas were most prevalent on the hands, arms, chest, and abdomen of poultry workers who came in contact with live chickens, particularly during the initial stages of dressing, thus confirming previous observations (5). Therefore, the present investigation was designed to examine the role of poultry bruises as a possible source of the staphylococcal infection encountered among poultry workers. This problem causes economic losses to poultry industry in terms of decreased manpower production as well as presenting a possible health hazard. MATERIALS AND METHODS Collection of samples. Forty-five hand swabs were collected on sterile cotton-stick applicators, and each was placed in a tube containing 10 ml of sterile saline. Thirty of these hand swabs were taken from processing line workers (trimmers) who handle the carcass birds prior to trimming of bruises and 15 hand swabs were obtained from those who handle normal healthy birds as well as birds that were trimmed of either bruised tissues or condemned parts of the carcass. The hands (back and palm) were swabbed thoroughly, including the fingernails and between the fingers. Fifteen other swab samples, obtained from sores, abcesses, and infected lacerations and lesions, were collected by the company 683 nurse. All samples were immediately cooled and subjected to bacterial analyses within 1 hr after collection. Poultry tissues were obtained from individual birds that were removed from the processing line at trimming stations. The skin over the tissue (normal or bruised) was swabbed with 70% alcohol, and the desired tissues were excised under aseptic conditions and immediately placed in sterile precooled petri dishes. Thirty-one bruised and 15 normal (control) tissue samples were collected and held at 2 to 4 C to prevent further growth of the organisms present. Each tissue sample was then aseptically minced and thoroughly homogenized (16,000 rev/min) with saline for 2 min in a precooled sterile Sorvall Omnimixer. Isolation and screening. Dilution measurements and bacterial isolation were executed by using standard bacteriological procedures. All tissue homogenates and hand swab samples were analyzed for both aerobic bacterial counts and staphylococcal population. The former was obtained by plating the proper dilution on tryptone-glucose extract-agar (TGEA), and the latter was made by plating on mannitol-salt-agar (MSA). Colonies appearing circular (1 to 2 mm in diameter), smooth, convex, glistening at the surface, with the entire edge surrounded by yellow or red zones on the MSA and exhibiting the morphological characteristics of staphylococci were examined and counted. Examination of Gramstained smears from suspected or typical colonies was always conducted to aid in the differentiation between Staphylococcus and Micrococcus. Enrichment techniques were employed to detect the presence of small numbers of staphylococci in samples. In this technique, 1 ml of saline suspension of the hand swabs or the swabs of the infected lacerations or lesions was inoculated into mannitol-salt broth containing 7.5% NaCl (to enrich selectively the staphylococci), and the tubes were incubated for 24 hr at 37 C. Growth from mannitol-salt broth was streaked onto MSA plates. The colonies on the plates were examined after incubation for 24 to 48 hr, and each staphylococcus isolate was again subcultured and stained (Gram-stained smears) to assure purity. All cultures were carried on TGEA slants and stored at refrigerator temperature until further analyses. The staphylococcal isolates were characterized by colony pigmentation on Staphylococcus medium 110 (Difco) after incubation at room temperature for 48 'ID HAMDY APPL. MICROBIOL. hr, by mannitol fermentation (anaerobically), by coagulase, lysozyme, deoxyribonuclease, gelatinase, and alpha hemolysin activities, and by bacteriophage typing. Mannitol fermentation was performed by stabbing freshly heated and cooled phenol red mannitol-agar tubes and observing for color changes. after 12 and 18 hr of incubation at 37 C. Determination of deoxyribonuclease was conducted on deoxyribonuclease test agar (Difco) by using the technique of Jeffries et al. (9). After incubation of the inoculated plates for 24 hr at 37 C, the plates were flooded with 1 N HCl and a clear zone (3 to 5 mm in diameter) was considered positive for deoxyribonuclease. The coagulase tube test was performed with reconstituted coagulase plasma (Difco) on all gram-positive staphylococci isolated and the tubes examined after 4 hr of incubation at 37 C. Any degree of clotting, however slight, was considered positive. Liquefaction of gelatin was tested by inoculating Chapman stone medium and examining for clear zones surrounding colonies after 48 hr of incubation at 30 C. The alpha hemolysin was detected by using rabbit blood-agar plates containing 2% blood. Lysozyme activity was determined by the plate method using a modified procedure previously reported by Grossgebauer et al. (4) and Jay (8). A 100-ml suspension of lyophilized cells of M. lysodeikticus (1 mg/ml) in 0.06 M phosphate buffer (pH 6.2), and 400 ml of Brain Heart Infusion agar (Difco) were prepared. The agar and suspensions were autoclaved separately at 15 psi for 15 min, cooled to 55 C, immediately mixed, and poured into sterile petri dishes. The plates were then inoculated with the test organism and incubated for 24 hr at 37 C. A clear zone (5 to 10 mm in diameter) surrounding the growth was considered positive for lysozyme activity. Bacteriophage typing. Coagulase-positive staphylococci were phage-tested by the method recommended by Blair and Williams (2) as modified by Blair and Parker (1). Each culture was streaked onto a Trypticase soy agar (TSA) plate and incubated overnight to check purity. A typical colony was then picked into Trypticase soy broth and incubated at 37 C for 4 to 5 hr. The broth culture was then spread over the surface of a sterile TSA plate and excess broth was removed. After the surface had dried, one drop of each phage at its routine test dilution (RTD) was placed on the seeded plate in a standard pattern. The plates were dried again (at room temperature) and then incubated overnight at 30 C. They were examined for lysed areas, and lytic reactions of 50 or more plaques were recorded. Weaker reactions were not reported unless they were contributory to establishing identity of patterns. If a culture was not lysed by RTD phage, it was retyped with phages at 1,OOOx RTD. Only strong (50 plaques or more) reactions were recorded. Phage patterns which differed by two or more strong lytic reactions were generally considered as different patterns. Phage typing was performed by P. B. Smith (Center for Disease Control, Atlanta, Ga.). RESULTS AND DISCUSSION A total of 170 cultures was isolated from the 106 samples collected. These cultures were characterized for colony pigmentation and other biochemical activities and then grouped into 6 distinct patterns (Table 2). Isolates exhibiting the reaction patterns of types 1 and 4 (42 cultures, 24.7% of total) represent S. aureus; types 2, 3, and-5 are characteristics of various strains of S. epidermidis (120 cultures, 70.6% of total); type 6 (8 cultures, 4.7% of total) may be considered a potentially pathogenic strain of S. epidermidis. Bronson (3), Grossgebauer et al. (4), and Hawiger (7) reported that coagulase-negative staphylococci which produced lysozyme were considered pathogens and are able to cause infection. Table 3 summarizes the results of bacterial counts (on TGEA and MSA) for all samples except those obtained from sores, abscesses, and infected lacerations. The data revealed that these samples contained high bacterial populations, the majority of which were salttolerant as indicated by the viable counts on MSA plates. McCarthy et al. (10) showed that bruised tissues, secured randomly from broilers on a commercial processing line and from experimentally inflicted bruises, contained a microbial count relatively high both aerobically and anaerobically compared to control samples. Many gram-positive micrococci were encountered on these plates. However, the predominant staphylococcal organism isolated from all samples (pattern 3) had white-pigmented colonies on staphylococcal medium 110, had no alpha hemolysin, did not ferment mannitol anaerobically, and exhibited no enzyme activities tested except deoxyribonuclease. These bacteria were found in 55 samples (51.9% of total) and are not considered ' From symmetrically located areas of the bruises on the same bird. ' Samples were not assayed. pathogens. The only other bacterium found in considerable numbers was a large gram-positive Bacillus which was encountered in eight of the samples examined. On the other hand, it was observed that 40 samples (37.7% of the 106 samples) contained large populations of pathogenic staphylococci (patterns 1, 4, and 6). Twenty-eight (70%) of these pathogens were S. aureus cultures which exhibited golden-pigmented colonies, fermented mannitol, and produced alpha hemolysin, coagulase, deoxyribonuclease, gelatinase, and lysozyme (pattern 1). It is of interest that pathogenic staphylococci (patterns 1, 4, and 6) were consistently isolated from 13 swab samples (86.6%) obtained from infected lacerations, abscesses, and sores, from 12 hand swab samples (40%) secured from workers who handle bruised birds and from 12 bruised tissue samples (38.7%). They were found in only 20% of samples obtained from normal tissue located symmetrically to the bruise on the same bird. None of the hand swab samples obtained from poultry workers who did not handle bruised birds contained staphylococcal organisms with the aforementioned characteristics. These results indicated that pathogenic staphylococci designated as patterns 1, 4, and 6 could be transmitted from bruised tissue to poultry workers who handle bruised birds and possibly to other healthy birds. To substantiate these findings, all the coagulase-positive staphylococcal isolates were phage-typed, and the results are recorded in Table 4. It was observed that three of the isolates obtained from poultry workers with infected lacerations, sores, and abscesses were of The results verify that bruised tissue can harbor S. aureus of the same phage type as found in infections and from hand swabs of workers who handle birds with bruises. Such staphylococci were not detected in hand swab samples obtained from workers who handle poultry only after the bruises have been trimmed away. The results strongly suggest that poultry bruises comprise a significant source of staphylococcal infections occurring among poultry workers. The finding of S. aureus in bruised tissue also may present a potential health-hazard to the consumer and suggests that more stringent measures may be required with respect to the further processing of bruised birds. When the authorities of one poultry processing plant in Clarke County were informed of the results obtained in this investigation, a new protocol was initiated among poultry workers. The protocol consisted of a thorough washing with sudsing antibacterial skin cleanser (pHisoHex, Winthrop Lab.) to all cuts, wounds, and scratches followed by Merthiolate painting and application of a triple antibiotic ointment (polymyxin B, bacitracin, and neomycin). When this treatment was followed, the incidence of staphylococcal infection was drastically reduced as evidence by the decreased number of infected lacerations and cuts among poultry workers examined during that time. Nine staphylococcal infections were reported among poultry workers of this plant during a 5-month period following the initiation of this protocol as compared to 35 cases during the 4day period prior to this treatment.

v3-fos

2020-12-10T09:04:13.099Z

1972-10-01T00:00:00.000Z

237230458

s2

Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies. The need for a faster method for the detection of salmonellae led to the development of the fluorescent-antibody (FA) technique. The FA technique can serve as a useful tool in screening raw materials, environmental samples, and finished products (1-4, 6-9, 11-14). Many organizations now testing for salmonellae employ the time-consuming (5-7 days) and laborious method of the Association of Official Analytical Chemists (AOAC). The objectives of this study were segmented into three phases. The first phase dealt with the development of a cultural method designed to enhance the rate of growth of low numbers of salmonellae and suppress the growth of organisms other than salmonellae. The following three-step cultural method was designed to satisfy these criteria: (i) a 7-hr pre-enrichment, (ii) a 17-hr selective enrichment, and (iii) a 5-hr modified M broth (Difco) elective enrichment. The second phase of the method was the development of a staining procedure which enhanced the fluorescence of the stained salmonella cell while retaining the maximum number of cells and flagella on the microscope slide. To facilitate reading the slide, the staining procedure was also designed to minimize background fluorescence. The third phase consisted of designing a serological scheme for the preparation of a commercially available conjugated salmonellae antiserum containing all 79 somatic and flagellar antigens for the 31 salmonellae serotypes identified in food poisoning in the United States for the years 1965 to 1970 (15). MATERIALS AND METHODS FA antisera. With the assistance of the Center for Disease Control and the approval of the Food and Drug Administration, we designed the coverage of the conjugated antiserum necessary for the task ( Table 1). As a professional courtesy, the Difco Corp. of Detroit, Mich., prepared and submitted two individual pools of conjugated salmonellae antisera conforming to the scheme. The first pool of conjugated antiserum (Difco 0) contained antibodies to the somatic factors of the 31 serotypes. The second pool (Difco H) contained antibodies to the 48 flagellar antigens. These antisera were rehydrated by adding 5 ml of sterile distilled water, as directed by the manufacturer. Quantities of 0.5 ml were dispensed into sterile vials and frozen until used. Before staining, the "O" and "H" antisera were thawed at room temperature and diluted to a working dilution of 1 :8 with pH 7.5 phosphate-buffered saline. A combination of "O" and "H" antisera (O+H) was prepared in this laboratory at a dilution of 1:16 for additional evaluation. This was later supplied by Difco and known as FA Salmonella Poly. The Sylvana Corp. of Millburn, N.J., submitted a conjugated antiserum having the same flagellar and somatic antigen coverage as Table 1, with the exception of the Vi antigen. This antiserum was tested at a 1:8 dilution. Another salmonella-conjugated antiserum was received for evaluation, as a professional courtesy, from E. M. Ellis of the National Animal Disease Center. The antiserum was kept under refrigeration and used at a 1:1 dilution. 645 Samples. The 250 naturally contaminated samples tested in this study were supplied by the Food and Drug Administration. These included 116 human foods and 134 animal feeds. Table 2 lists the types of samples tested and their most probable number values. Sampling procedure. The samples were aseptically divided into 1,000-g quantities, placed into presterilized, 1-gal receptacles, and sealed. The test material was mixed to achieve homogeneity by tumbling end-over-end 100 times. A 25-g test sample was retrieved. Pre-enrichment phase. A 25-g sample was placed in 225 ml of preheated (35 + 2 C) modified M broth. The modified M broth was composed of tryptone (Difco), 10 g; yeast extract (Difco), 5 g; glucose, 2 g; sodium citrate, 5 g; sodium chloride, 5 g; dipotassium phosphate, 5 g; manganese chloride, 0.14 g; magnesium sulfate, 0.8 g; ferrous sulfate, 0.04 g; Tween 80, 0.75 g; and distilled water, 1 liter. The pH was adjusted to 7.0 with 4 to 6 N HCl prior to autoclaving for 15 min at 15 psi. The pH of the modified M broth plus sample was adjusted to 6.8 to 7.2 with 5 N NaOH when necessary. Blending of samples was performed when necessary to obtain a homogeneous suspension. The pre-enrichments were incubated in a shaker water bath (ca. 34 oscillations/min) for 7 hr at 35 4 Selective enrichment phase. After incubation, the pre-enrichment broth was removed from the water bath, and the particulate matter was allowed to settle for 5 min. Fifty milliliters was withdrawn from the top one-third layer of the culture and transferred to 450 ml (35 2 C) of selenite-F broth (BBL). The selenite-F culture was incubated at 35 + 2 C for 17 hr in the shaker water bath. AOAC cultural method. The remaining portion of the pre-enrichment culture was placed in a still-air incubator for an additional 17 hr at 35 2 C for simultaneous evaluation by the AOAC method (10). Elective enrichment phase. After incubation, the selenite-F broth culture was removed from the water bath and allowed to stand for 5 min. Two milliliters was withdrawn from the top third of the culture and transferred to 18 ml (35 2 C) of modified M broth. The pH of the elective modified M broth and sample was adjusted to 6.8 to 7.2 with 5 N NaOH prior to incubation for 5 hr in a water bath at 35 ±fi 2 C. FA slide preparation. Nonfluorescing glass slides were washed with sudless soap and rinsed in FA SLIDE METHOD FOR SALMONELLAE DETECTION distilled water. Slides were soaked for 5 min in chromic acid, rinsed in distilled water, and air dried. A drop of glycerin was placed on each etched circle and immediately sprayed with Teflon. The slides were rinsed in tap water to remove the glycerin and placed in 0.5% NaOH for 5 min, followed by distilledwater rinses and air drying. The etched circles were coated with 0.3% purified agar using a water color brush, and the slides were dried at 55 C. The slides are stable in this form indefinitely, when stored in a dust-free environment. Duplicate smears were made on the prepared slides by withdrawing two 3-mm loopsful from the top one-third of the elective enrichment. The smears were dried on a slide warmer at 35 2 C. FA staining procedure. The dried smears were fixed for 3 min in modified Kirkpatrick ethyl alcohol-chloroform-Formalin (60:30: 10) solution, followed by immersion in ethyl alcohol (95%) for 1 min, and then forced-air dried. The slides were placed in a staining chamber containing moistened filter paper, to prevent the stain from drying on the smear. The smears were stained by placing two drops of antiserum on each smear, allowing 30 min at 35 2 C for the reaction to take place. The slides were then rinsed with phosphate-buffered saline (pH 7.5) followed by two 5-min soaks in phosphate-buffered saline (pH 7.5), 1 min in distilled water, 1 min in ethyl alcohol (95%), and finally air dried. All baths used in the staining procedure were changed daily. All stained smears received one drop of mounting fluid (5 ml of Difco FA mounting fluid, 0.5 ml of carbonate buffer, pH adjusted to 9.2 with 5 N NaOH), a no. 1 cover slip, and one drop of Cargille, type A immersion oil. Microscopy examinations. FA-stained smears were viewed on a Wild Heerburg microscope equipped with an Osram HBO-200 mercury arc burner, 50x and 100 x immersion fluorite objectives; BG-38 heat-absorbing filter; a BG-12 blue pass filter and an OG-1 blue absorbing eyepiece filter; a darkfield condenser and 15x wide-angle eyepieces. Darkfield microscopy was used to distinguish bacteria from debris. In this work, the criteria for interpreting the degree of fluorescence of a smear were subjectively defined as follows: 0, no visibly fluorescing rods; 1+, faintly fluorescing rods without discernible lumen; 2+, faintly fluorescing rods with discernible lumen; 3+, strongly fluorescing rods with discernible lumen; and 4+, intensely fluorescing rods with discernible lumen. The criteria for a positive reaction, as viewed on a prepared microscope slide, are (i) typical salmonella morphology, with or without attached flagella under darkfield and ultraviolet light and (ii) cells yielding a 3+ or 4+ degree of fluorescence under ultraviolet light. FA cultural. After the smears were prepared from the modified M-broth elective enrichment, a cultural confirmation in addition to the AOAC procedure was performed by streaking a 3-mm loopful on selective media plates, as outlined in the AOAC method. The elective enrichment broth was then held under refrigeration. If the streak plates failed to detect salmonellae, 1 ml of the modified M broth was bNA, not available. spread on plate count agar (Difco) and another series of selective enrichment plates were inoculated. If this second series of selective plates failed to yield salmonellae, the plate count agar plate was flooded with 10 ml of H broth (Difco) and streaked onto three additional selective plates. The FA slide readings were compared with the results obtained on the final series of selective plates. Tables 3 and 4 compare the FA slide results, FA cultural results, and the standard AOAC method results. Table 3 a Abbreviations: AOAC+, any sample producing an isolate that exhibited cultural, biochemical, and unconjugated serological reactions typical for salmonellae in accordance with the AOAC published method; AOAC -, any sample confirmed as salmonellae-negative following AOAC published methodology; FAC + (FA cultural positive), any modified M-broth elective enrichment (from which the slides are made) confirmed as containing salmonellae following AOAC published methodology; FAC -(FA cultural negative), any modified M-broth elective enrichment negative for salmonellae following AOAC published methodology; Slide+, any smear showing rods of (i) proper morphology (with or without attached flagella) under darkfield and ultraviolet light, and (ii) a 3+ or 4+ degree of fluorescence under ultraviolet light; Slide-, any smear not conforming to the above; FA slide agreement, occurs when (i) a positive slide is confirmed by an AOAC positive or FAC positive, or both, (ii) when the FA slide and AOAC and FA cultural methods are negative concurrently; FA slide false positive, any slide positive which cannot be culturally confirmed; FA slide false negative, any instance in which the slide was negative and the AOAC culture or FA cultural method, or both, yielded salmonellae. antiserum may be due to the 1:16 dilution of the antiserum. Because all of the antisera were not tested an equal number of times on all the samples, the above percentage agreements for each antiserum from Table 3 cannot be directly compared. RESULTS AND DISCUSSION Therefore, Table 4 offers the results of the three antisera tested the most extensively and reports only the results obtained with the antisera when tested on the same 65 samples concurrently. The This study helped to demonstrate that the FA technique may be successfully used in rapid screening for salmonellae in foods and feeds. The advantages of the application of the FA method are: (i) 32-hr elapsed time, (ii) increased sensitivity over the cultural method through the detection of low numbers of salmonellae (5), (iii) increased specificity through the use of a designed antiserum, (iv) an economic saving by releasing product sooner, thereby freeing expensive storage space, (v) the ability to rapidly determine the effectiveness of sanitation procedures in industrial processes, and (vi) a shorter testing time in which more samples can be checked, thus giving greater protection to the consumer. With at least two commercial sources of conjugated salmonellae sera for use in the direct procedure, the application of the FA technique may increase in industry. There are, however, several aspects of the procedure that require further development. These include: (i) increasing the specificity and sensitivity of the antiserum, (ii) cultural improvement, and (iii) microscopy definition in the diagnostic phase, to minimize subjective interpretations. The somatic antigens of the salmonellae are shared, to some extent, with other Enterobacteriaceae. Concomitant microflora-possessing antigens common to salmonellae may produce FA slide false positives in some sample types. Intensification of the selective and elective enrichment phases to stimulate salmonellae growth with the suppression of nonsalmonellae may be required to reduce the number of false positives. Also, the number of false positives can be minimized by use of higher dilutions of the antiserum. The sensitivity and selectivity of the FA method may vary, as slide false negative results seem to occur more frequently in various specific sample types. These false-negatives may be due, in part, to the degree of background fluorescence of some foods which tend to mask the fluorescence of the salmonellae cells. A uniform nomenclature and criteria for the microscopy detection of salmonellae should be established. The relationships between the number of cells and the culturally positive samples should also be established. We hope to continue our efforts in studying the advantages of the combination or the integration, or both, of microbiological samples for use as a broad-base screening technique, fortified by ultimate confirmation with the standard AOAC method.

v3-fos

2019-03-20T13:04:13.642Z

1972-01-01T00:00:00.000Z

237231602

s2

Automated Slide Staining Machine A machine is described which can perform the Gram stain. Comparison of slides stained by machine versus hand revealed no difference in reproducibility or accuracy. In addition to providing clean, dry, uniformly stained slides, the machine saves 24 sec per slide when compared with a hand staining technique. MATERIALS AND METHODS Slide staining machine. The slide staining machine is a patented (A. N. Pedersen, U.S. Patent 3,507,292, 1970) device for transporting slides through a number of troughs containing staining solutions. After heat fixation of the specimen, the slide is inserted in a wire carrier which is then connected to a conveyor chain. During transport, slides are carried at a slight angle. When contact is made with the "downstream" wall of a trough, the angle of carriage permits the slide to be lifted above the trough wall and to pass to the succeeding jar. The angle of carriage during passage from one trough to another also permits the staining solutions to drain from the slide. A counterweight on the slide holder minimizes the force required to raise the slide. Figure 1 is a drawing of part of the slide staining machine. It shows three of the staining troughs and two slides, in their carriers. Slide 1 has been forced to rise up upon contact with the wall of a trough. Slide 2 is passing through the staining solution in a trough. Troughs containing staining solutions are followed by one or more troughs of running water for rinsing the slides between stains. This continuous water flow is controlled by a solenoid valve that automatically is activated by starting the machine. The water enters the lower section of the trough via tubing and exits through a notch in the upper side wall. At the completion of staining, the slide carriers are moved off the conveyor onto a rail. There they accumulate in front of an air blower which dries them. Accordingly, the operator of the apparatus need only be concerned with the input to the apparatus; the output end can be ignored until a large number of slides accumulate. Figure 2 is a picture of the staining machine in actual operation. Stains. To perform the Gram stain with this machine, the solutions are arranged in troughs as follows: (i) crystal violet, (ii) running water, (iii) running water, (iv) iodine, (v) running water, (vi) acetone alcohol, (vii) acetone alcohol, (viii) acetone alcohol, (ix) running water, (x) safranin, and (xi) running water. Optimal Gram staining is achieved by immersing the slides in each trough for 15 sec. Since staining time with this machine is determined by the width of the trough, each trough is of identical width. Total staining time is 2 min and 45 sec. The compositions of these solutions are as follows. 1, Crystal violet contains 2 g of crystal violet, 20 ml of 95% ethanol, and 80 ml of 1% ammonium oxalate. 2, Lugols iodine (1% aqueous iodine) contains 1 g of iodine, 2 g of potassium iodide, and 100 ml of deionized water. 3, Acetone alcohol contains 40% acetone and 60% alcohol. 4, Safranin contains 0.5 g of safranin and 100 ml of deionized water. The stain solutions are changed daily, and their troughs are cleaned thoroughly with acetone alcohol. Reproducibility studies. Four slides from each of 50 specimens (sputa, urine, etc.) were stained: two by machine and two by hand. They were then examined microscopically with an oil immersion lens. Slides were graded on a scale of 16 for perfect correlation. They were read as 0 to 4+ in quantity for the four categories of bacteria (gram-positive cocci and rods; gram-negative cocci and rods). Gradings were based on the number of bacteria seen per oil immersion field: 1+, <1 bacterium seen; 2+, 1 to 5 bacteria seen; 3+, 5 to 30 bacteria seen; 4+, >30 bacteria seen. One point was deducted for each lack of correlation. Results represent the mean scores for 50 specimens. All slides were coded and read in a "blind" manner by four different observers. False results. The slides read in the study of reproducibility were also evaluated for false results. Slides stained by manual or machine techniques were evaluated separately and within the two groups for absolute discrepancies, i.e., the frequency with which one slide of a pair showed no organisms, whereas the companion slide was positive for bac-17 teria. These results were then compared to the culture reports. Detection of contamination of staining solutions. All stain solutions as well as the troughs of running water were cultured for bacteria on three separate occasions with a pour plate technique with Trypticase soy yeast (TSY) agar. The stain solutions were also deliberately contaminated with 108 cells of Pseudomonas aeruginosa, Enterobacter species, Streptococcus fecalis, and Candida albicans and were subsequently cultured to study their capacity to support microbial growth. Expenditure of time in gram staining. During a portion of two separate days, the total time spent on Gram staining slides by hand was determined. On two other days, a similar calculation was made for the time spent in attaching the slides to and removing them from the slide carrier and for engaging the carrier on the conveyor. Table 1 indicates the reproducibility of the hand and machine techniques for Gram staining and compares the techniques with each other. The readings of the two slides stained by the machine are as comparable as those for the two slides stained by hand. Discrepancies were similar when slides stained by hand were individually compared with slides stained by the machine. Table 2 is an attempt to study the potential for false smears resulting from acquisition or loss of bacteria on a slide while it is being moved through the Gram staining machine. There were 21 readings in which all four slides were negative for bacteria. Twelve of those specimens were positive on culture, usually with light growth. On two occasions, both of the slides stained by the hand technique were positive, whereas both of the machine-stained slides were negative. The converse situation was not encountered. There were four readings when only one slide, of four, was positive; two FIG. 2. Slide staining machine in operation. The lid has been raised to activate (i) the flow of running water, (ii) the conveyor belt, and (iii) the air blower for slide drying. The slide holders are hung on the arm located on the right side of the machine. Several slides are passing through the stain solutions; still others have accumulated in front of the air blower. At the technologist's convenience, these slides may be removed from their holders and examined microscopically. b Four slides from each of 50 specimens (sputa, urine, etc.) were stained: two by machine and two by hand. Slides were graded on a scale of 16 for perfect correlation. They were read as 0 to 4+ in quantity for the four categories of bacteria (gram-positive cocci and rods, gram-negative cocci and rods). One point was deducted for each lack of correlation. Results represent the mean scores for 50 specimens. All slides were coded and read in a "double-blind" manner. Data presented include mean, standard deviation, and, in parentheses, the total score recorded out of a possible 400 for perfect correlation. c For each of the 50 specimens, one slide stained by hand was compared with one slide stained by machine. (0) indicates no bacteria seen. b Plus (+) indicates if any bacteria were seen. of these were hand-stained slides and two were machine-stained slides. Also, there were two instances when the only negative reading of the four matched slides was a machine-stained slide and one when the negative slide was stained by hand. In all instances of discrepancy, cultures were positive. To evaluate further the potential for false "positivity," cultures of trough fluids were obtained. The specimens were negative, except on one occasion when the safranin solution contained 109 Pseudomonas species per ml. This was traced to contaminated deionized water which was used for the preparation of the stock solution. After correction of this problem, daily culture of the safranin solution has been negative over a period of 3 months. Deliberate contamination of the stain solutions with three different species of bacteria and C. albicans revealed that only the safranin solution was capable of supporting bacterial growth. Furthermore, only gram-negative bacteria were able to survive. Table 3 reveals the time saved in the clinical microbiology laboratory by the use of this slide staining machine. The period of time studied represents only a part of the entire day's output, but on the average it takes slightly more than 2.5 times as long to stain by hand as by machine. Even for the relatively small number of slides stained, an average of 21 min was saved per day. DISCUSSION The described machine is a simple and inexpensive apparatus for staining slides. The studies reported here indicate that slides stained by machine are equivalent in reproducibility to those stained by hand. The reproducibility within the methods is matched by reproducibility between the two techniques. There is a theoretical possibility of acquiring bacteria in the staining solutions either because of contaminated solutions per se or via transfer of bacteria from previously processed slides. The reproducibility data suggest that this does not occur, at least not more so than in the hand techniques. When the question of "false positivity" was more carefully analyzed (Table 2), it appeared that the problem is not more frequent with machine staining. Cremer (1) also studied this question with the Shandon-Elliot staining machine. No transfer of bacteria from positive unfixed slides to negative ones occurred during either the Gram or auramine stain. Our cultures of the staining solutions were also reassuring in this regard. It is important, however, to note the precaution of using fresh stain solutions daily and for cleaning of the troughs, particularly the one containing safranin, with acetone alcohol. With its demonstrated potential for contamination, daily culture of the safranin solution should be continued. There are several advantages of such a ma-chine. One can extrapolate from the time saved in our laboratory to other workloads, but in a year we would save at least 128 hr of a technologist's time. Since each slide is stained in a uniform manner, there is much greater consistency in the appearance of bacteria. In contrast, hand staining of batches of slides leads to unequal exposure to stains and inconsistent staining. To enhance uniformity of staining, especially decolorization, with the machine, thin smears should be made. Manually stained slides often have residual crystals of stain on both sides which require removal before reading. The machine consistently produces clean, uniformly stained, dry, and ready to read slides. Also, the availability of such a machine encourages the laboratory and the house staff to perform direct Gram stains on all appropriate specimens for bacterial culture. Although we have demonstrated the application of this machine to the Gram stain, it should also be suitable for other procedures such as the auramine-rhodamine, Kinyoun, Wright, and hematoxylin-eosin stains. Negotiations are currently underway with a leading medical equipment manufacturer for the production and distribution of this apparatus. ACKNOWLEDGMENTS The technical assistance of George Nielsen and Janet Nelson is gratefully acknowledged.

v3-fos

2018-04-03T02:38:28.622Z

1972-08-01T00:00:00.000Z

34418254

s2

Factors Affecting the Stability of Transmissible Enteritis Virus of Turkeys Abstract The effects of environmental factors on the stability of transmissible enteritis virus of turkeys were studied, using an assay system of poult inoculation. Viral infectivity persisted for less than 6 hr at 37 C in nutrient broth. Survival of virus was enhanced in the presence of cysteine and nicotinamide-adenine dinucleotide, and at pH 5.5. Survival was also enhanced in a growing culture of intestinal microflora and could be observed as well in a culture of Streptococcus fecalis in medium with reduced oxygen tension. These results were compared with findings in experiments with several other intestinal viruses, and it is suggested that some intestinal viruses may be adapted to the conditions of low pH and redox potential that are normal in the intestine. The effects of environmental factors on the stability of transmissible enteritis virus of turkeys were studied, using an assay system of poult inoculation. Viral infectivity persisted for less than 6 hr at 37 C in nutrient broth. Survival of virus was enhanced in the presence of cysteine and nicotinamide-adenine dinucleotide, and at pH 5.5. Survival was also enhanced in a growing culture of intestinal microflora and could be observed as well in a culture of Streptococcus [ecalis in medium with reduced oxygen tension. These results were compared with findings in experiments with several other intestinal viruses, and it is suggested that some intestinal viruses may be adapted to the conditions of low pH and redox potential that are normal in the intestine. Transmissible enteritis of turkeys is a disease that occurs as explosive outbreaks in flocks. Affected birds show depression, enteritis, and loss of weight. Mortalities may be high. Recent studies indicate that this disease is caused by an enveloped virus that bears some morphologic similarities to the myxoviruses.! Although the disease has been studied for many years [1,2], it has not been possible to isolate the causative virus in cell cultures [2,3]. Because the virus is so difficult to grow in vitro, the effect of the environment on its stability was investigated in the hope of improving techniques of isolation. Materials and Methods Source of virus. The Minnesota strain [2] of transmissible enteritis (TE) virus was propagated in turkeys and used for the experiments. Turkey poults were inoculated perorally with infective intestinal contents and killed three days later. Intestinal contents were diluted with an equal volume of phosphate-buffered saline, clarified by centrifugation at 8,700 g for 10 min, and passed through a 0.3-flm membrane filter. The filtrate was shown to be free of bacteria and viruses cytopathogenic for turkey-embryo-kidney cells by methods described previously [2]. Assay system. The only reliable assay for infectivity of TE virus is the development of clinical signs and loss of weight in challenged poults [2]. One-day-old poults were acquired from local hatcheries in which there was no history of infection due to TE virus; they were raised in isolation on antibiotic-free feed. Before oral challenge, poults were weighed in groups of four and placed in modified Horsfall-Bauer isolation units. Each poult was inoculated perorally with 0.5 ml of the material to be tested. Because of the cumbrous assay system, dilutions of inoculum were not titrated, except in experiments using the survival medium described below. After inoculation, consumption of feed and water was observed for three to five days, and the birds were then killed and weighed. Infection with TE virus was determined on the basis of failure to gain weight, coupled with clinical signs of anorexia, depression, and reduced body temperature. All experiments were repeated to ensure validity. Survival medium. The basal medium used in experiments on viral survival consisted of mycoplasma medium [4] containing 100 mg of nicotinamide-adenine dinucleotide/liter, 100 mg of Results Figure 1. Period of survival of infectivity of transmissible enteritis virus diluted I: 30 in tryptose-phosphate broth at 37 C. Results are expressed as the percentage of weight change from the initial weight of groups of four inoculated poults, four days after challenge. Inoculated (+) and uninoculated (-) control groups are presented. Virus survived overnight in the survival medium described above only when cysteine and nicotinamide-adenine dinucleotide were present. Furthermore, it was necessary for the pH of the medium to be near 5.5 ( figure 2B). Effect of other modifications. Other substances added to the survival medium in the hope of enhancing stability of the virus included 1 mg of catalase/rnl, 2.5 mg of maltosez'ml, 5 flg of sodium deoxycholate/rnl, 30flg of menadione/ ml, 8 mg of arginine/ml, and 5 % of a filtrate of feces from uninfected poults. None of these additives substantially enhanced persistence of infectivity. Sera from turkeys, rabbits, or swine at a concentration of 5 % allowed optimal persistence of infectivity. Concentrations of serum of 10% or more inhibited survival of the virus, and omission of serum also prevented overnight survival. Effect of intestinal microfiora. A 10-ml volume of tryptose-phosphate broth was inoculated with a loopful of unfiltered intestinal contents from a bird infected with TE virus. After overnight incubation at 37 C, the broth contained a luxuriant growth of gram-positive and gramnegative bacteria, which when inoculated into poults caused clinical signs of infection with TE. Similar results were obtained if the broth contained 0.075% thallium acetate, except that in this case, only streptococci grew in the broth. Three streptococcal colonies, differing in hemolytic pattern, were isolated from the intestine of an uninfected turkey poult. Broth cultures were prepared, and three 10-ml volumes of tryptosephosphate broth were inoculated with 0.1 ml of the respective streptococcal cultures. Two hours later, each tube was inoculated with 0.3 ml of infective intestinal filtrate. After incubation overnight at 37 C, only one of the tubes was capable of causing infection with TE in susceptible poults ( figure 2C). The Streptococcus used in this tube was identified as Streptococcus [ecalis. Compared with the streptococci used in the noninfectious tubes, the S. [ecalis reduced tetrazolium more rapidly during growth and produced a more acidic medium. The final pH in spent medium from S. [ecalis was 5.9, compared with 6.2 for the other two streptococci. Infectivity was better maintained if the broth medium was boiled and allowed to cool imme- <.> 0 cysteine/liter, and 5 % heat-inactivated rabbit serum. Acetic acid was added to adjust the pH of the medium 5.5. A 10-ml volume of this medium was inoculated with 0.3 ml of infective intestinal filtrate for studies of survival of infectivity, and the culture was incubated overnight before inoculation into poults. Since this was further diluted 1: 100 before inoculation of the poults, the final dilution of original material was 10-3 • Rate of viral inactivation at 37 C. A 2-ml volume of undiluted intestinal filtrate was incubated at 37 C and assayed at different times for infectivity. In various experiments, infectivity generally vanished within 6 hr (figure 1), although it once persisted for 8 hr. Similar results were obtained with intestinal filtrate diluted 1: 30 in nutrient broth. Effect of pH and redox potential. Considerable experimentation was required to arrive at the survival medium described above. Modifications were made in order to develop a suspending medium that would allow infectivity to survive for 18-24 hr (overnight) at 37 C. Discussion In experiments concurrent with this work, the average titer of TE virus in an intestinal filtrate was 10 4 turkey-infective doses/nil [2]. In experiments using the survival medium, intestinal filtrate had a final dilution of 10-3 , after incubation overnight before inoculation into poults. Thus in the survival medium, the virus lost less than 1 log of activity after 18 hr at 37 C, compared with a loss of 4 logs of activity after 6 hr in a neutral medium. It was believed that S. iecalis and the general intestinal microflora acted by lowering the pH and redox potential of the medium, causing conditions similar to those in the survival medium. The removal of oxygen by boiling aided this action. Similar experiments have been carried out using the virus of hemorrhagic enteritis of turkeys [5], which differs from the TE virus [6]. It was found that survival of the hemorrhagic enteritis virus was also enhanced in the presence of a growing culture of S. [ecalis. Other workers have observed a stabilization of poliovirus by cysteine or cystine [7][8][9][10], by lowered pH [8], and by the removal of oxygen [8,9]. Reducing agents have stabilized an ECHO virus and a Coxsackie B virus, as well as poliovirus [8]. Thus, there are at least five viruses of the intestinal tract that have shown enhanced stability at lowered redox potential, lowered pH, or both. In contrast to their effect on enteric viruses, reducing agents decreased the stability of vaccinia virus, Newcastle disease virus, and three arboviruses [11]. It was suggested that the presence of a viral envelope might be correlated with lability to reducing agents. It has been shown, however, that TE virus is enveloped [2] and that it is stabilized by reducing agents. Thus, the presence of a lipid coat does not determine sensitivity to lowered redox potential. It is possible that viruses that normally inhabit the intestinal tract, a site of low redox potential, have become adapted to their habitat. On the other hand, the characteristic of stabilization by low redox potential may not be limited solely to enteric viruses. Zinsser et al. [12] have suggested that an encephalitic strain of herpesvirus may be stabilized by reducing agents. It has been suggested that viral enteritis is common in man [13], but there is difficulty in associating known viruses with the diseases observed [14]. Several syndromes have been described in which the causative agents cannot be grown outside the natural host, man [15,16]. In this, they resemble transmissible enteritis of turkeys. The possibility that these viruses may have some sensitivity to oxygen, as has been demonstrated for all intestinal viruses so far examined, has implications for efforts aimed at viral isolation.

v3-fos

2020-12-10T09:04:17.266Z

1972-02-01T00:00:00.000Z

237229184

s2

Heat Resistance of Salmonellae in Concentrated Milk The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level. a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vacuum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level. It is well known that bacterial cells are more resistant to dry heat than moist heat. There are several papers that amply demonstrate an increase in bacterial heat resistance as the solute concentration of the heating menstruum increases (1,2,11,14,15). This increase in resistance has been suggested to be a consequence of reduced water activity, and, undoubtedly, this is an important factor. However, Goepfert et al. (11) reported that the chemical nature of the solute controlling the water activity was more influential on the heat resistance of salmonellae at water activity levels above 0.75. This was later confirmed by Baird-Parker et al. (1) and Moats et al. (14). Because of this, it becomes necessary to experimentally determine the heat resistance of salmonellae in each individual test material rather than extrapolating data derived from experiments on similar but nonidentical products. In 1968, McDonough and Hargrove (13) reported that survival of two species of Salmonella was greater in concentrated milk (50% solids) than in skim milk heated at the same temperature. This study was undertaken to extend the observations of McDonough and Hargrove and to investigate the factors (growth temperature, growth medium, reduced pressure) that influence the heat resistance of salmonellae in a single food material, i.e., concentrated skim milk. It was hoped that the 415 data generated by this study would enable the dry milk industry to assess their current practices with regard to efficiency of the process in destroying salmonellae and to add to present knowledge about the heat resistance of salmonellae in dry and semidry environments. MATERIALS AND METHODS Bacterial cultures. All cultures in this investigation, i.e., Salmonella typhimurium, Salmonella alachua, and Escherichia coli (0104: H7), were obtained from the culture collection of the Food Research Institute, University of Wisconsin. The strain of S. alachua had been originally isolated from nonfat dry milk. Stock cultures were stored at room temperature on nutrient agar slants (NA) in screw-cap tubes. Working cultures were transferred every 24 hr in Trypticase soy broth (TSB) and incubated without agitation at 35 to 37 C, except as noted below in the study of the effect of growth medium and temperature on the heat resistance of cells. Milk solutions. Skim and concentrated milk solutions were prepared from nonfat dry milk powder as described previously (6). Heating of microorganisms. (i) When heated at atmospheric pressure, milk solutions at solids levels of 10, 30, 42, and 51% (w/w) were placed in stainlesssteel mixing cups. The cups were placed in a water bath at test temperature in such a manner that the level of the bath was 2 to 3 inches (ca. 5.1 to 7.6 cm) above the level of the milk in the cup. After equilibration of the milk solution to the test temperature, the inoculum (24-hr-old TSB-grown cells) was added. After inoculation and throughout the trial, the heating menstruum was agitated by a mechanical stirrer. Test solution and water bath temperature were also monitored throughout the trial. (ii) When heated under reduced pressure, milk solutions were placed in the laboratory scale vacuum pan described previously (7). After equilibrating to the test temperature and pressure, the inoculum (1 to 2%, v/v) was introduced through the port designed for this purpose. Throughout the trial, the inflow of pretempered, sterile, distilled water was matched to the outflow of condensate to maintain a constant solids level. Enumeration of microorganisms. In most trials, survivors were enumerated by surface-plating procedures. At appropriate intervals, 1-ml samples were taken from the heating menstruum and added to 9 ml of 0.1% peptone-water. One-tenth milliliter quantities of the appropriate peptone-water dilutions were surface plated on Trypticase soy agar supplemented with 0.2% yeast extract. Plates were examined after incubation at 35 to 37 C for 48 hr. When the inoculum was grown in concentrated milk prior to heating in a fresh concentrate, the three-tube most-probable-number (MPN) procedure was employed to enumerate survivors. This entailed pre-enriching 1-ml samples of the appropriate dilutions in nutrient broth for 6 hr at 35 to 37 C prior to transferring 1-ml portions to tubes containing 9 ml of tetrathionate broth and selenite-cystine broth. The enrichment media were incubated at 35 to 37 C for 24 hr. One loopful of each broth was streaked onto Salmonella-Shigella agar plates which were then incubated at 35 to 37 C for 48 hr. Typical colonies were confirmed as salmonellae by appropriate serological tests. MPN values were calculated on the basis of the pattern of positive dilutions in the series. Calculation of D values. The number of survivors were plotted (ordinate) against time (abscissa) on semi-logarithmic graph paper. All trials were conducted for a period of time sufficient to result in a 5-log cycle drop in viable cells. In some instances, the plots were diphasic, i.e., there was an initial phase of rapid death followed by a phase in which death proceeded at a slower rate. In such cases, the decimal reduction time (D) value was obtained from the portion of the plot describing the slower rate. Instances in which diphasic curves were obtained are so indicated in Table 1. RESULTS AND DISCUSSION Previously, McDonough and Hargrove (13) showed that growth of salmonellae occurred in milk concentrates containing 60% solids. We confirmed these observations and delineated the temperature limits within which salmonellae would grow in milk concentrates (6). Thus, it was decided to investigate the processing parameters that would suffice to ensure the destruction of salmonellae in concentrated milk. The results of the trials in which S. typhi-murium, S. alachua, and E. coli (all grown at 35 to 37 C) were heated in milk solutions are shown in Table 1. It is clearly evident that increased solid levels result in an increased resistance to heat destruction by all of the organisms tested. This was not unexpected and confirms the earlier observations of Mc-Donough and Hargrove (13). The basis for the increased resistance to heat is not known. It is unlikely that lactose is affording protection since Fay (9) reported no increase in heat resistance when cells were heated in concentrated-lactose solutions. We have confirmed (unpublished data) this observation. Moats et al. (14) reported that casein added to phosphate buffer did not significantly protect S. anatum from heat destruction, but the concentration of casein was low (i.e., 1%); these data tell us little regarding more concentrated casein solutions. Kadan et al. (12) reported that addition of fat (up to 14%) did not influence the heat resistance of Staphylococcus aureus in skim milk. However, the addition of 30% serum solids did effect approximately a 45% increase in the D60 c value for staphylococci in skim milk. Further investigation is needed to determine the nature of the substance(s) in skim milk that is responsible for the increased heat resistance of salmonellae and escherichiae in concentrated milk. The relative heat resistance of the test organisms was found to vary with the solids level of the heating menstruum. Thus, although the strain of E. coli was more resistant than both strains of salmonellae in 10% milk, the situation was reversed in the 42 and 51% solids milk. The consequence of the relative heat resistance values in concentrated milks is that a heat treatment given these products might render the product coliform-free but leave it contaminated with Salmonella. Therefore, the value of a coliform test as an indicator of enteric contamination in concentrated milk is rather minimal. The relative susceptibility of E. coli and salmonellae to spray drying must be determined before the value of the indicator organism analyses can be accurately assessed. Such an investigation is currently in progress. It was interesting to note that S. alachua was more heat-resistant in concentrated milk than S. typhimurium. It would be easy to speculate that perhaps this resistance enabled the S. alachua strain to survive the processing treatment inherent in the manufacture of the dry milk product from which it was isolated. Without additional information, this would only be speculation. However, a correlation between resistance to environmental condi- tions and frequency and source of isolation has been suggested (8) and may in fact warrant further attention. Figure 1 shows the thermal destruction curves for S. typhimurium (grown at 35 to 37 C) heated in the various milk solutions. It can be seen that the Z value increases as the solids level in the milk is increased. The same behavior was noted for E. coli (Z = 4.6, 4.9, 6.3, and 7.9 C at 10, 30, 42, and 51% solids, respectively) and S. alachua (Z = 4.1, 6.2, and 6.9 C at 10, 42, and 51% solids, respectively). An increase in Z value was previously reported by Goepfert and Biggie (10) in their investigation on the heat resistance of salmonellae in chocolate. Similarily, Cotterill and Glauert (3) noted a Z value of 9.1 C for S. oranienburg heated in egg yolk containing 10% NaCl. This behavior underscores the necessity for determining the heat resistance of an organism in situ rather than extrapolating from data derived in experiments conducted using laboratory media or dilute-food suspensions. In a study of the heat resistance of salmonellae in laboratory media, Ng et al. (16) reported a profound influence of prior (to heating) growth temperature on the heat resistance of two strains of salmonellae. The influence of growth temperature on the heat resistance of S. typhimurium in 10 and 42% solidscontaining milk solutions was investigated. Figure 2 shows the thermal destruction curves VOL. 23, 1972 IOC tion increased when cells were incubated for % SOLIDS various periods in 50% sucrose prior to heating. 10 -Similarly, Calhoun and Frazier (2) reported 30 that Pseudomonas fluorescens grown in broth 50 4 containing glucose (a, = 0.97) was more re-\ *" \ 5I sistant to heating in reduced (by glucose) water activity solutions. Goepfert et al. (11) found that prior growth of S. tivity) solutions has resulted in increased re-TEMPERATURE (CC) sistance when exposed to heat in a concen- FIG. 2. Influence of growth temperature on the trated solution. For example, Fay (9) noticed thermal resistance of S. typhimurium in 10 and 42% that the resistance of E. coli to heat destrucmilk solids. surviving salmonellae were enumerated by the MPN procedure. A mean D55.1 c value of 20.0 min for cells so treated was obtained. Comparing this with a D,5.1 c value of 18.3 min for cells grown in TSB at 35 to 37 C would indicate that the heat resistance of this one strain was not significantly enhanced by growth in 42% solids-containing milk solution. However, Fay (9) reported that only brief exposure to 50% sucrose was necessary to enhance the heat resistance of E. coli, and his data indicate that protracted exposure (i.e., >7 hr) resulted in a return to normal resistance. Cotterill and Glauert (4) reported that increase in thermal resistance of S. oranienburg which occurred during storage in egg yolk containing 10% NaCl was temperature-and time-dependent. Their data show that maximum thermal resistance was attained after 12 to 24 hr at 32 C and at 12 hr at 40 C. At 40 C, the thermal resistance after 24 hr of storage was nearly equivalent to that possessed by salmonellae that were not exposed to the salt-containing yolk prior to heating. Interestingly, prior exposure to yolk containing 10% sugar did not enhance the thermal resistance of the salmonellae that were heated in this product. It is therefore possible that the very similar thermal resistance of TSB-grown and 42% milk-grown S. typhimurium is due to (i) the absence of any effect due to prior exposure or (ii) too long an exposure to the concentrated milk prior to heating. Experimentation employing cells that were exposed to the concentrate for shorter periods of time prior to heating would demonstrate whether an enhancement of heat resistance followed by a return to normal resistance was actually occurring. It is also possible that concomitant growth by the microflora of the milk influenced the heat resistance of the salmonellae. This could only be negated by employing a sterile product in the experiment. In a previous paper (6), we reported that vacuum concentration of skim milk was lethal to salmonellae and E. coli only when the vapor temperature exceeded the maximum growth temperature of the organisms. These observations were extended to compare the heat resistance of S. typhimurium and E. coli in 10 and 30% milk solids heated at atmospheric pressure and under reduced pressure. The data are shown in Table 2. It is quite evident that reducing the pressure is an effective means of reducing the heat treatment necessary to destroy salmonellae in concentrated milks. Apparently, not only was the D value of each organism reduced, but the protective effect of higher solids concentration was also minimized. Similar effects of reduced pressure on the heat resistance of salmonellae were noted by Ballas (cited in reference 5). His research resulted in USDA acceptance of an alternate method of pasteurizing egg whites. In this process, 17 to 20 inches of vacuum are applied to the liquid whites prior to heating to 56.7 C for 3.5 min. The explanation for this reduced heat resistance under partial vacuum is not known but is of significant importance to merit further examination. It is apparent that there are a number of factors that affect the heat resistance of enteric bacteria in food products. It is also clear that these factors cause significant differences in the behavior of salmonellae in broth and food menstrua. Experience has taught us the futility of attempting to predict behavior of salmonellae in food products based on data derived from experiments performed in laboratory media and buffers. It is hoped that more studies of salmonella behavior in situ will be performed so that data are available to enable food processors to design adequate processing schedules to destroy salmonellae.

v3-fos

2019-04-03T13:05:58.804Z

1972-01-01T00:00:00.000Z

91920879

s2

Simplified dissection as an aid in carcass evaluation on the landrace and Yorkshire breeds Information on carcass quality obtained from dissection and conventional carcass evaluation was studied on the Landrace and Yorkshire breeds. Progeny testing pigs (n = 196) were slaughtered at a weight of c. 90 kg. After conventional carcass evaluation, the carcass half was dissected. The objects of study were the most valuable parts of the half carcass (= ham -Jcarre -p back -p fore back -p shoulder -p kidney fat) and its skin-p fat and meat-pbone components. The possibility of restricting the dissection to the ham and the back was also examined. By means of the least squares methods the following results were obtained: The effect of the slaughter weight on the skin+fat, the meat -pbone and the valuable part was very significant. The variation due to age was not significant. The carcass quality on the gilts was better than that of the castrates. By stepwise multiple regression procedures estimations were derived for the skin -pfat component, the meat-p bone component and the most valuable part of the carcass. By dissection of the ham and the back more information was generally obtained about the slaughter quality of the most valuable part than was obtained by the conventional carcass evaluation. The index A A (= X X 10) was calculated, in which A = weight of the meat-pbone component, B C B = age in days and G = weight of the half carcass. The index correlated with the skin-f-fat component and its percentage as follows: r = —o.34** o.ss***, and with the meat+ bone component and its percentage: r = 0.77*** —o.7B***. Possibilities of developing the index were examined. While the production of pigment has increased, the demand has increasingly centred on fat-free and low-caloried red meat. For years, the price paid to the producer has consequently been determined by the carcass weight and the thickness of the back fat. In the retail trade and the meat industry, however, the value of the carcass is affected not only by its fattiness but also by the relative proportions of various parts of the carcass and by the quality of the meat. Typical of the development is the increase in the value of back, hams, shoulders and even side, and the decrease in the value of head, trotters, shank and belly. Pig breeders have tired to adjust their methods of carcass evaluation to this situation by a) analysis of the anatomic composition of the carcass by dissection (Blendl 1966 a, Blendl 1966 b, Weniger et al. 1967, Pedersen 1968, Lohse et al. 1969, Clausen et al. 1970), b) connecting carcass quality more closely to its economic value (Böckenhoff et al. 1967, Sych & Horst 1969, Schön 1970), and c) replacing the subjective points evaluation, e.g. of the ham, with objective measurements (Blend 1966 b, Partanen 1969). back more information was generally obtained about the slaughter quality of the most valuable part than was obtained by the conventional carcass evaluation. While the production of pigment has increased, the demand has increasingly centred on fat-free and low-caloried red meat. For years, the price paid to the producer has consequently been determined by the carcass weight and the thickness of the back fat. In the retail trade and the meat industry, however, the value of the carcass is affected not only by its fattiness but also by the relative proportions of various parts of the carcass and by the quality of the meat. Typical of the development is the increase in the value of back, hams, shoulders and even side, and the decrease in the value of head, trotters, shank and belly. Pig breeders have tired to adjust their methods of carcass evaluation to this situation by a) analysis of the anatomic composition of the carcass by dissection (Blendl 1966 a, Blendl 1966 b, Weniger et al. 1967, Pedersen 1968, Lohse et al. 1969, Clausen et al. 1970, b) connecting carcass quality more closely to its economic value (Böckenhoff et al. 1967, Sych & Horst 1969, Schön 1970, and c) replacing the subjective points evaluation, e.g. of the ham, with objective measurements b, Partanen 1969). Complete dissection of the carcass of test pigs is a laborious and expensive process. An alternative approach is a simplified dissection where the carcass is divided into retail parts some of which are further dissected to assess the leanness. The objectives set for the present study were approached as follows. 1) An examination was made of the possibilities of restricting dissection analysis to individual parts of the carcass. 1) An analysis was made of the effects of carcass weight, age and sex on the most valuable part of the carcass and its components. 3) An investigation was made of the suitability of identical dissection models for the Landrace breed and the Yorkshire breed. 4) Information obtained by conventional evaluation of the carcass was compared with information obtained by partial dissection. 5) An examination was made of the position of the »new index» as a measurement of the fattiness and the leanness of the carcass, although the presentation of a final test index will not be necessary until the completion of a test period lasting several years, when, for instance, the heritability of individual parts of the carcass has been determined with relative certainty. Material Landrace (n = 119) and Yorkshire (n = 77) progeny testing pigs were reared at the Pohjanmaa litter testing station to a live weight of c. 90 kg. The feeding was the standard mixture for progeny testing pigs (Partanen 1969 Methods The least squares procedures (Harvey 1966, SCC 1968) were used to analyse overall variances of the most valuable part of the carcass and its components in order to ascertain the role of the following factors in the variation: linear regression on slaughter age and slaughter weight, sex, breed, their interaction and the years 1967 1969 within breeds. The following model was used to analyse the variances; y ijkl = a -(-a, +bj + C Jk + (ab),j +d. x ijki + g-z ijki + Cjjki jin which a i illl effect of sex, bj = j!*i effect of breed, c Jk = effect of kill year within the jit breed, (ab) (J = interaction of the iit sex and the j* breed, x lJkl and z lJk , = independent continuous variables, d and g = partial regression coefficients, and e iJkl = random error. Stepwise multiple regression analysis (SCC 196 8). The results were processed by stepwise multiple regression analysis as follows: a) preliminary analyses (n = 174) and b) analyses by breed after supplementation (n = 119 Landrace and 77 Yorkshire pigs) of the material. The results provided the correlation matrix, the multiple correlation coefficients (R), the coefficient of the multiple determination (R 2 ) and the partial regression coefficients, etc. The partial regression coefficients are not shown, for any of the explanatory variables are linearly dependent on one another. Results and discussion Preliminary procedure (174 pigs). The possibilities of measuring the most valuable part of the carcass and its skin+fat and meat+bone components were analysed by stepwise multiple regression analysis. It should be pointed out thant the most valuable part of the carcass excludes the head, feet, shanks, neck, belly and sides. The sides may indeed be regarded as being included in the most valuable part of the carcass, but its dissection into its skin+fat and meat+bone components is time-consuming and rather laborious, and the side was consequently excluded for reasons of expediency. Reduction of the proportion of the listed relatively less valuable parts of the carcass is one of the aims of breeding, and it is hoped that this will be attained by selection for the biggest possible valuable part of the carcass and for its meat+bone component. Table 1 shows an estimation for the measurement of the variance of the skin+fat component of the most valuable part of the carcass. The skin+fat model in the six first steps of the regression analysis included the following characteristics in the order listed: the skin+fat of the back, ham, shoulder and carre, the kidney fat and the skin+fat of the fore back. The R 2 = 0.96. The Table moreover Effects of age, carcass weight, breed, sex and years. Tabic 3 shows the variation ratios and the statistical significances of the effects of certain factors influencing the variances in the most valuable part of the carcass and its components and their percentages. The variation due to differences in slaughter weight in the most valuable part of the carcass and its skin -• fat and meat-j bone components was found to be statistically very significant. The effect of differences in slaughter weight was almost completely eliminated when the proportions of the most valuable part of the carcass and of its skin+fat and mcat + bone components in the carcass were calculated. The same result can probably be arrived at by carrying out a correction bases on a linear regression on carcass weight. There was no need for correction by age in addition to correction of slaughter weight. The effect of sex on the skin+fat and meat + bone components of the most valuable part of the carcass and on the percentages of these was statistically very significant. The females were found to be less fatty and more meaty than the castrates. The effect of the years on the most valuable part of the carcass was almost significant, and on the relative proportion of the most valuable part it was very significant. The relative proportion of the most valuable part declined from year to year (1967 -1969). The reason for this phenomenon is explicable in terms of the following factors: a) the decline in the fattiness of the carcass when selection was made for thin back fat, b) changes occurring in the manner of dissection, and c) the conditions in the piggery. Analyses by breed. Skin + fat of the most valuable part. An estimation for the measurement of the skin+fat component of the most valuable part of the carcass and its percentage is shown for the Landrace by stepwise multiple regression analysis in Table 4. In the order listed, the six first steps of the model for the skin+fat of the most valuable part included: the skin+fat of the back, ham, shoulder and carre; the kidney fat, and the skin+fat of the fore back. The model measuring the percentage of the skin+fat component of the most valuable part included first the thickness of the back fat (r = o.73***) and then the percentages of the skin+fat of the ham and of the back; the skin+fat of the shoulder; the kidney fat; and the percentages of the skin + fat in the carre and in the fore back. Table 4 shows the results of the same analysis for the Y orkshire breed, too. With the first six steps the estimations measuring the skin+fat component of the most valuable part and its percentage included the weight units of the following parts of their percentages, in the order listed; the skin+fat of the back, ham, carre and shoulder, the kidney fat; and the skin+fat of the fore back. The R 2 percentage in the models amounted to 96-99. The correspondence of the estimations of the skin+fat component and its percentage can be regarded as good for the Landrace and especially so for the Yorkshire breed. This facilitates the carrying out of the measurement in practice when attempting to select for a small skin+fat component and its percentage. The models show differences between the breeds, but the undisputed position of the skin+fat components of the back and the ham emerge in both breeds. Meat + bone of the most valuable part. The estimation for the Landrace of the meat+bone component of the most valuable part and its percentage included in five steps of the regression analysis, in the order listed: the meat+bone parts or their percentages of the ham, shoulder, fore back, back and carre (Table 5). The R 2 percentages amounted to 98 and 96. Attention is drawn to the introduction of the shoulder and the fore back after the ham. The information provided by the elements of the forecarcass concerning the meatiness of the most valuable part of the carcass was greater than that provided by the back, but there was no difference between the correlations of these three elements to the meat+bone component of the most valuable part. Mention should also be made of the very significant correlation of the index with the meat+bone component of the most valuable part (r = o.79***) and its percentage (r = o.77***). The respective estimation for the Y orkshire breed of the meat+bone component of the most valuable part and its percentage is shown in Table 5. The estimation of the meat+bone component included in five steps the meat+bone parts of the ham, back, shoulder, fore back and carre, in the order listed. After this, the R 2 percentage was 100. However, Restriction of dissection analysis to ham and back. On the basis of the above analyses it is possible to examine the chances that exist for restricting dissection analysis to a smaller part of the carcass, e.g. to the ham and the back. It would then be possible without extra cost to dissect the carrd too, although the information provided by this is slight in comparison with the information provided by the parts mentioned above. The estimation for the skin+fat component of the most valuable part of the carcass or its percentage (Tables 1 and 4) generally included first the skin+fat of the back or its percentage and second the skin+fat of the ham or its percentage. The R 2 percentage was thereafter 72-77. The estimation of the meat+bone component of the most valuable part of the carcass or its percentage (Table 5) included first the meat+bone of the ham or its percentage (R 2 % = 66-72). It is found on the basis of the correlation coefficients that the meat+ bone of the ham is a better measurement than any other individual result of conventional carcass evaluation or of dissection when the meat+bone component of the most valuable part of the carcass is to be assessed. Earlier, Uusisalmi (1971 b) obtained the results that 64-69 per cet of the overall variance of the skin+fat component of the most valuable part of the carcass or its percentage could be explained by means of conventional carcass evaluation, as could 56-65 per cent of the variance of the meat+bone component or its percentage; n = 153. By comparing those results with the results now obtained, we find that the dissection of the back and the ham gives a picture of the fattiness of the carcass which is as good as or better than the picture obtained by total conventional carcass evaluation (see Fig. 1). Information obtained on the shoulder-)-fore back and the whole back by dissection of the ham and the back. In both breeds only 17 per cent of the variance in the skin -ffat of the shoulder-|-fore back was explicable in terms of the variances in the skin-f fat parts of the ham and the back. In the Yorkshire breed it was not possible to assess even satisfactorily the meat-j-bone of the shoulder and fore back by means of the meat-j--bone of the back (R 2 % = 34). But in the Landrace a good picture of the variance in the meat-fbone of the shoulder and fore back was obtained from the meat-fbone parts of the ham and the shoulder (R 2 % = 62). It can be concluded from the results that restriction of dissection to the ham and the back will weaken the assessment of the carcass quality of the forepart of the carcass (Tables 6 and 7). The fattiness of the whole back (= fore back -f back-fcarr£) could be well assessed in both breeds from the skin-ffat of the back (R 2 % 71-81), however, dissection on the ham performed in addition to dissection on the back did not improve the result in this respect. In the Yorkshire breed 76 per cent of the variance in the meat-fbone of the back parts could be explained from the variance in the meat-)-bone parts of the back and the ham. In the Landrace it was found that the variance in the meat-j-bone components of the ham and the shoulder explained 37 per cent of the variance in the meat -f bone of all the back parts. Simplified dissection and conventional carcass evaluation. If dissection of the most valuable part of the carcass is reduced merely to dissec-tion of the ham and the back, the emphasis in selection will be transferred to these parts of the carcass. The ham and the back admittedly amount to c. 35 per cent of the half carcass, and in the retail trade they are among the parts which are most in demand and most valued. Thus their dissection into skin-)-fat and meat+bone components also involves a considerable expenditure on testing, in the form of reduction in the value of the half carcass. As the picture of the quality of the entire carcass becomes somewhat biased in some respects owing to the restricted dissection analysis, it would be worth while to maintan, or at least occasionally to employ, conventional carcass evaluation for back fat or 5.0.1., area of m. long, dorsi and length of carcass. These characteristics would also be control measurements of the development. Increasing attention should also be paid to the measurements of the development. Increasing attention should also be paid to the measurement of meat quality (e.g. colour of meat). Measurement notes should likewise be made of daily gain and feed consumption.

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Fifteen years of observations on the dwarf gene in the domestic fowl ed in World Poult. Sci. J., 1971 , 27, 279 -81and in the present journal (1971 , 3). RSCOTT G. H., R ACHAPAETAYAKOM P., $ ERNIER P. E., I9 6 Observations on gross requirements for certain nutrients in dwarf White Leghorn hens. Poult. Sci., 40, 13 72-3. ARSCOTT G. H., RACHAPAETAYAKOM P., BERNIER P. E., ADAMS F. A., 19 62. Influence of ascorbic acid, calcium and phosphorus and specific gravity of eggs. Poult. Sci., 41, 485-8. ARSCOTT G. H., B ERNIER P. E., Ig68. Effect of dietary protein on performance of dwarf White Leghorn layers. Poult. Sci., 47, 165 2. ARSCOTT G. H., BERNIER P. E., 1970 . Protein and amino acid needs of the mini-hen. Proc. 5th Ann. Pacific Northwest Animal Nutrition Conference, Richland, Washington, November Iz-I 3, I970, 5, 52-59. BERNIER P. E., 1953 . A dominant lethal in S. C. White Leghorns. Poult. Sci., 32, 88 9-0 . ERNIER P. E., ig6o. A spontaneous chromosome aberration in a S. C. White Leghorn. Poult. Sci. 39 , 1234. BERNIER P. E., I9 6o Midget layers-a progress report. Proc. Eighteenth Annual Oregon Animal Industry ConJerence, 19 60, 23 -24. BERNIER P. E., ARSCOTT G. H., 19 60. Relative efficiency of sex-linked dwarf layers and their normal sisters. Poult. Sci., 39, 1234 -235. BERNIER P. E., ARSCOTT G. H., 19 66. Growth and feed requirements of dwarf White Leghorn pullets compared to their normal-size sisters. Poult. Sci., 45, io7o. BERNIER P. E., ARSCOTT G. H., 19 68. Some economical and nutritional implications of minilayers. Proc. 3rd Ann. Pacific Northwest Animal Nutrition Conference, Harrison Hot Springs, B. C., Canada, November 7-8, 19 68, 3, 47-53. BIRD S., SINCLAIR J. W., 1939 . A study of the energy required for maintenance, egg production, and changes in body weight in the domestic hen. Sci. Agri., 19, 542-55 0. BYERLY T. C., 1941 . Feed and other costs of producing market eggs. Maryland Agric. Exp. Station Bulletin AL (Tech.). COLE R. K., 19 66. Hereditary hypothyroidism in the domestic fowl. Genetics, 53, 1021 -33 . CREW, F. A. E., MUNRO S. S., 193 8. Gynandromorphism and lateral asymmetry in birds. Proc. Roy. Soc. (Edinburgh), 58, Pt. II, 114 -3 CREW F. A. E., MUNRO S. S., 1939 . Lateral asymmetry in the fowl. Proc. 7th World’s Poultry Congress (Cleveland), 61-6 4. FESTING M. F., NORDSKOG A. W., 19 67. Response to selection for body weight and egg weight in chickens. Genetics, 55, 219 -31 . HOLLANDER W. F., 1944 . Mosaic effects in domestic birds. Quart. Rev. Biol., 19, 285- 307 . HUTT F. B., 1949 . Genetics of the fowl, MacGraw-Hill. HUTT F. B., 1953 . Sex-linked dwarfism in the fowl. Genetics, 38, 670 . HUTT F. B., ig 5g. Sex-linked dwarfism in the fowl. J. Hered., 50, 209 -21 . KEARL C. D., I957 Layer feed utilization efficiency-where is it? Farm Economics (Cornell), zoz : 557 6. ME RAT P., GUILLAUME J., 19 69. Etude d’un gene de nanisme lie au sexe chez la poule. II. Fonctionne- ment thyroldien. Ann. Genet. Sel. Anim., 1, 131 -3 . MIROSH L, W., BERNIER P. E., ELLINGTON E. F., ARSCOTT G. H., ROWE K. Ed 1972. Some hor- monal factors in sex-linked recessive dwarfism of the White Leghorn chicken. Probably Poultry Science. MIROSH L. W., I9 67 Endocrinology of sex-linked recessive dwarf White Leghorn chickens, Gallus domes- ticus, with special reference to the thyroid., Oregon State University. M. Sc. Thesis. NORDSKOG A. W., BRIGGS D. M., 19 68. The body weight egg production paradox. Poult. Sci., 47, 498-504. RYAN W. C., BERNIER P. E., 19 68. Cytological evidence for a spontaneous chromosome translocation in the domestic fowl. Experientia, 24, 623 -64 . TAYLOR A. G., xg 3o Report of the ninth and tenth annual Canadian National Egg Laying Contests. Canada. Department of Agriculture Bulletin 139 (n. s.). TIENHOVEN van A., WILLI MSON J. H., TOM LIN SN M. C., MAC INNE S K. L., 19 66. Possible role of the thyroid and the pituitary glands in sex-linked dwarfism in the fowl. Endocrinology, 78, 950 -7 . WALLACE H. B., x97 o. The egg industry. A look into the 70’s. Canadian Poultry Review, 94, 23 -4 , 26-7 , 38, 39-42. production and egg weight significantly and to levels comparable to those observed with a 2 per cent protein diet. Currently a low level of Protamone is being fed to layers reared with and without a Protamone supplement, but the level had to be cut in half to .oi6! per cent as it depressed egg production. Physiological studies have involved observation of the hypophyses, thyroid, adrenals and ovaries of both growing and mature dwarf females, and also a bioassay of hypophyseal homogenates in immature hypophysectomized rats. Although the thyroids of dwarf females were found to be smaller than those of normal birds at 7 -13 months of age it could not be ascertained from the bioassay that alterations in the pituitary thyrotropic activity are responsible for the thyroid disturbances in the dwarfs. The pituitary bioassay results do not support the contention of accumulation of somatropic hormone in the pituitaries of dwarf birds. Observations on ACTH and on gonadotropin potency of the dwarf pituitaries were also made. Observations on dwarfism at the Oregon Agricultural Experiment Station go back to 1952 when one of us (PEB) obtained a dwarf male (!E 4 , 001 ) in the progeny of a cross between a S. C. White Leghorn male and a Cornish hen. Dwarf female segregates have also been observed in our Cornish line. In ig 5 6 a dwarf Leghorn female was observed in the progeny of a cross between our OSU Production Line and a male of another strain. The comparatively large egg laid by that hen ( 2 6 oz -!-) aroused our interest in the economic potential of dwarfism in layers. This interest had been first kindled by the studies of BIRD and S I rrCr,mx ( 1939 ) and of BY!R!, Y (1941) which demonstrated the tremendous influence of body size on feed requirements for egg production. The brief report of K EARL ( 1957 ), in which he showed that labor efficiency and increased egg production per hen were the main reasons for increased efficiency in egg production, and that actually there had been little if any improvement in feed efficiency of egg production in the preceding twenty years, firmed up this interest in investigating the economic potential of dwarfism still further. It was reasoned that one would possibly expect diminishing returns in searching for additional eggs and increased labor efficiency but that reduced feed consumption was definitely a most profitable avenue to explore. Decreased body size is of course assurance of decreased feed consumption. This has been taken advantage of through the gradual monopoly of Legho y ns on egg production as well as the decreasing body size of many strains of layers. One day while explaining these facts to a group of visiting poultrymen and showing them some of our midget chickens, one of them, Mr. Barry Brownell, mentioned that he had a few such small chickens himself and that he would gladly give them to our station. This gift increased the size of our population of miniature chickens and really launched us on what has become an increasing research commitment at the Oregon Agricultural Experiment Station. Serendipity thus led us to what we think is a promising avenue in the continuing search for greater efficiency in egg production. The dwarf gene appears to have a relatively high mutation rate judging from our observations and the reports and correspondence received from various countries, the most recent being from Australia. It remains to be determined whether we are dealing in all these instances of sexlinked dwarfism with genes identical by descent or simply alike in state. H U TT ( 1959 ) predicted the dwarf gene should eventually be found in many breeds of Bantam fowl into which the mutant was probably incorporated over the years in light of its recurrence. We have continued to obtain a few dwarf segregates from an inbred line which, to our regret, because of limited facilities, we were forced to mix with our other dwarfs. We have subsequently hypothesized that some of these dwarf segregates were probably of the hypothyroid type described by COLE ( 19 66). They have complicated our selection program. Today we would like to review some of our findings under the headings of genetics, reproduction, genetic selection, nutrition and physiology. GENETICS In the first year of our more intensive studies, in 1957 , we established that the dwarf trait we were working with was determined by a sex-linked recessive gene, possibly homologous to that described by HuTT ( 1949HuTT ( , 1953 in the New Ham!shiye breed. Correspondence with Dr H UTT indicated that he had yet another dwarf gene under study, this time in Single Comb White Leghorns. We have since determined that it was likely of the same origin as some of the birds donated to the Oregon Agricultural Experiment Station. H UTT ( 1959 ) in reporting his studies on dwarfism in both the New Ham!shire and the Single Comb White Leghorn expressed skepticism on the economic future of the dwarf layer because of its reduced egg production and egg size. However he recognized the possibility that its feed consumption might compensate for the lower egg production and egg size. When this study of H UTT was published we already had an experiment under way to compare the performance of 120 normal and 120 dwarf sister segregates from a mating of males heterozygous for dwarfism with normal females. We found, B!RrTIER (ig6o), and B ERNIER and A RSCOTT (i 9 6o), Table i, that dwarf hens did not lay as well as their normal size sisters and that their eggs were not as large but that their reduced feed consumption, when expressed on a per dozen of 24 oz. basis, made the dwarf layers not only competitive but superior. These results encouraged us to continue our studies and to expand them by undertaking a genetic selection program as well as an investigation of the nutritive requirements of dwarf layers. We reasoned that the reduction in body size caused by dwarfism meant a reduction in maintenance requirements and therefore that a greater proportion of the feed intake would have to satisfy the requirements for egg production. In the first few years we had to combine our nutrition and selection studies because of limited facilities and numbers but as our enthusiasm grew we decided to undertake nutrition studies on a larger scale by substituting dwarf for normal size layers in our regular nutrition research program in the Poultry Department. It was also decided to produce the larger number of dwarf layers necessary for these nutrition studies by mating dwarf males to normal size females. This program has been continued since and in one year we were presented with a gift in the form of an unusual bird which we would like to introduce to you at this time. MOSAIC FOR DWARFISM A mosaic for dwarfism and normal size, this bird, we would like to remind you, is the progeny of a dwarf male and of a normal female. Phenotypically if it is a female it should be a dwarf but to our surprise we found it to be a half-sider, a mosaic or a case of lateral asymmetry as the illustrations clearly show (fig. I a-i b). The mosaic is without question normal on its right side but dwarf on its left side. It has not been an exceptionally good layer but it has laid well and when mated to a dwarf male it has given exclusively dwarf progeny, both male and female in a I : I ratio. The large spur on the right leg has developed this last year. There is a sizeable catalog of mosaics in the poultry genetics literature which was reviewed by H OLLAND E R (i 944 ). What are the possible explanations for our half and half mosaic ? The most generally accepted hypotheses for lateral asymmetry in the domestic fowl are those proposed by CREW and Murrxo ( 193 8,i 939 ) ; a slight discrepancy in size, less than 4 p. ioo, would be explained by the elimination of an autosome at the first cleavage division whereas a major discrepancy ( 10 -15 p. 100 ) would be the result of non-disj unction at the same stage, an autosome being lost from one side and gained on the other. We cannot accept CREW and Murrxo's hypothesis of non-disjunction of an autosome to explain our case. The sex chromosome carrying the gene for normal size needs to be reintroduced into the zygote at the first cleavage. Non-disjunction of Z d w at the first cleavage would result in the absence of Z d W on one side and it would probably be lethal whereas Z d w Z dW would probably not be a simple dwarf female. So far we favor one hypothesis and Dr R. N. S HOFFN T R , University of Minnesota (Personal communication) has offered another that is a possibility but a remote one in our estimation because of the two chance events it requires. The hypothesis we favor is that the polar body carrying the large sex chromosome, Z + , and therefore the dominant sex linked gene for normal body size, since the dam was a normal-size female, reunited with half the zygote after the first cleavage. This would result in triploidy for the autosomes and diploidy for the Z sex chromosome on the normal side, AAAZ + Z d w, and simple diploidy on the dwarf side, or AAZä w W. The second possibility, suggested by Dr R. N. S HOFFNER , is that of non-disjunction of the ZW at the first metaphase (first polar body extrusion) resulting in the female gamete with a set of autosomes, A, and Z + W sex chromosomes. This female gamete, AZ + W, fertilized by a dwarf male gamete, AZ r lW, would result in a zygote diploid for autosomes but trisomic for the sex chromosomes, AAZ + Z d wW. At the first or possibly some very early cleavage the chance segregation of the sex chromosomes could be Z d wW to one daughter cell and Z + O to another daughter cell which followed by unilateral proliferation of these cells would result in one side, Z d w, being dwarf as well as a genetic female, while the other side, Z + O, would be a genetic « neuter » of normal size. The endocrine control would originate from the left functional gonad or ovary. These two possible explanations are illustrated in fig. 2 . The critical observation will of course be to determine the karyotype of the mosaic on each of its two sides. So far a limited study of feather follicles and of blood cell cultures have not shown departure from the normal female karyotype of ZW. We plan a detailed cytological study whenever we undertake an autopsy on this mosaic bird. SEX-LINKED RECESSIVE LETHAL In our genetic selection in the dwarf line, in i 9 6i, we observed a sex-linked recessive lethal which probably would have gone unnoticed had it not been for [some unusual circumstance. Earlier we referred to the fact that we innocently had mixed birds possibly carrying hypothyroidism and sex-linked recessive dwarfism. As our intensive selection program progressed we necessarily inbred and this resulted in the segregation of the polygenes for hypothyroidism. These segregates were manifestedly poorer layers and many were non-layers while the males were often obese and difficult if not impossible to ejaculate. Chance favored us with a male that was an exceptionally heavy semen producer as well as easy to ejaculate. This male was used extensively in inseminating 5 o dams and it sired 437 chicks. Throughout the brooding season we noted that there appeared to be an unusually large number of red combs in that brood of chicks. It turned out that the sex distribution was 297 males and 140 females or 68 : 32 or very close to the 2 : i ratio expected on the basis of a sexlinked recessive lethal. The affected female zygotes apparently die very early, so early that it is often difficult to identify the egg as infertile or as early dead. It has not been possible as yet to determine the relationship of this sex-linked recessive lethal to the gene for dwarfism. Male ;! 6 2 66 was also heterozygous for the spontaneous chromosome translocation we have described earlier, BE RNI E R ( 1953 , ig6o) and R y arr and B!RNWR (i 9 68), and it is likely that this heterozygosity may explain the excellent semen production. Table 2 shows the results obtained with male ;!6 2 66. REPRODUCTION We can now report on reproduction in different types of matings. First, when dwarf females are produced by mating dwarf males with normal females, we indicated earlier this is how we produce the females needed in the nutrition studies. It is the easiest method to produce dwarf stock in a large breeding organization. Only a small number of males need to be bred to allow the production of a very large number of dwarf pullets. Results over a period of years are illustrated in table 3 . The first year of this program in 19 6 5 we did not have a sufficient number of males to mate in natural matings with the females available and necessary to produce the number of pullets required. We therefore resorted to artificial insemination. In 19 66 the number of males was again insufficient but we decided to use the small number available in groups that we rotated from pen to pen every three days with a period of three days in each cycle of nine days without males. In 19 6 7 we had enough males to mate in the ratio of i : r 5 but the fertility (8 0 .8 p. ioo) was not considered satisfactory. In 19 68 and since, the male-female ratio was lowered to I : 10 but only this year in 197I has fertility reached a high level and rapidly enough to be considered satisfactory. What may be a simple explanation for this improvement is that until this year the males, kept in the dark to reduce fighting, were moved directly from dark holding pens to the breeding pens. Apparently it took a period of time for them to produce satisfactory amounts of semen, to adjust to their new surroundings and to mate with all the females. This year we moved the males from darkness to a lighted pen approximately one month before they were to be placed with the hens. We have plotted the rate of increase in fertility in the different years in the different pens and the results are illustrated in fig. 3 a-As indicated earlier we are now satisfied that good fertility can be obtained in matings of dwarf males and normal females but we would like to increase, if possible, the I : 10 male-female ratio we have used in recent years. We have no explanation for the low fertility observed in ig6g not only in these dwarf-normal matings but also in our regular matings as we note in Table 4 other than some lighting problem which one cannot identify more completely. Second, dwarf birds can of course be reproduced as a line which we can submit to selection for economic traits. Table 4 shows the fertility and hatchability observed in recent years in our selection program in the dwarf and in the normal size lines for comparative purposes. It is evident that the reproductive performance of our dwarf line is comparable to that of our normal size Production Line. GENETIC SELECTION Observations on our dwarf layers in the first few years revealed that their principal defects were delayed sexual maturity, smaller eggs and lower egg production. Limited facilities did not allow us to test as many families as would have been desirable, so we compromised. We raised more dwarf pullets than we had room to test for the entire production period and we placed heavy selection pressure on sexual maturity and on initial egg size. We rigorously culled individuals and families that matured late and that laid small eggs. Although a convenient compromise it unfortunately prevented us from obtaining comparative performance data in the early years except in the nutrition investigations. In more recent years we have been able to allocate more cage space to the dwarf selection program and we can report on the relative performance of the dwarf line and of our OSU Production Line. Table 5 gives those comparative data for recent years. Sexual maturity (SM) expressed in weeks was as high as 32 . 5 weeks in the dwarf line in 19 66-6 7 but it has gradually decreased to 24 . 3 weeks in the current generation which is only i. 7 weeks later than that of our OSU Production Line. Hen-housed egg production (HH) which was disappointingly low in 19 6 5 -66, 19 66-6 7 , and 19 68-6 9 , began to show improvement in 19 68-6 9 and in the current generation it is 8 3 . 4 p. 100 as high as that observed in the OSU Production Line. We should perhaps indicate that the dwarf layers in this selection project are fed a commercial complete breeder 15 p. I oo protein ration which we have shown in our nutrition investigations to be inadequate in protein or methionine for them. This inadequacy provides an additional stress factor which hopefully increases the sensitivity of our selection. Body weight as measured at io and at 20 weeks of age, when the layers are housed, has not changed in the dwarf layers but only because of the restriction placed on it in our selection. It would very likely have increased as a result of a correlated response to our selection for egg size. We have not always been able to measure body weight of all the dwarf layers at 20 weeks because of the limited housing facilities in prior years. Egg size, as we have indicated earlier, is critical for dwarf layers and we have placed emphasis on this trait in our selection. We are making progress and the fact, as demonstrated by F ESTING and N ORDSKOG (ig6 7 ), that egg size can be manipulated somewhat independently of body weight and which is so well demonstrated historically in the modern Leghorn is certainly justification for optimism. I,ivability in the dwarf line has usually been superior to that observed in our OSU Production Line. NUTRITION STUDIES Our nutrition studies began in 195 8 with the association of GHA with the research project. That first year we placed 120 dwarf and 120 of their normal size sisters in individual cages. We kept individual records of feed consumption, of all eggs produced and of their weight. We compared the effects of two calcium-phosphorus levels ( 3 . 0 p. I oo Ca-o. 9 p. I oo P and 2 . 25 p. ioo Ca-o.6 p. I oo P) on corn-soybean mealfish meal rations, with similar calorie protein ratios (8 4 , 9 -8 7 . I ), containing r5 p. 100 protein with either the normal or double that level of a vitamin trace mineral supplement (DVTMS) and 1 8 p. 100 protein with double the normal vitamin trace mineral supplementation. These results were reported by A RS C O T T , R A C HAPA E TAYAKOM and B!RNIER (ig6i) and are summarized in table 6. The additional calcium and phosphorus improved egg production and shell quality of dwarf layers more than those of their normal sisters. The additional vitamin trace mineral supplementation also benefited egg production and shell quality in the dwarf layers. The r8 p. ioo protein level did not bring an improvement in egg production but it resulted in a marked decrease in feed intake which may have masked the possible improvement in egg production. Egg weight was not affected by any of the treatments. Our next two experiments conducted in ig6i and 19 6 2 compared different levels of calcium and have not been reported as yet. In general they confirm the results obtained in the first experiment showing improved shell quality from higher calcium levels. In our third investigation we studied the influence of ascorbic acid, calcium and phosphorus on egg shell quality as measured by specific gravity. This experiment was reported by A RS C O TT, RAC H A PA ET AY A K O M , BERNI!R and A D A M S ( 19 6 2 ) and the results are illustrated in table 7 . No improvement in egg shell thickness was apparent with either normal size or dwarf White Leghorn fed rations containing ascorbic acid. A marked improvement in shell thickness, accompanied by an increase in blood calcium and phosphorus, was obtained in normal size birds in the presence of 3 p. 100 calcium and a similar improvement in shell thickness was obtained with the dwarfs fed the rations containing 3 p. 100 calcium. Increasing the phosphorus level to 0 . 9 p. IOO appeared to reduce shell thickness and to increase blood phosphorus. This latter observation has since been reported by others. In our fourth nutrition investigation we measured the growth and feed requirements of developing dwarf pullets compared to their normal size sisters. We used two populations of 700 day old chicks of each type which were each brooded in duplicate groups. Both types of birds were fed the same rations. During the first 8 weeks they were fed a high energy corn-soybean meal-5 p. IOO fish meal starter diet with 20 p. 100 protein and i 370 kcal. metabolizable energy/lb. From the eighth through the 23 rd week the birds were fed a medium energy corn-barley-soybean meal-2 p. 100 fish meal grower diet with i 4 . 9 p. 100 protein and 1 2 85 kcal. metabolizable energy/lb-Body weight and feed consumption were recorded at 4 -6 week intervals. The results were reported by B ERNIER and A RSCOTT ( 19 66) and are illustrated in Tables 8 and 9 and fig. 4 a-4 b for both body weight and feed consumption. Feed conversion as shown in table io consistently favored the dwarf pullets. Mortality, Table ii, was heavier in the first 8 weeks of life in the dwarfs possibly because they were not rigidly culled at hatching time because of limited numbers. Table 12 illustrates the fact that even after allowing for the delayed sexual maturity of the dwarf pullets they still required much less feed to reach laying age. Our fifth investigation involved two experiments comparing the development and feed requirements of developing dwarf pullets fed a 20 p. 100 protein starter for the first four or the first eight weeks followed in both instances by a i 5 p. ioo protein developer. The results have not been published as yet but they are satisfactory and furthermore the egg production of the pullets is not impaired by the lower protein level fed during the development period. The sixth investigation compared different protein levels in two separate 2 8 0day periods using corn-soybean meal base diets for the dwarf layers, supplemented by higher calcium and vitamin trace mineral levels than those fed the normal layers-3.7 p. 100 vs. 2.8 p. ioo Ca and 0 . 33 p. 1 00 vs. 0 . 25 p. ioo vitamin trace mineral supplement respectively. The protein levels ranged from 12 to 2 i p. 100 for the dwarf layers and from 12 to 1 8 p. I oo for their normal sisters. The results of these two experiments have been reported by A RSCOTT and BE RNI E R ( I g68) and BE RN IER and A R scoTT ( I g68) and are shown in Tables 13 -1 6. Egg production and feed per dozen eggs for the dwarfs both plateaued at the z 5 p. IOO level of protein while in normals this occurred at 14 p. 100 . The dwarfs laid i 3 p. 100 fewer eggs but the feed required per dozen eggs was 1 8 p. 100 less than in normals. Egg size increased with protein levels to 21 p. I oo in dwarfs but in normals it plateaued at 1 6 p. IOO protein. Feed consumption of dwarfs increased with protein levels in Experiment i but it plateaued at z 5 p. IOO in Experiment 2 . Dwarfs fed 21 p. I oo protein had a protein intake of y . 3 grams while normals had a protein intake of y . 2 grams when fed 1 6 p. 100 protein. The addition of . 05 p. ioo dl-methionine to the z2 p. I oo protein diet of dwarfs increased egg weight significantly but . 05 p. I oo lysine was without effect. Our last reported nutrition investigation also involved two experiments each of ten 2 8-day periods and in which a ration of i 5 .2 p. ioo protein, with and without different levels (. 025 , . 05 , . 1 p. ioo) of dl-methionine, a level (. 05 p. 100 ) of lysine and a level ( 2 p. 100 ) of safliower oil, alone and in combination, as well as a ration containing 20 . 9 p. zoo protein, were fed to duplicate lots of dwarf layers and compared to a ration of 15 .8 p. 100 protein fed to duplicate lots of normals. This study was reported by A RSCOTT and B!RNI!R ( 1970 ) and table 17 shows that in Experiment i only the higher protein level resulted in a significant improvement in egg production of the dwarfs although . 05 p. I oo dl-methionine resulted in an intermediate response. In Experiment 2 no difference was observed between the two protein.levels or. the various levels of methionine or combinations of methionine (. 05 p. ioo), lysine and safliower oil. There was a tendency towards higher production in the presence of . 05 p. Ioo methionine or 20 . 9 p. 100 protein. Table 1 8 shows that in Experiment i the addition of . 05 p. I oo dl-methionine and the 20 . 9 p. I oo protein rations both brought about a significant increase in egg weight. In Experiment 2 increasing levels of methionine resulted in increased egg weight but only the .i p. 100 level of methionine showed a significant improvement over the negative control. The dwarf group fed the 20 . 9 p. I oo protein diet also laid significantly larger eggs than the control group. Combinations involving . 05 p. 100 dl-methionine and 2 p. IOO safflower oil with or without . 05 p. 100 I-lysine also brought about a significant improvement in egg weight. Table I g shows the effect of protein level and supplements on feed per dozen eggs. The normals produced eggs less efficiently than the dwarfs. The most efficient performance was that of dwarfs fed the supplement of . 05 p. 100 dl-methionine in Experiment 2 . The various treatments had no significant effect on final body weight of dwarfs. Normals weighed significantly more than dwarfs or 3 . 9 pounds as compared to 3 . 0 pounds. It thus seems that supplementation of a 15 . 2 p. I oo protein diet with . 05 and . dl-methionine improved egg production and egg weight in dwarfs to levels comparable to those observed with the ,2o.g p. 100 protein diet. However, dwarfs still laid fewer and smaller eggs than their normal size sisters although there was progress attributable to both genetic and nutrition influences. We have conducted an additional experiment on supplementation of a i 5 5 p. I oo protein diet for dwarfs with levels of up to 0 . 15 p. IOO methionine which has not been reported yet. There was no apparent beneficial effect from levels above 0 . 05 p. 100 dl-methionine. Fish meal at the level of 3 p. I oo apparently meets the increased requirement for methionine in the diet of dwarf layers. Currently we are conducting an experiment comparing diets based on different cereals with and without a supplement of 0 . 033 p. I oo Protamone. Dwarf pullets fed diets supplemented with Protamone grew faster and ate more in the first four weeks than even normal size pullets but the effect gradually decreased and was negligible at 1 6 weeks of age. The level of 0 . 033 p. 100 Protamone in the diet of layers depressed egg production and was subsequently reduced by one half. PHYSIOLOGY In the early years of our work with dwarfs we realized that a better understanding of their physiology, more specifically, the hormonal factors involved, would more precisely define their potential. We therefore undertook some studies with the kind cooperation of a colleague endocrinologist in the Department of Animal Science, Dr E. F. F,I, LINGTON , now at the University of Nebraska. The studies were begun in 19 66 and when completed the results were presented as a thesis by one of our graduate students, Mr. I,. W. M IROSH , ( I g6 7 ) recipient of the Chester M. Wilcox Memorial Fellowship in Poultry Science. Two manuscripts incorporating the major results will be submitted for publication shortly (M IROSH , BE RNI E R , E I ,I,I N GT ON , A R scoTT and RowE, 1971 ). The study was conducted on I g normal size and 21 dwarf pullets 2 -3 months of age and 20 normal and 20 dwarf pullets 7 -i 3 months of age. It was in two parts. One involved measurements of various endocrine glands and certain anatomical structures as shown in Tables 20 -22 . The second part involved the bioassay of the hypophyses of both types of birds at the two ages using hypophysectomized rats and the results are reported in Tables 23 -25 . Normal chickens in both age groups were found to have significantly heavier hypophyses, thyroids, adrenals and ovaries than the dwarfs. These differences tended to disappear when the endocrine weights were expressed as a function of body weight, either absolute or metabolic body weight, except for the following instances. The relative weight of the hypophysis was significantly smaller in the older normals than in the other three groups of birds which could also 'indicate that the older dwarfs had a larger than expected hypophysis on a relative basis. The adrenals were significantly heavier in the older normals than in the other three groups which would indicate that the dwarfs did not have as large adrenals as would be expected. The relative weight of the thyroid was significantly heavier in the older normals than in the young normals while the reverse held for the dwarfs which had a relatively smaller thyroid as they grew older. The weight of the thyroid in the normal chickens almost doubled as they passed from the 2 -3 month to the 7 -i 3 month stage while that of the dwarfs changed very little which would lead to the hypothesis of hypothyroidism. This hypothesis is also supported by the histological characteristics of the thyroid. The epithelial cell height of the older dwarfs was significantly lower than that of the older normals and the young chickens of both types. The diameter of the colloid and the follicle were significantly higher in normals than in dwarfs of both ages. It is tempting to explain sex-linked [recessive dwarfism by hypothyroidism. However the finding that such dwarfism is not overcome by the feeding of iodinated casein (van T I E NHOV E N et a l 19 66 ; B!RNI!R, 19 65, unpublished) nor by the injection of thyroxin (B ERNIER , i 9 6 5 , unpublished) would argue against hypothyroidism as the the principal or sole factor involved. Notwithstanding these facts hypothyroidism as an explanation for sex-linked recessive dwarfism gains support from the histology of the thyroid as observed in this study and that of van TI!NHOV!N et al. (i 9 66). More recently, MA RA T and G UILLAUM E ( 19 6 9 ) have reported smaller thyroids relative to body weight, a lower metabolism, reduced thyroid secretion, a higher percentage of abdominal fat, and a calmer disposition in dwarf birds than in normal size birds which led them to hypothesize hypothyroidism as associated with sex-linked recessive dwarfism. Whether or not alterations in pituitary thyrotropic activity are responsible for the thyroid disturbances observed in dwarfs could not be fully ascertained from the bioassay results of the hypophyses with hypophysectomized rats in this study. Of the four doses of chicken pituitaries, ranging up to as much as four pituitaries, only the highest dose originating from the normal chickens of both ages and from the older dwarfs brought about a significant weight response in the thyroids of hypophysectomized rats. Either the sensitivity of the rat thyroid to chicken thyrotropic stimulating hormone is low or the pituitary thyrotropic stimulating hormone is at a relatively low level in the chicken. On the basis of pituitary cytological studies with sex-linked recessive dwarfism, van T IENHOVEN et al. ( 19 66) have proposed that sex-linked recessive dwarfism is the result of a failure in the release of somatropic hormone from the pituitary. They found that the anterior pituitaries of dwarf chickens showed frequent recurrence of small cells with secretory droplets which they speculated to be the result of an accumulation of somatropic hormone because of a failure of its release into circulation. The pituitary bioassay results in our study do not support the contention of such an accumulation of somatropic hormone. Total pituitary STH content of dwarfs did not differ significantly from that of normals at either age studied. Nevertheless it is still possible that a decreased pituitary secretion of STH which would not neces-sarily be detected by a bioassay of only the pituitary glands could be a factor in dwarfism. The possibility that ACTH and the adrenals may be involved in sex-linked recessive dwarfism must not be overlooked. The significant involvement of adrenal hormones in pathways of carbohydrate, protein, and fat metabolism in mammalian species is well known and the same general conclusion is probably also valid for birds. As compared to the normals, the pituitary ACTH activity of dwarfs was lower at 2 -3 months of age but comparable at 7 -13 months of age. Incidentally pituitary ACTH concentration for both types of birds appeared to be higher at the younger age than at the older one, as did STH concentration which may well be a reflection of involvement at a time when growth is rapid. The absolute adrenal weight was significantly less in the dwarfs than in the normals at the younger age but not at the older one. The difference at the younger age was removed by expressing the weight Gonadotropin content of the hypophyses of dwarf and normal chickens appeared comparable at both ages as evidenced by ovarian and uterine weight responses together with ovarian histological responses in the assay rats. There were no apparent differences in ovarian weight and in comb weight and area, comb size being an indirect reflection of ICSH activity. It is also of interest to note that van T IENHOVEN et al. ( 19 66) were unable to find differences in the presumptive gonadotropic cells between the two types of chickens upon histological examination. It would appear from the result of this study that sex-linked dwarfism may possibly involve more than one endocrine disturbance, possibly other endocrine structures not considered in the present study such as the pancreas. The development of procedures, such as radioimmunoassay, that allow detection of circulating levels of hormones from the pituitary will greatly facilitate progress in solving the problem. Hormone administration, as has been done with thyroidal principles (van T IENHOVEN et al., ig66 ; B ERNIER , 19 6 5 , unpublished) would appear to be a promising approach to study the endocrine basis of dwarfism. Because of the species specificity problem existing for protein hormones, it would be advisable to obtain such hormonal materials from chickens for experimental purposes. Unfortunately these materials are not, to our knowledge, readily available at the present time. So much then for the evidence we have accumulated on the economic potential of dwarfism in egg production. Another area of great promise is that of management, more particularly housing density, or to paraphrase H. B. WA!,r,AC! (zg 7 o), oviduct density per square foot. We have not worked in that area but we were pleased to note a significant study by N ORDSKOG & B RIGGS ( 19 68) which needs to be placed in its proper perspective to be fully appreciated. Before the advent of the broiler industry the profitability of an egg enterprise depended not on lyon egg production but also on the salvage value of body weight which meant reduced depreciation. There was also a general belief amongst poultrymen that body size was indicative of stamina because larger birds appeared to stand up better under conditions of stress. The evidence as reviewed by Hu2!r ( 1949 ) appeared to show a curvilinear relation between body weight and egg production altough the extensive data of Tny!,ox (ig 3 o) collected in Canada supported a linear relation in Legho y ns. In the light of the popular belief and the experimental evidence relating heavier egg production in heavier Leghorns, the recent study of N ORDSKOG and B RIGGS ( 19 68) elucidates the body-weight paradox as they termed it. They found that heavier birds stand up better under environmental stress but that smaller birds are genetically superior in egg production. There is thus an antagonism between the genetical and the environmental effects. In itself this antagonism justifies the trend to controlled environmental housing for egg production. We have noted in our work that minilayers withstand short periods of hot weather better than our normal size layers while the reverse is true in periods of cold weather.

v3-fos

2020-12-10T09:04:16.829Z

1972-02-01T00:00:00.000Z

237234963

s2

Effect of Selected Herbicides on Bacterial Growth Rates Specific growth rate constants were used to evaluate the effects of selected herbicides on Erwinia carotovora, Pseudomonas fluorescens, and Bacillus sp. Comparison of growth rate constants permitted the identification of either stimulatory or inhibitory effects of these substances. E. carotovora was inhibited by 6,7-dihydrodipyrido(1,2-a:2′-c)pyrazinediium (diquat) and 4-hydroxy-3,5-diiodobenzonitrile (ioxynil) at 25 μg/ml; 1,1′-dimethyl-4,4′-bipyridinium (paraquat) at 50 μg/ml; and pentachlorophenol (PCP) at 10 μg/ml. P. fluorescens was inhibited by paraquat and PCP at 25 μg/ml and by 4-amino-3,5,6-trichloropicolinic acid (picloram) at 50 μg/ml. Stimulation of P. fluorescens was observed with 4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline (nitralin) at 25 μg/ml. The Bacillus species was inhibited by diquat (25 μg/ml), ioxynil (10 μg/ml), and paraquat and PCP (5 μg/ml). No significant effect of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin), or 1,1-dimethyl-3-(α,α,α-trifluoro-m-tolyl)urea (fluometuron) on growth rates of the bacteria was observed at 25 and 50 μg/ml. Growth flasks (250-ml Bellco side arm) were inoculated with a 4.4-ml inoculum of the organism giving a final volume of 40.4 ml and were shaken at 30 C in water bath for 108 oscillations per min (WCLID model C-2156). Optical density (OD) at 600 nm was determined at regular time intervals in a Bausch and Lomb Spectronic 20. The OD.6,0 values from the growth curve were converted into dry weight values from a standard dry weight curve for each organism. The slope of the straight-line segment of the plot of log dry weight versus (Table 1) was observed with diquat, ioxynil, paraquat, and PCP. No response was observed with nitralin or picloram. The growth rate of P. fluorescens (Table 1) was inhibited by paraquat, PCP, and picloram. Inhibition of the growth rate of the Bacillus sp. (Table 1) was observed with diquat, ioxynil, paraquat, and PCP. Inhibition of the growth of B. megaterium and B. subtilis by ioxynil and bromoxynil (3,5-dibromo-4-hydroxy-benzonitrile) has previously been reported (7). An apparent stimulatory effect on P. fluorescens was observed in the presence of nitralin (Table 1). This may reflect a utilization or alteration of the compound by the bacterium. Atrazine, trifluralin, cotoran, and diuron did not significantly affect the growth rate of any test organism. These chemicals are relatively nonpolar in nature, and thus they may be unable to penetrate the bacterial cell or to inhibit any membrane or cell wall surface activities. In situations in which growth of the specific organism was not completely inhibited by the test chemical, long lag phases followed by growth were observed. This may have been due to the adaptation of the organism to the herbicide or to mutant selection. E. carotovora in the presence of ioxynil, PCP, and diquat; P. fluorescens in the presence of ioxynil, paraquat, diquat, PCP, and trifluralin; and Bacillus in the presence of ioxynil and diquat exhibited this type of response. The technique reported in this study utilized a growth rate constant which is characteristic of the conditions of the growth of the bacterium in liquid culture. These values were used to evaluate the effects of selected herbicides in terms of stimulatory or inhibitory responses. Results obtained in this study are being investigated further in terms of adaptation to, and degradation of, specific compounds. Herbicides used in this study were provided by the following companies: Amchem Products, Inc.; Chevron Chemical Co.; CIBA Agrochemical Co.; Dow Chemical Co.; E. I. du Pont de Nemours and Co.; Elanco Products Co.; Geigy Agricultural Chemical Co.; and Gulf Research and Development Co. The technical assistance of Jannie Huffman is greatly appreciated. F.W.B. was supported by The Belle W. Baruch Foundation.

v3-fos

2018-04-03T02:15:51.794Z

1972-04-01T00:00:00.000Z

32756471

s2

Ruminal bacterial degradation of benzo(b)-thien-4-yl methylcarbamate (Mobam) and effect of Mobam on ruminal bacteria. Mixtures of ruminal bacteria degraded benzo(b)thien-4-yl methylcarbamate (Mobam) to 4-hydroxybenzothiophene, CO(2), and polar product(s). The metabolite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrin-hydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3',6'-diacetate. lite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrinhydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3', 6'-diacetate. Benzo(b)thien-4-yl methylcarbamate (Mobam) is an effective pesticide that exhibits broad spectrum insecticidal activity coupled with an apparent low mammalian toxicity (2,7). Ruminants administered "IC-Mobam as single oral doses excreted 87 to 96% of the 14C dose via the urine in 24 hr (10). Cleavage of the methylcarbamate moiety of Mobam was evident in the production of two primary metabolites, 4-benzothienyl sulfate and 4-benzothienyl sulfate-i-oxide. The purpose of this investigation was to determine whether ruminal bacteria degrade Mobam. The effect of Mobam on ruminal bacteria was also considered. MATERIALS were obtained by stomach tube from calves (>70 days of age, free of ruminal ciliated protozoa) maintained on a pelleted ration of alfalfa, grain, and wheat bran (10:5:1) (13). Bacteria from the ruminal samples were concentrated by centrifuging the contents at 3,000 x g for 8 min and then centrifuging the resulting supernatant fluid at 13,000 x g for 20 min at 5 C. The sedimented bacterial cells were made up to one-fourth the original volume with basal medium (14) and incubated with "4C-Mobam (100,000 to 250,000 dpm/ml of medium with Mobam concentration adjusted to 72 ug/ml). Preparations of cell suspensions of pure cultures were obtained from each culture incubated in RFTY liquid medium for 48 hr at 39 C. The cultures were centrifuged, diluted in basal medium (14), and incubated with "C-Mobam as above but for 18 hr. Cell densities for each culture were diluted so that a 1:10 dilution of 745 on March 19, 2020 by guest http://aem.asm.org/ Downloaded from the incubating cell preparation had an absorbancy reading of 0.4 at 600 nm in 1.2-cm-diameter cuvettes. Assays. Thin-layer chromatograms were prepared in the laboratory with Silica Gel G (250 ulm thick, Brinkmann) on glass plates (5 by 20 by 0.35 cm) and spotted with "4C-material. The TLC plates were developed ascendingly with hexane-ethyl acetateacetic acid (70:30:2) and scanned for "4C distribution (14). Samples were dissolved in dioxane or toluene (12) scintillation solution. The dioxane formulation consisted of 7.0 g of PPO (2,5-diphenyloxazole), 100 g of naphthalene, 300 mg of POPOP [1,4bis-2-(5-phenyloxazoyl)-benzene], and enough reagent-grade dioxane to bring the volume to 1 liter. Counting of radioactivity was done with a liquid scintillation counter. Counting efficiencies were determined by internal standardization with toluene-1-"4C or by channels ratio standardization. Counting times ranged from 2 to 30 min, depending on the level of radioactivity in the samples. Radioactive "CO2 produced in "4C-Mobam culture experiments was monitored as previously described (12). Gas production of ruminal bacteria in the presence of Mobam (100 to 500 ug/ml) was determined manometrically (14). Bacteria were assayed for carboxylesterase activity by incubating the preparations with 1-naphthyl acetate (NA) for 15 min at 39 C. The reaction was stopped by addition of 10% lauryl sulfate solution, and the 1-naphthol produced was coupled with fast garnet GBC (CI 37210, 4-amino-3, 1'-dimethyl azobenzene) and read photometrically at 560 nm (9). Similarly, fluorescein-3',6'-diacetate (FDA) was used as a substrate for bacterial carboxylesterases, with fluorescein production determined with a spectrofluorometer (4). Protein content of sonically treated bacterial extracts was determined by the method of Lowry et al. (8), with bovine serum albumin (Sigma Chemical Co.) as the standard. Metabolite detection. Bacterial cultures containing "C-Mobam products were extracted with methylene chloride. The extract was evaporated to dryness, taken up in methanol, and applied to a column of Sephadex LH-20 (0.9 by 40 cm) in methanol. The column was eluted with methanol, and the radioactive fractions were analyzed by TLC and gasliquid chromatographic (GLC) techniques. Metabolites were recovered from TLC plates by stripping "IC-containing bands and eluting with methylene chloride. The "C material was taken to dryness under N, and derivatized with 1 gliter of pyridine and 100 Mliter of acetic anhydride. The sample was then analyzed by GLC on an instrument equipped with a "C monitor and effluent splitters to facilitate simultaneous flame ionization detection and trapping with glass capillary tubes (14). A 2% SE-30 Chromasorb W (60/80 mesh) column [6 ft (1.8 m) by 4 mm] programmed from 100 to 200 C at 5 C/min was used. Injection port and detector temperatures were set at 300 C. Helium (55 ml/min) was used as a carrier gas. Metabolites trapped were analyzed by infrared and mass spectrophotometric analysis as previously reported (14). Culture techniques. Effect of Mobam on growth of mixed ruminal bacteria was determined as follows. Gauze-strained ruminal contents (0.1 ml) were inoculated, in duplicate, into 13-mm cuvettes each containing 4.9 ml of RFTY medium under CO, with either 0, 2, 10, 50, 100, or 200 Mg of Mobam/ml of medium. The cuvettes were flushed with CO, sealed with neoprene stoppers, and incubated at 39 C for 24 hr. Absorbancy readings were taken at 600 nm. Readings (at 0, 4, 8, and 24 hr of incubation) were compared with uninoculated medium containing the appropriate concentrations of Mobam. Mixtures of bacteria from ruminal fluid (300 ml) and newly isolated ruminal bacterial strains (see below; no. 29, 53, and 88, each grown 48 hr in 300 ml of RFTY liquid medium) were separately concentrated by centrifugation and suspended in 7.5-ml volumes of basal medium (14). These cells were then sonically treated at maximum intensity (Fisher probe model BP-5; generator model CW-5) for 3 min at 5 C and assayed for NA carboxylesterase activity (9). Agar well-diffusion experiments were run with the sonically treated preparations as follows. A medium of 1.5% agar in 0.2 M sodium phosphate buffer at pH 6.8 was poured into petri dishes containing Mobam dissolved in a minimum volume of ethanol. Final concentration of Mobam was 500 Mg/ml of medium. Fifty to 75 Mliters of the sonically treated cell preparations was added to each 8-mm-diameter well cut in the solidified medium. The preparations were incubated at 39 C. At 0, 4, 8, and 24 hr of incubation, separate preparations were flooded with 10% ferric chloride solution and observed for development of dark blue-black zones due to the presence of 4-hydroxybenzothiophene. These sonically treated cell preparations were compared to sonically treated mixedand pure culture-cell preparations treated to temperatures of near 100 C for 5 min or treated with 15% ethanol. In some instances, FDA (100 Mg/ml) was used as substrate. FDA degradation was indicated by development of a fluorescent zone surrounding the wells when viewed under ultraviolet light. "4C radioactivity in fraction 1, when removed from TLC plates and acetylated, was released from the GLC column when the GLC column oven reached a temperature of 145 C. The retention time of the compound compared favorably with an authentic sample of 4-hydroxybenzothiophene (which was acetylated according to the method used for the metabolite). Infrared and mass spectra of the metabolite were identical to the authentic sample. The metabolite was therefore identified as 4hydroxybenzothiophene. The "4C-compound in fraction 2 was degraded during GLC. It had an infrared spectrum identical with high-purity Mobam. 4C-Mobam degradation. Degradation of "4C-Mobam by mixed-ruminal bacterial preparations is shown in Table 1. Carbonyl-labeled Mobam was progressively degraded with release of 30.7% of the total "4C as "CO2 in 18 hr. Mobam, at RF 0.27 on TLC, accounted for 59.8% of the radioactivity, and 7.2% of the "C remained at RF 0 to 0.1. Total "4C recovered in CO2 and methylene chloride extractions was 97.7%. With ["4C-methyl]Mobam, only a trace of "4CO2 (0.9%) was found at 18 hr. Mobam accounted for 62.0% of the "4C, and the remaining radioactivity (15.1%) remained at the origin. Total "4C recovered was 78.0%. Attempts to account for the loss of the methyl "C were not successful. With "4C-RL-Mobam, no "CO2 was produced, and 33.8% of the "C co-chromatographed with 4-hydroxybenzothiophene at RF 0.49. Remaining "C (65.1%) was at the same RF as Mobam. Total "C recovered was 98.9%. Since no "4C was found at the origin when "C-RL-Mobam was incubated with mixed bacterial preparations but was found when incubated with Mobam labeled in the carbonyl or methyl positions of the methylcarbamate moiety, it indicated that only the methylcarbamate moiety was the source of carbon for production of polar product(s) by the bacteria. These products were not identified. Mobam-degrading bacteria. A number of bacterial isolates from 10-6 and 10-' dilutions of ruminal samples were effective in repeatedly showing zones of hydrolysis in tributyrin emulsion-Mobam medium. Three isolates from this medium were found to degrade Mobam. These obligately anaerobic bacterial strains (no. 29, 53, and 88) are gram-negative motile curved rods that appear similar to isolates of Hobson and Mann (5). These cultures on tributyrin emulsion-Spirit Blue agar medium showed colonies with clearing zones of hydrolysis surrounded by a dark blue coloration. Sonically treated cell preparations of these strains and mixtures of ruminal bacteria (2. 'Remaining 14C in the preparations was methylene chloride extracted and quantitated by liquid scintillation methods. The percentage of 14C recovered was then assigned to the RF values according to thin-layer chromatography (TLC) scanning information for each preparation. WILLIAMS AND STOLZENBERG 159, H-18, and D-32) did not degrade Mobam. Whether these strains could be adapted over a long period of time to degrade Mobam is unknown. With soil bacterial species, adaptation to chlorophenyl-carbamates is necessary before degradation of these substrates occurs (6). Mobam inhibition of bacteria. Manometric experiments with mixtures of ruminal bacteria showed no suppression of endogenous gasses produced in 80 min of incubation in the presence of Mobam concentrations of up to 500 ,ug/ml. Values obtained at the 500-,ug level were 2.86 Aliters of gas produced per min per 38 mg of bacteria (dry weight) from the experimental preparations and 2.65 Mliters of gas produced per min per 38 mg of bacteria (dry weight) from the control preparations (without Mobam). Gas production by the 10 characterized ruminal strains was also not inhibited by Mobam. Growth of mixtures of ruminal bacteria in RFTY liquid medium containing Mobam up to 200 gg/ml suggested that the actively growing members of the mixed population apparently were not suppressed. Evidence that growth could be inhibited by Mobam was demonstrated with a gram-negative, tributyrin-degrading bacillus which was isolated and maintained in Mobam-free medium. This obligately anaerobic ruminal bacterial isolate was inhibited by Mobam and an organophosphate, Phosdrin, as shown in Table 2. Both pesticides at 10-4 M concentrations and above were inhibitory at 72 hr of incubation. This strain (no. 102) was one of only two strains out of ten isolated on Mobam-free medium that was inhibited by Mobam. Strain 53, which was isolated on a medium containing Mobam, as expected, showed a resistance to Mobam at the levels tested in Table 2. Also, strains L-34 and HD-1 showed resistance to these Mobam concentrations. No other strains have been tested at this time. Suppression of ruminal bacterial populations showing zones of tributyrin hydrolysis was demonstrated when numbers of colonies growing in the presence of Mobam (600 ug/ml) were compared to counts growing in the absence of Mobam. At 2 weeks of incubation, 1.2 x 107 colonies/g of ruminal contents showed zones in the presence of Mobam. In comparison, 4.6 x 10' colonies/g of ruminal contents showed zones in the absence of Mobam. Total culturable counts on 0.25% glycerol-2% tributyrin-RFTY-agar medium with and without Mobam, respectively, were 1.4 x 109/g and 1.8 x 109/g of ruminal contents. [On RFTY-agar medium containing glucose, cellobiose, and starch, culturable counts ranged from 7.5 to 14 x 109/g ruminal contents (13)]. When Mobam (500 ug/ml) was incorporated in the agar well preparations with sonically treated preparations of ruminal bacteria, fluorescein production was suppressed. To determine the extent of Mobam inhibition, suspensions of ruminal bacteria were incubated with NA and FDA as shown in Table 3. Mobam, at a concentration of 6.5 x 10-4 M, inhibited 65.5% of the production of 1-naphthol. However, only 5% inhibition was observed with FDA in combination with Mobam at a concentration of 9.7 x 10-2 M. With isolate no. 53, 69.5% inhibition of FDA hydrolysis was observed with Mobam at a concentration of 13.4 X 10-4 M. With isolate no. 29, 35.7% inhibition of FDA hydrolysis with Mobam at 9.7 x 10-2 M was obtained. The levels of Mobam used in these experiments are higher than one might expect in a contaminant of ruminant rations. The rate of Mobam degradation by ruminal bacteria appears to be adequate to detoxify the insecticide. The biological activity of the hydrolysis product (4-hydroxybenzothiophene) is unknown. Data presented on the effect of Mobam on ruminal bacterial endogenous gas production, growth in RFTY liquid medium, and total culturable counts suggested that ruminal bacteria were not inhibited by Mobam. However, colony counts of bacteria showing zones of hydrolysis on tributyrin differential media were reduced in the presence of Mobam, and Mobam inhibited hydrolysis of aromatic ester compounds by pure cultures of ruminal bacteria. The relative importance to rumen metabolism of the bacterial reactions inhibited by Mobam presently cannot be assessed. Further basic knowledge on ruminal bacterial carboxylesterases in relation to water-insoluble aromatic ester compounds and rumen metabolism may be helpful in evaluating Mobam inhibition of microbial reactions.

v3-fos

2020-12-10T09:04:13.098Z

1972-11-01T00:00:00.000Z

237230254

s2

Identification of the Predominant Volatile Compounds Produced by Aspergillus flavus A culture of Aspergillus flavus grown on moistened wheat meal was homogenized with a blendor, and the resulting slurry was vacuum-distilled at 5 mm of Hg and 35 C. The aqueous distillate was collected in traps cooled to -10 to -80 C. The culture volatiles were extracted from the distillate with CH2Cl2, and, after removal of the bulk of the solvent, the concentrated volatiles were examined by packed-column gas chromatography. Nineteen peaks were observed, and coupled gas chromatography-mass spectrometry was employed to identify the larger components. The compounds identified were: 3-methyl-butanol, 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, and cis-2-octen-1-ol. The two octenols were the predominant compounds, and sufficient sample was trapped from the gas chromatograph for infrared analyses; this confirmed the mass spectral identifications and permitted the assignment of the cis designation to 2-octen-1-ol. Both oct-1-en-3-ol and cis-2-octen-1-ol are thought to be responsible for the characteristic musty-fungal odor of certain fungi; the latter compound may be a useful chemical index of fungal growth. flavus exhibits a strong lipolytic activity and, under certain conditions, also proteolytic and amylolytic activities. A. flavus can cause discoloration of products high in fat (8) and has been stated to be the chief aflatoxin-producing microorganism (7). During storage of grain, A. flavus is capable of penetrating the bran, giving rise to interior microflora (13,17). It should be stressed that the odor defined as musty or musty-fungal is ascribed to A. flavus, and before the development of microbiological methods the sense of smell was the only tool available to detect this mold. The purpose of this study was the isolation and identification of the odorous volatiles produced by A. flavus. MATERIALS AND METHODS Microorganisms. A. flavus was isolated from wheat grain and maintained on Czapek-Dox agar slants at 3 C until used. Culture media and growth conditions. The culture medium used was coarse, wheat meal sterilized ' ml of suspension for 100 g of wheat meal. To prepare the suspension, the surface of a well-developed sporing culture of A. flavus grown on 5% brewer's extract agar in a 750-ml flat-bottom Erlenmeyer flask was taken and suspended in physiological saline. After inoculation with the suspension, the wheat meal was mixed thoroughly, after which a 1-to 1.5-cm layer was formed. The mold was incubated at 28 to 30 C and 92 to 96% relative humidity. After 3 to 4 days, the conidia covered the whole area with mycelium. For each use a 2-kg culture medium was prepared. The purity of the cultures was checked by microscope observation. Isolation of volatiles from the medium. About 1,200 g of the coarse wheat meal incubated with A. flavus was cooled to -10 C and homogenized in a Waring Blendor with redistilled ice-cold water (in the ratio 1: 3). The resulting slurry was transferred to a 10-liter round-bottom flask and subjected to vacuum distillation in an all-glass apparatus (14). The distillation step lasted for 4 hr and was done under nitrogen at 5 mm of Hg. The temperature of the water bath was 35 C, whereas that of the cold traps in which the distillate was collected ranged from -10 to -80 C. The extraction of volatiles with an organic solvent was carried out, with the distillate being collected in traps cooled to -40 and -80 C. Portions (350 ml) of the distillate were extracted with CH2Cl, by liquidliquid extraction in an all-glass apparatus. The con-KAMIN'SKI ET AL. centration step took about 8 hr. The resulting CH2Cl2 extracts were dried with anhydrous Na2SO4 and concentrated with a Vigreux distillation column down to a volume of 100 Mliter (14). The concentrate thus obtained was then transferred to a glass ampoule and sealed. The same technique was used for the isolation and concentration of the volatiles from noninoculated medium, i.e., a control consisting of the normal, coarse, wheat meal autoclaved at 1 atm for 90 min. Gas chromatography. The separation of the volatile substances in the concentrated distillates was carried out with a Willy Giede model GCHF 18.3 gas chromatograph equipped with a flame ionization detector. The columns were stainless steel (3 m long; outer diameter, 3 mm) packed with 15% Carbowax 20 M terminated with terephthalic acid on 80 to 100mesh, acid-washed, dimethyldichlorosilane (DMCS)treated Chromosorb W. Nitrogen was used as the carrier gas at a flow rate of 20 ml/min. Samples (2 gliter) were applied to the column which was held isothermally at 120 C. Trapping of pure compounds from the distillate for infrared analysis. The separation step was performed by means of the same gas chromatograph equipped with a katharometer. The columns were stainless steel (2 m long; outer diameter, 6 mm) packed with 5% free fatty acid phase (FFAP) on 80 to 100-mesh, acid-washed, DMCS-treated Chromosorb W. Nitrogen was used as the carrier gas at a flow rate of 40 ml/min. The column was operated isothermally at 100 C. The main peaks were trapped as they left the detector in small glass traps containing 1 ml of carbon tetrachloride cooled to -20 C. The concentration and purity of each compound in carbon tetrachloride were checked by means of the gas chromatograph equipped with a flame ionization detector. Identification of the predominant volatiles by mass spectrometry. Coupled gas chromatographymass spectrometry (GC-MS) was carried out at the Department of Food Science and Technology, Oregon State University, Corvallis. The apparatus and operational parameters have been described (16), except for the addition of a single-stage Llewellyn helium separator to the GC-MS interface. Compounds were identified by comparing the mass spectra of the unknowns to a file of standard spectra (1) and to original literature (4). Tentative identifications were verified by GC-MS of the compounds identified and also by GC retention time. IR spectrometry. Infrared (IR) spectra of the isolated fractions trapped from the distillate, as well as those of the standards, were examined by means of an IR 20 Zeiss apparatus. The spectra were taken in spectrally pure CCI4 using cells 0.43 mm thick. The chart speed in the frequency range 700 to 4,000 cm-' was 200 mm per min per 100 cm-'. RESULTS AND DISCUSSION The gas chromatograms obtained from the concentrated distillate of A. flavus and the medium are presented in Fig. 1 and Fig. 2, respectively. The concentrated samples of A. flavus exhibiting a strong odor appeared complex in nature, yielding 19 gas chromatographic peaks, most of them homogeneous. A list of the volatiles identified in the concentrated distillate from A. flavus is presented in Table 1. These compounds were also detected in head space samples taken above the molds. 2-octen-1-ol isolated from the mold was found to differ from the synthetic preparation (Compagnie Parento, Inc.) within the IR range at 700 to 1,050 cm-I and at 1,650 to 1,670 cml ( Fig. 4 and Table 3). According to the data obtained, the synthetic preparation is in the trans form, and that isolated from the mold is of the cis form. The IR spectra of these compounds at wave numbers 2,700 cm-1 to 3,030 cm-1 gave a very intense band; therefore to obtain an onscale spectrum at these wave lengths, a 0.1-mm cell was used instead of 0.43 mm. Pure 1-octen-3-ol yields a strong characteristic fungal-resinous odor. In concentrations close to the threshold value, 0.01 ,g/1 ml of water, the odor resembled that of mushroom. 2-octen-1-ol exhibits a characteristic and strong musty-oily odor. The recognition threshold of this compound in water is 0.1 gg/1 ml. According to results obtained in this laboratory, these two compounds, i.e., 1-octen-3-ol and 2-octen-1-ol, are produced by molds other than A. flavus; however, the quantities and their relative abundances are different. A. flavus was also cultivated on corn, barley, oats, rice, soybeans, and rape. In the head space samples, in all the cases there were more and less volatile substances. The cereal grains were found to be richer in volatiles than the oil seeds. In all cases, 1-octene-3-ol and 2-octen-1-ol were found, only in different amounts. In wheat, some authors stress the absence of such compounds (12,15). In our experiments, however, in which strongly concentrated distillates were used, these two compounds were found in the control samples (Fig. 2). In the sample from the A. flavus culture, however, the quantity of these compounds was about 1,000 times higher. The occurrence of these two compounds even in sound wheat grain may be due to the infection of the grain by interior microflora (17). Several papers on 1-octene-3-ol are in the literature; it has been detected in milk and milk products (10), soybean and its products (18,19), and potatoes, in which it occurs as the main component of the volatile fraction (5). 1-Octen-3-ol was also found in clover, in which it also comprises the main component of the volatiles (11), and it is also found in cranberries (3) and black currents (2). 1-Octen-3-ol was first isolated from a Japanese mushroom by Murahashi (Chem. Abstr., 32:3755), and it has also very recently been identified in another mushroom, Agaricus bisporus (6). There is scant information available on the occurrence of 2-octen-1-ol in foods, although it has recently been reported in A. bisporus. The occurrence of these two compounds in food products is due, first of all, to the development of microflora. The molds, as well as their spores, are ubiquitous on foodstuffs, and under conditions favoring their development they produce these compounds. The possibility cannot be completely eliminated that these compounds are produced in a purely chemical way through oxidation of linoleic acid (9). In certain foodstuffs, these compounds may come from animal feed. According to Honkanen and Moisio (11), 1-octen-3-ol entered the milk via the bloodstream and originated from the fodder of the cows. Generally speaking, the occurrence of 1octen-3-ol and especially 2-octen-1-ol in agricultural products can indicate their contamination by molds, although in certain products the origin of 1-octen--3-ol may be quite different.

v3-fos

2018-04-03T01:13:20.362Z

1972-11-01T00:00:00.000Z

28511554

s2

Degradation of chlorbromuron and related compounds by the fungus Rhizoctonia solani. The ability of the soil fungus Rhizoctonia solani to degrade phenyl-substituted urea herbicides was investigated. The fungus was able to transform chlorbromuron [3-(3-chloro-4-bromophenyl)-1-methyl-1-methoxyurea] to the demethylated product [3-(3-chloro-4-bromophenyl)-1-methoxyurea], which was isolated and identified. Evidence was obtained that further degradation of chlorbromuron occurred. Several other phenylurea compounds (chloroxuron, diuron, fenuron, fluometuron, linuron, metobromuron, neburon, and siduron) were also metabolized by the fungus, indicating that R. solani may possess a generalized ability to attack this group of herbicides. Biochemical activity of the soil microflora is assumed to be a major factor for removal of phenylurea herbicides from soil (4,6), and several reports have been made concerning the microbial degradation of these compounds (2,3,7,8). However, there is a noticeable lack of detailed microbial and biochemical studies on transformations of the phenylurea herbicides and especially on the participation of fungi, which constitute the largest portion of the total microbial protoplasm in most cultivated soils (1). Hill and McGahen (3) reported first on the involvement of fungi, Penicillium and Aspergillus spp., in the transformation of monuron [3-(p-chlorophenyl)-1, 1-dimethylurea ]; they claimed that it can be used as a carbon source in an agar medium. The first specific data on fungi were presented by Tweedy et al. (7), who found that Talaromyces wortmanii and Fusarium oxysporum metabolize metobromuron [3-(p-bromophenyl)-1-methoxy-1-methylurea1. They isolated 1-(p-bromophenyl)-3-methoxyurea and 1-(p-bromophenyl)-3-methylurea which indicated a dealkylation and a dealkoxylation reaction. They were also able to identify p-bromophenylurea as an intermediate. From the finding of p-bromoacetanilide, they concluded that an acetylation reaction is involved in the detoxication of metobromuron. The present study was designed to investigate the ability of a selected soil fungus to de-'This research was authorized for publication as paper no. 4247 in the journal series of the Pennsylvania Agricultural Experiment Station. grade chlorbromuron [3-(3-chloro-4-bromophenyl)-1-methoxy-1-methylurea] and other phenylurea herbicides. mg per liter. Chlorbromuron and other herbicidal compounds were dissolved in absolute ethanol, sterilized by membrane filtration (0.22-,um pore size Millipore filter), and added to the growth media. The ethanol concentration in the final growth medium was 1%. All cultures were grown in 250-ml flasks containing 60 ml of media and were incubated at 30 C on a rotary incubator shaker (250 oscillations/min). At least two replicates were included in each treatment, and each experiment was repeated two or three times. Larger cultures from which chlorbromuron breakdown products were isolated consisted of 750 ml of media in a 2-liter flask. All compounds except 3-chloro-4-bromoaniline were made visible with ultraviolet (UV) light. The aniline was detected by spraying the plate with an aromatic amine specific reagent composed of 1 g of dimethylamino benzaldehyde dissolved in 180 ml of 1-butanol, 30 ml of ethanol, and 3 ml of HCl. For routine thin-layer analyses, 5 ml of filtered media was extracted twice with an equal volume of ether. The extracts were then combined, concentrated fifty times by heating in a water bath at 40 C, and spotted on the thin-layer plates. Breakdown products of chlorbromuron were isolated by extracting the growth media twice with an equal volume of ether. The ether extract was then evaporated to dryness with a flash evaporator at room temperature. The resultant residue was dissolved in 2 ml of ether and analyzed on a preparative thin-layer plate. The compound being isolated was removed from the plate and purified two more times by thin-layer chromatography (TLC). Growth media samples were prepared for gas chromatographic examination by extracting twice with equal volumes of ether. The extracts were then combined and adjusted to give a theoretical concentration of 5 jg/ml. Mass spectra were taken with a model 902 mass spectrometer (Associated Electrical Industries, Ltd., England) by using a direct probe technique; the samples were subjected to an ionization potential of 70 eV. RESULTS After screening a number of soil fungi for their ability to attack chlorbromuron, it was found that Rhizoctonia solani was most active in transforming the herbicide. When the fungus was grown in a medium containing nonradioactive and "C-carbonyl-labeled chlorbromuron at a total concentration of 10 ,g/ml, approximately 80% of the radioactivity initially present was still detected in the medium after 10 days of growth. Ether extracts of the growth media were analyzed by TLC to determine the distribution of the remaining radioactivity. After 3 days of incubation, only 14% of the radioactivity initially present was found in the RF area of chlorbromuron. Concurrent with this disappearance was the appearance of new "4C-containing spots with RF values of 0.63 ("metabolite X") and 0.23 ("compound Y") ( Fig. 1). In addition, small amounts of radioactivity were detected at the origin of the thin-layer plate and in the aqueous medium after ether extraction, but, due to the low levels of radioactivity, no attempt was made to characterize or identify these compounds. Downloaded from Metabolite X accumulated during the initial period of growth and 3 days after inoculation reached a maximum level, accounting for 67% of the radioactivity present in the growth medium; then it gradually decreased in concentration. This metabolite was isolated and purified by preparative TLC. Based on its chromatographic characteristics and breakdown pattern obtained by mass spectral analysis (Table 1), metabolite X was identified as 3-(3chloro-4-bromophenyl)-1-methoxyurea, or as the demethylated herbicide. The spot with an RF value of 0.23 was designated compound Y, because it was determined that the production of this compound did not depend on the biochemical activity of the fungus, but was produced as a result of nonbiological phenomena acting on the demethylated herbicide. The chromatographic characteristics of this herbicide derivative did not coincide with any of the suspected and available metabolites of chlorbromuron. Equal amounts of compound Y were formed when the demethylated herbicide was incubated for 6 days at a concentration of 10 gg/ml in sterile media or growth media inoculated with R. solani. Mass spectral analysis indicated that compound Y had a molecular weight of 336 and possessed the characteristic breakdown pattern of a molecule containing one Cl and one Br atom ( Table 1). The ability of R. solani to transform the proposed metabolites of chlorbromuron was tested by growing the fungus in the presence of 10 ug of the following compounds per ml: 3-(3chloro-4-bromophenyl)-1-methoxyurea (metabolite X); 3-(3-chloro-4-bromophenyl)-1-methylurea; 3-(3-chloro-4-bromophenyl) urea; and 3chloro-4-bromoaniline. The pattern of fungal degradation of these compounds in comparison to sterile controls after TLC analysis is shown in Fig. 2. Chlorbromuron was completely transformed by the fungus to metabolite X and compound Y as well as a spot with an RF value of 0.29. This spot also appeared with the authentic demethylated herbicide after TLC and was therefore ignored. TLC analysis of the cultures containing the demethylated herbicide (metabolite X) revealed similar patterns of degradation in sterile and nonsterile samples; compound Y and an RF 0.29 spot appeared with equal intensity in both analyses. The demethoxylated herbicide remained intact during incubation in the sterile control, but was partially transformed to a compound with RF values equal to 3-(3-chloro-4-bromophenyl) urea. This urea derivative was partially degraded by R. solani, as indicated by reduction in size and intensity of the urea spot as compared with the sterile control. 3-Chloro-4-bromoaniline disappeared completely due to fungal activity. Since this compound appears only faintly under UV light, its transformation was followed by spraying with p-dimethylamino benzaldehyde as a chromogenic reagent. A weak UV-positive spot with i-layer chromatography with methylene chloride-complete and rapid disappearance of 3-chloro 'onitrile (4:1, v/v) as solvent system. 4-bromoaniline provides an explanation as to why this probable metabolite was not detected RF value of 0.78 appearing as a result of in the culture media. gal growth does not react with the chromo- The appearance of compound Y has been tic reagent and was not investigated further. shown to be independent of the biochemical )nce it was established that R. solani is activity of the fungus, but the production of able of degrading one of the phenylurea compound Y from the demethylated herbicide bicides, its ability to transform related may be of significance, if it results from the Lpounds was examined. The fungus was inherent instability of the metabolite. wn in the presence of 10 Ag of the following The apparent ability of the fungus to attack ipounds per ml: (i) chloroxuron; (ii) diuron; a large number of phenylurea herbicides in fenuron; (iv) fluometuron; (v) linuron; (vi) spite of variations in ring structures, ring subtobromuron; (vii) neburon; and (viii) sid-stituents, and alkyl moieties is notable. The n. fact that one organism is capable of attacking Lfter growth of the fungus, the growth such a diverse group of substrates suggests hia were filtered, extracted, and examined that the fungus R. solani may possess a gener-TLC analysis. The percentage of herbicide alized ability to degrade this class of comisformed was estimated by the size and pounds. The data demonstrate that microbial mnsity of the herbicide spot on the thin-degradation of chlorbromuron and a number of Nr plate relative to a sterile control (Table other phenylurea herbicides takes place and In this preliminary study, no attempt was indicates the role of soil fungi in this process. de to identify the resultant metabolites, LITERATURE CITED but the use of p-dimethylamino benzaldehyde indicated that aniline intermediates did not accumulate.

v3-fos

2020-12-10T09:04:11.324Z

1972-09-01T00:00:00.000Z

237231428

s2

Fungi Isolated from Flue-Cured Tobacco Sold in Southeast United States, 1968-1970 Flue-cured tobacco leaves, from low- and middle-stalk positions, offered for sale in each of two markets, within each of five tobacco types, were evaluated for moisture content (MC) and filamentous fungi during August through October in 1968, 1969, and 1970. Alternaria alternata, Penicillium cyclopium, Aspergillus niger, Aspergillus repens, and Aspergillus flavus were most frequently isolated from cultured tissue. Other filamentous fungi that grew from the tissue included species from four genera of field fungi and seven species of storage fungi. Although the MC ranged from 11.0 to 22.5%, it averaged 16.4, 16.8, and 15.9% for samples taken in 1968, 1969, and 1970, respectively. Average populations of fungi per sample over the three years ranged from 0 to 1,528,500 colonies/g of tobacco. Several species of storage fungi (mainly species of Aspergillus and Penicillium) commonly are isolated from marketed, damaged, and nondamaged flue-cured tobacco, but rarely are isolated from tobacco leaves immediately before or after curing (8)(9)(10) or from tobacco inoculated in the field with these fungi (7). As with cereal grains (1,2), tobacco is invaded by "field" fungi before and "storage" fungi after harvest. In an earlier report (8) differences in moisture content (MC) and in the numbers and kinds of fungi isolated from marketed tobacco were reported for samples from the Middle and Old Belt Markets in North Carolina. It was speculated then that the 3.6% difference in tobacco MC was responsible for the different species of fungi observed growing from cultured tobacco. To test this, a broader study was initiated to compare systematically the fungi and MC associated with two grades of tobacco, sold in each of two markets, within each of the five tobacco types, in each of three consecutive years. There are five types of flue-cured tobacco having certain common characteristics and closely related grades. The five types are described elsewhere (5,6). Briefly, type 11 is tobacco grown in northcentral North Carolina (N.C.) and southern Virginia; type lla is grown in central N.C.; type 12 is grown in eastern N.C.; type 13 is grown in southeastern N.C. and northeastern South Carolina (S.C.); and type 14 is grown in southeastern Georgia and northern Florida. There are 94 markets in the flue-cured tobacco-producing areas (6). Markets sampled were in Danville and South Boston, Va. (type 11); Durham and Warrenton, N.C. (type lla); Kinston and Rocky Mount, N.C. (type 12); Lumberton, N.C., and Conway, S.C. (type 13); and Valdosta and Waycross, Ga. (type 14) (Fig. 1). These samples were taken from August to October, 1968October, , 1969October, , and 1970. A sample consisted of 5 to 15 leaves pulled from the center of each of five piles (weighing about 175 lb [80 kg]) of tobacco graded in either group B (leaf) or X (lug). B-grades grow in the middle of a 20-leaf plant. X-grades grow 2 or 3 leaves from the botton of the plant. Each sample was put into a separate plastic bag, sealed, and transported to the laboratory for analysis. Samples not immediately evaluated were stored at 3 to 4 C until tested. For each type both warehouses were visited and samples from both grades were collected the same day. The fungi associated with the leaves in 1968 were determined by two methods. In one, 25leaf discs (9-mm diameter) per sample were cultured on Czapeks agar with 6% NaCl according to a procedure previously described (7). The Aspergillus colonies were identified as to species according to Raper and Fennell (4). Fungi not sporulating were recorded and tabu- lated as unknown filamentous fungi. The percentage of the 25 discs which yielded a given fungus was determined for each sample. The overall percentage of discs from which a given fungus grew was computed on the basis of the total number of discs cultured from all samples which yielded the fungus. Fungal populations in each sample for 1968, 1969, and 1970 were determined by chopping 10 g of tobacco in 500 ml of 0.15% agar in a food blender and making subsequent cultures from the dilutions. Results are expressed as the geometric average number of colonies per gram for all samples which yielded the indicated fungus. This technique has been used to determine the numbers and kinds of fungi associated with tobacco (9, 11) and is a slight modification of procedures used to determine the fungi associated with stored cereal grains (1). The geometric average was used rather than the arithmetic average because it reduces the distortion from averaging colony counts in the millions with those in the thousands. Percentage MC (wet-weight basis) was determined by drying 6.47 g at 100 C for 16 hr (3). Because comparison of the data showed similar MC and fungal populations for tobacco samples from different stalk positions, among markets, types, and years, all data were combined for this report. The fungi growing from cultured leaf discs in 1968 are presented in Table 1. Alternaria alternata (Fr.) Keissl (A. tenuis Nees), Pencillium cyclopium Westling, Aspergillus niger Van Tiegham, Aspergillus repens de Bary, and Aspergillus flavus Link occurred most frequently. Although some species grew from 100% of the cultured discs of individual samples, the overall percentage was 42.3% or below. Not all samples were invaded by fungi; the same fungi did not grow from all samples; some samples were invaded by only one fungal species. Bacteria and yeasts did not grow well on this medium and their frequency was not recorded. The populations of fungi (expressed as colonies per gram of tobacco) determined for samples collected in 1968, 1969, and 1970 are presented in Table 2. Although not shown in the table, 31 of 300 samples had populations that reached or exceeded 10,000 colonies/g; 23 of which were between 10,000 and 99,000 colonies/g; 3 of which were 105,000, 135,000, and 160,000 colonies/g; and 5 ranged from 3 million to 22 million colonies/g. Of the fungal populations over 1 million, three were P. cyclopium, one was A. repens, and one was Aspergillus ruber. Many samples had mold counts so low that no colonies grew in the initial dilution cultures of 10-3 and this accounts for the failure to detect fungi in so many samples. The average and range in MC of tobacco graded X was 16.1% and 11 to 20.8%, 16.5% and 13.5 to 22.2%, and 15.5% and 12.3 to 19.5% in 1968, 1969, and 1970, respectively. The average and range in MC of tobacco graded B was 16.8% and 13.6 to 22.5%, 17.1% and 14.9 to 21.6%, and 16.2% and 14.2 to 21% in 1968, 1969, and 1970, respectively. The two grades of tobacco sold in these years in the five tobacco regions (types 11-14) had essentially the same ranges in the numbers and kinds of fungi and in MC. Further study of the fungi associated with marketed tobacco does not seem jusitified because the species of fungi associated with such widely varying samples were similar. Moreover, studies with stored tobacco (11) and other crops (2) have shown that the storage environment (temperature and relative humidity) determines to a large extent which fungi will grow and at what rate. Further studies on the factors that influence growth of storage fungi seem justified and are in progress. I thank Terrylyn D'Orio and Clementine Zimmerman for technical assistance, and C. S. Hoges, Jr., and Dorothy I. Fennell for assistance in identifying Penicillium cyclopium.

v3-fos

2016-05-12T22:15:10.714Z

1972-01-15T00:00:00.000Z

14783219

s2

Genetic aspects of meat quality in pigs Danish investigations have shown that environmental conditions like season of year, temperature during transport, and carcass weight have, on an average of a larger sample of individuals, only a slight effect at least on the variance of meat colour in the muscle. Investigations within many breeds of pigs, including those given in tables 7, 8, and 10 , give estimates of additive gene effects of h2 = 0.3 to h2 = 0.4 for values of muscle colour and 45 mn pH values. The genetic correlation between sexes in meat colour and in two other characters is given. The phenotypic and genetic variability of characteristics related to meat quality are given together with objective carcass measurements, and the phenotypic and genetic relationship between these are discussed. The possibility of being able to master, through a selection program, the problem of meat quality and the problem of stress adaptability related to this, is discussed. INTRODUCTION Problems on meat quality have been reported as early as in 1 88 3 , and in connection with a pork exhibition in Berlin HE R TE R and W IL S D O R F (IC!I4) dealt with problems like meat with a pale, moist surface creating processing problems. These authors also dealt with breed differences in muscle colour. Already in the early days of the Danish bacon production the meat colour was taken into consideration, as the first director of the Danish pig progeny testing stations reported of complaints from Great Britain, indicating that occasionnally the Danish bacon had a poor, pale colour (BECK, 1931 ). A regulation was made requiring that all test carcasses In their description of acute heart problems associated with sudden death in pigs, F RED E ( 192 6) and H UPKA ( I939 ) used the designation « muscular degeneration » for the most pronounced cases of muscles with discolouration. I,uDVIGS!N ( 1953I,uDVIGS!N ( , 1954 described muscular discolouration in the Danish Landyace in connection with processing and canning problems and claimed that this discolouration involved both nutritional and genetic factors. W I S M E R -PE D E R SE N ( 1959 ) found a phenotypic correlation of Y p = &mdash; 0.!1 between the pH measurement in the m. L. do y si behind the last rib 45 minutes after killing and the water-holding capacity of this muscle. He also found a correlation of r p = &mdash; 0 .86 between the same pH value and the corresponding concentration of lactic acid. C LAUS F N and N6 R T OF T T HOMS E N (ig6o) associated a high acid content of the meat with pale colour characteristics and reported a correlation of r p = -! 0 .6 between these traits. W ISM E R -PE D E RS E N and B RISK E Y ( 19 6 1 ) were able to produce pale, moist meat by delaying the temperature fall in the carcass after killing. They concluded that the fast process of chilling the carcass post mortem caused a partial reduction in pale colour characteristics of porcine muscle. H AI ,I, UNG ( I g6 2 ) confirmed these results. Because of the relationship shown by I,uDVIGS!N ( 1953I,uDVIGS!N ( , 1954I,uDVIGS!N ( , 1955 between the colour changes in the skeletal muscles and the concentration of lactic acid in the same muscle, C LAUSEN and NB R T O FT ( 195 6) introduced an arbitrary colour scale of I o classes to be given on the cross section of the m. L. do y si cut at the tip of the last rib. This score was introduced in January 1954 and is still used on all test pigs slaughtered. During the period 1954 -19 65 certain environmental and genetic causation factors have been investigated. As earlier mentioned, W ISM E R -P ED E RS E N ( 1959 ) found a phenotypic correlation of r p = &mdash; 0 . 71 between the pH value in the m. L. dorsi behind the last rib 45 minutes post mortem and the water holding capacity in the same muscle. Furthermore he found Y p = &mdash; 0 .86 between this 45 minutes pH value and the corresponding concentration of lactic acid ; consequently the 45 minutes pH value was measured on all Danish test pigs from 195 8 to 19 6 2 , but due to missing data only the material from the test year I g 5 8-5 g could be analyzed. Table I shows the relationship between the Danish colour score and the 45 minutes pH, both taken at the tip of the last rib. ENVIRONMENTAL FACTORS AFFECTING THE MEAT COLOUR I,UDVIGS!N ( 1954 ) and W ISM E R -PE D E RS E N and R I E MANN ( 19 6 0 ) discussed the importance of preventing the pigs from fighting and biting during transport to the bacon factory in order to reduce the incidence of pale, moist muscles. In I g 59 9 the Danish Meat Research Institute developed a halter to be placed on the pigs before delivery at the bacon factory (W ICHMAN -J6 R GE NS E N 1959 , I9 6 I ). This halter was tested on pigs from the three progeny testing stations from January to July 19 6 0 , and caused a general improvement in the mean of the muscle colour score of the test pigs. (« Sjaelland » : P ! 0 . 20 , « Fyn « : P ! o.ooi, and « Jylland » P < 0 . 20 ). Using halter, the phenotypic variance within the same day of delivery was also decreased, but most in the uncastrated animals, the gilts (table 2 ). It is clearly shown that the procedure of using halter decreases the variance between pigs delivered on the same lorry, and that the gilts are the most sensible sex, their variance in meat colour score being app. io per cent larger than that of the castrates. A remarkable effect of the distance (km to bacon factory) on the intra week variance in meat colour is demonstrated. To repeat this examination of the effect of transport stress on both the colour score and the 45 minutes pH value, an experiment was carried out with 54 test groups (each test group consisted of two castrates and two gilts) to be delivered from the newly established fourth test station to the bacon factory, driving distance being 0 . 5 km. One gilt and one castrate from each of the 54 test groups were killed in their individual pens and transported dead to the bacon factory, whereas the other gilt and castrate litter mates were transported alive to the factory (N 6RTO rr THOMS!rr, 10 6 1 ). The results are shown in table 3 . The muscle colour mean was improved in both the castrates and the gilts killed in the pen. The standard deviation within litter and sex decreased in both sexes, but most in the gilts. However, only the standard deviation of the 45 minutes pH value in the muscle differed significantly in the gilts killed in the pen from that of the gilts killed at the factory (P < 0 . 04 8, table 3 ). To investigate the effect of transportation on the meat quality in order to standardize the treatment during transport of not only the test pigs, but also ordinary bacon pigs from commercial producers, the Danish Meat Research Institute has set into operation an experiment in 1971 (W I C HMANN -J 6R GE N SE N , 1971 ). The effect of season on both the meat colour score and 45 mn pH in the m. L. dorsi is investigated examining their variances. The effect of the 3 months-season is approximately 5 per cent of the phenotypic variance. In table 4 is given the relative variance of the month and the day of delivery and of the individual test pig for each of six station-sex subgroups within the test year 195 8-59. It is clearly shown that 8 5 to I oo per cent of the phenotypic variance is deu to the difference between the individual reaction among pigs delivered to the bacon factory on the same lorry on the same day. The influence of outside temperature on the muscle colour score was estimated as the linear regression of the mean muscle colour score on the temperature, measured outside the test stations at noon in Co for all test stations and sexes over four years. The mean temperature of year fluctuated from 7 . 2 C o to II . O C°, and the regression estimates fluctuated from -fo.ooo 02 to -0 . 02 . 24 out of 2 8 regression coefficients showed a negative influence of the outdoor temperature, the delivery distance of 14 km showing the strongest influence. Eftect of chilled carcass weight on muscle colour and 45 mrc PH in m. I,. dorsi Table 5 shows the repeatability of the consistently negative effects of chilled carcass weight on both muscle colour and pH though these effects are only slight, being of the order between o. 3 per cent and 4 .8 per cent of the phenotypic variance. Similarity in relationship to other characters for the 4.5 mn PH value and for the meat colour both in the m. I,. dorsi at the tip of the last rib In the previous tables 2 , 3 , 4 , and 5 it is shown that these two characters have reacted quite similarly against the causative effects mentioned. The reactions of these two characters with a third one are also very similar which is shown in table 6. The correlation of the residual sector is a measure of the covariation within litters after the elimination of the additive gene effect. The effects of the inter-(epistatic) and intra-(dominant) allelic gene action are included in this correlation. Meat colour and pH in the muscle are negatively correlated with fast gain and positively with feed consumption rate. At constant gain the covariation between feed consumption and these two characters is not changed. Length of the pig and all fat measurements are positively correlated with colour and pH in the muscle whereas meat content is negatively correlated. None of these correlations are strong. The intra-station phenotypic correlation between the two characters was estimated at + 0 .68 and + 0 . 71 for castrates and gilts, respectively. However, it is of more importance that in the same material of 1 7 IS castrates and i 8m gilts, the genetic correlation was estimated at r G = -f-0 .86 ( 4 8 0 The partitioning of the phenotypic variance of the meat colour score and two other important carcass characteristics are based on the data from 195 6 to 1965 (table 8). This should include a sufficient number of test yeartest stationsex subgroups, one year of test comprising material from four test stations, and therefore eight subgroups including app. go degrees of freedom for sires. The hierarchical structure of these estimates from 195 8 to 19 65 is : two test pigs of the same sex per test group, five test pigs of the same sex per sire half-sib family, and 12 test pigs of the same sex per breeding centre (elite herd). Within the elite herds relationships of y . 5 p. I oo were found between dams mated to the same sire, and 2 .6 p. IOO between sires standing at the same breeding centre. Thus the data in tables 7 and 8 are corrected for the effect of the relationship between sires and for that between dams ; further for the influence of chilled carcass weight, seasons of year, progeny test stations, and year of test. The pH value, measured 45 minutes after killing, is included in the international recognized criteria for classifying a pig carcass to be either normal or PSE (e. g. DuTSOrr et al., 1971 ). In table 7 are given the results of the only complete test year in respect to data set including this criterion for meat quality. This is the same set of data as used for the results in table 6. An essential fact in table 7 is the characteristic higher heritability in both criteria for the gilts than for the castrates in this sample. However, this dramatic difference between sexes is undoubtedly due to sampling variation as it is not found over the 9 years period 195 6-19 6 5 for the meat colour score (table 8 ; castrates : h 2 = 0 . 27 J; 0 . 0 6, gilts : h 2 = 0 . 3 6 J; 0 . 0 6). As an average between the two sexes, an estimate of h 2 = 0 . 32 for the 45 mn pH value (table 7 ) is a moderately high heritability, indicating that the additive gene effect is controlling the structural conditions in the pig ; W ISM E R -P ED E RS E N having demonstrated a fairly high phenotypic correlation of Y p = &mdash; 0 . 71 between this 45 mn pH value and the water holding capacity. The best estimate of the partitioning of the phenotypic variance in the Danish Landrace pig in the period prior to 19 6 7 is given in table 8 for three characters which are important for the selection of bacon type. PE D E RS E N ( 19 6 4 ) found that the m. L. do y si area controls app. 25 p. 100 of the lean meat content in the carcass ( Y p (castrates) = + 0.44 and Y p (gi l ts) = + o.aC!), whereas he found that the side fat measurement controls 50 p. I oo of the lean meat content in the carcass (Y P (castrates) _ -0.71 and Yp(!,it.) _ -0.68). That is the reason why these two characters are included in table 8 together with the meat colour score. Difference between sexes is not found in the colour score mean like in the two other characteristics, but the phenotypic standard deviation as well as the genetic one differ between the two sexes in all characters as demonstraded in the tables 7 and 8. This difference in variance is about 8 p. I oo, and a similar difference is found in the score for nasal alterations : In table 8 it is clearly demonstrated that under a system of test, where it is necessary to restrict the material so that only a little more than five pigs per sire half-sib family and only 12 pigs per herd is obtained, it is necessary to include a number of test years to get sufficient unbiased estimates of the population parameters in the breed. This agrees with the theory given by Ro B ER ' rsorr ( I9 6 0 ) about experimental design on the measurement of heritabilities. Besides having a sufficient number of individuals per subgroup to get unbiased estimates of the different intra-class correlations, the years must cover some sire generations because the sample of paternal half sib groups sent to the test station per year is not necessarily representative for the potentialities of zygotes from the breeding centres as a whole. Sex differences in the heritabilities are not demonstrated in the muscle arae and the side fat measurement in the overall estimates within test stations and years. In the points for meat colour it should be concluded that the sire component estimated from the castrates data tends to be decreased and, therefore, the « litter environment » and the error variance is correspondingly increased. In a breeding program it should, therefore, be more efficient to base the selection on data from uncastrated animals. It has been shown previously that the effects test stations, seasons of year, and chilled carcass weight are affecting the meat colour only slightly (tables 5 and 7 ). Table 8 shows that the only two causations which matter for the meat colour is the heritability and the residual error. If only gilts are included in the selection program, it is realistic to work with a heritability of 0 . 4 and a residual error of o.6. If both sexes are included in the test group, the heritability is 0 . 3 and the residual error is o. 7 . A rather strong maternal effect is found in the muscle area. This could to some extent be due to mothering abilities of prenatal nature. GENETIC COVARIATION BETWEEN THE TWO SEXES IN POINTS FOR MEAT COLOUR AND TWO OTHER TRAITS The test groups of the litters from the state recognized breeding centres consist of 2 castrated males and 2 females. Because of the uncertainty of the genetic variance of the meat colour in the castrates and also because the genetic improvement of a character is increased per year when only using 2 instead of 4 litter mates due to the increased selection intensity, it was of interest to investigate the genetic correlation between the two sexes with respect to their performance in the three carcass characteristics (table 9 ) (J ONSSON , 1071 b). The variance components for the interaction between sire half-sib families and the two sexes were very small in the meat colour score and in the side fat measurement, 0.003 4 points 2 and o.002 8 cm 2 , respectively ; the F-quotients were 1 , 09 and 1.IS, respectively. This is the reason why the corresponding genetic correlations are not unity. In the muse. Long. do y si area, however, the F-quotient was consistently beneath unity in the different test year-test station subgroups, so no sire-sex interaction is found in this character. The two Danish carcass evaluation centres were started in 19 6 7 to investigate the new characters for carcass quality which were to be recorded at these centres. The material comprised 1 403 gilts and 1 400 castrates. As this first investigation on the new carcass characteristics was planned so that sires would be tested on at least two test groups (two gilts and two castrates per test group ; it was not possible, as originally planned, to set the limit at three test groups), it was impossible to include the variation between breeding centres in the hierarchical classification. So any carry-over effect from breeding centres will be included in the sire component. The hierarchical structure of the analysis was as follows : The standard errors for the heritability estimates were computed according to the method given by B. Woo!,x (FALCONER, 19 6 3 ) as shown in Jorrssorr (i g y a). The per cent of lean meat in the entire carcass side (character 9 ) is predicted by io individual carcass measurements and weights, including cold carcass weight and sex. The side fat measurement is a prominent x-variable in the prediction equation, controlling app. 5 0 per cent of the lean content in the entire side. R = 0 .8 7 (C LAUSEN e1 al., 19 68). Table I gives the phenotypic and genetic population parameter estimates in the Danish Landrace pig for six traditional and six new carcass quality characters introduced at the two carcass evaluation centres. For the average backfat thickness the magnitude of heritability given in table I o seems to be more reasonable, and for the m. L. dorsi area the value given in table 8 seems more reasonable. An estimate of h2 ! 0 .6 2 seems too high for the area of m. L. dorsi ; this should also be the case for characters nr. 6, 8 and 9 in table io. One reason for this must be the lack of including the class for « breeding centres » in the hierarchy and the lack of having corrected for seasonal differences. The period of investigation is perhaps also a little short. The elite breeders have given considerable attention to the three characters m. L. dorsi area, side-fat measurement and per cent lean meat in the entire side from 19 6 7 and onwards. This means that the effect of selection is included in the differences between sires' half-sib groups. Having included more years and corrected for effects from seasons and years, and having included the classification breeding centres in the hierarchy, a probable upward bias will be corrected for. But the estimates for the additive gene effect in table io show that no lack of additive genetic variability in the breed ecists, which is also confirmed by the estimates given by ST AUN ( 19 68) and ST AUN and JE NS E N ( I970 ) for the same breed. Their heritability estimates for the same characters, estimated from the data from the pig progeny testing stations, rank from o. 4 6 to 0 . 7 8. The magnitude of the genetic standard deviation and the coefficient of variation for the important characters is striking. Heritability estimates for colour values reported from other breeds range from 0 . (F!.ocx, 19 68). These estimates are similar to the present estimate given for the character 6. In Denmark cross breeding experiments between the pig breeds Large White and Danish Land y ace are planned. One of the main subjects to be investigated is the hypothesis of dominant gene effect on meat quality characteristics. S YBFSMA ( I970 ) has suggested that Cross breeding different breeds is a very promising means of improving meat quality. WrsM!R-P!D!RS!rr ( 1959 ) found a correlation of y r = o.y between 45 mn pH and 24 hours water holding capacity, both characters giving a reasonable accurate measure of structural conditions in the tissue. W!NiG!x et al. ( 1970 , reported from W E I SS , 19 6 7 ) found heritability estimates of h 2 = 0.37 ! 0 . 14 for WHC (centrifuging) and h 2 = 0 . 19 ::!:: o.io for 45 mn pH value. The estimates of heritability given among others by WENiG!R et al. ( I970 ) for the WHC of h 2 = 0.37 ! 0. 14 and that for the 45 mn pH value of h 2 = 0 . 32 from table 7 are supporting the hypothesis that structural conditions in the muscle as a meat quality criterion is controlled by additive gene effect, and, therefore, can be included in a selection program for improvement of meat quality. In table m, the traditional meat and carcass quality characters are given. The genetic correlations are given below the diagonal and the phenotypic correlations are given above the diagonal. It is doubtful, whether the positive genetic correlation between colour brightness and muscle size still exists in the Danish Landrace pig, as reported by J ONSSON (i g y a). This change in sign could have been forced by a change in gene frequency in the Danish Land y ace pig because of a more direct selection for meat content during the recent years. Table i This correlation is slight, but indicates a negative trend. The negative trend between muscle colour and size of muscle agrees with estimates from other breeds, e. g. with that given by F!ocx ( 19 68), who estimated r p = &mdash; 0 . 2 8 and r G = &mdash; 0 . 5 6 between these two characters in the German veredeltes Landschwein breed. DISCUSSION In tables 2 , 3 , !, and 5 it is demonstrated that the average environment has only a slight effect on the meat colour in the carcass. For the Danish Land y ace pig table 7 , 8 and io show together with estimates from other breeds that brightness of colour is influenced to a moderate degree by additive gene action, heritability estimates being of a magnitude of h2 ! 0 . 3 for colour values. The high genetic correlation between sexes in meat colour as well as in other characters important for value of the carcass tells that including only one in the family selection for carcass and for meat evaluation ensures equal genetic progress in the other sex. However, the problem of structure in meat tissue is not connected with muscle colour. It is, however, significantly related to the stress syndrome as reported e. g. by L UD VIGSE N ( 19 68 a and 19 68 b), JUDGE ( 19 6 9 ) ( 19 68), and many others. Undoubtedly, it is only a matter of technique to be able to obtain sufficient accurate and repeatable WHC values to ensure reasonably high heritability estimates to be included in selection programs for improvement of the meat structure, genetically. But all these characters have only a secondary effect on the adaptability of the pig to environmental stress conditions. The main problem of the future must be to lay open the characteristics of the stress syndrome in the pig and the genetic effect behind these adaptation characters. In this way the question can be answered, whether the problem of quality and death losses, as reported from many countries (e. g. by W ENIGER e1 al., 1970 ) can be fought against by means of selection within the breed populations and/or by means of crossing between breeds utilizing a probable heterosis, or may be be mastered through environmental measures. GERRiTS ! at. ( I9 6 9 ) have shown that intense selection for meatiness in pigs has a significant correlated response to growth hormone concentration. Considerable need exists for more selection experiments like this cited to lay open these problems. RÉSUMÉ ASPECTS GÉNÉTIQUES DE LA QUALITÉ DE LA VIANDE CHEZ LE PORC Les recherches effectuées au Danemark ont montré que l'influence des conditions ambiantes, telles que la période de l'année, la température pendant le transport et le poids de la carcasse, sur un assez grand nombre d'animaux examinés est faible, du moins sur la variance de la couleur de la viande dans le muscle. On donne la corrélation génétique entre les sexes en ce qui concerne la couleur de la viande et deux autres caractères. On donne en outre la variabilité phénotypique et génétique des caractéristiques relatives à la qualité de la viande ainsi que les mensurations objectives de la carcasse, et on examine la relation phénotypique et génétique existant entre ces caractéristiques.

v3-fos

2014-10-01T00:00:00.000Z

1972-10-15T00:00:00.000Z

9397307

s2

Chromosomes of chicken-pheasant hybrids a majority of the embryos carry a haploid set each of chicken and pheasant chromosomes. Three of the male embryos resulting from chicken female-pheasant male crosses were cytogenetically indistinguishable from male domestic chicken whereas two embryos obtained from a similar cross, were chimeras composed of chicken cells and hybrid cells. The presence of hybrid embryos of chicken karyotype is attributable either to parthenogenesis in the hens or to fertilization of the hens ovum by rooster spermatozoa from previous matings, surviving in the female reproductive tract. It is postulated that the chimeric hybrids may be the result of double fertilization of the ovum and its retained polar body, and that the spermatozoa from the semen invested during this investigation and the rooster spermatozoa remaining viable in the hens oviduct from previous matings, were probably involved in this process. Studies on intergeneric hybrids obtained by crossing ring-necked pheasant and Columbian Rock chickens have shown that only less than thirty percent of the eggs laid were fertile (B ASRUR , ig6g ; B H AT N AGA R , 19 68). It was observed that among the fertile eggs incubated, over fifty percent died at various times before hatching (B ASRUR , ig6g). The exact reason for the high mortality rate in chickenpheasant hybrids is not known although it is generally noted that the hybrid progeny are at great developmental disadvantage if the two species involved in hybridization are dissimilar in their cytogenetic make up (B ENIRSCHKE ,et al.,ig6 5 ). Higher fertility has been reported in matings of domestic chicken roosters to pheasant females than that noted in reciprocal crosses (A SMONDSON and Lox!rrz, 1957 ) although hatchability was noted to be much lower in the chicken male-pheasant female crosses (B ASRUR , 19 6 9 ). The present report concerns our preliminary observations during an investigation on the cytogenetic basis of embryonic mortality in chicken-pheasant hybrids. MATERIALS AND METHODS Nineteen embryos belonging to ring necked pheasant femalechicken male crosses, and fifteen embryos resulting from domestic chicken femalering-necked pheasant male crosses were used for karyotype analysis. The eggs were incubated for i6 to 20 days prior to removing the embryos for sexing and for tissue culture. The embryos were first decapitated and the anatomic sex of each embryo was recorded on the basis of the number of gonads : paired in males and single in females. For the histological confirmation of the sex, gonads were removed and fixed in 10 p. 100 neutral buffered formalin and processed according to the routine haematoxylin and eosin (H and E) method. The hind limbs of each embryo were removed asceptically and were freed from skin and bones before rinsing in sterile phosphate buffered saline (PBS). The tissue was chopped with a pair of sterile Bard-Parker knives and the pieces were triturated in o.2g p. 100 trypsin at room temperature on a Magnastir, for i 5 minutes. The mince was passed through a glass funnel fitted with cheese cloth filter and the f ilterate was spun at 6 00 r.p.m. for 8 minutes. The centrifugation was repeated replacing the trypsin solution with PBS containing a drop of calf serum, and the cell button was suspended in growth medium H 597 supplemented with 20 p. 100 calf serum and antibiotics (B ASRUR and G ILMAN , i 9 6 3 ). Leighton tubes, seeded with approximately 200 ooo cells per ml of growth medium were incubated at 37°C. The day-old cultures were replenished with fresh growth medium and on day two, the mitotic cells were accumulated by the exposure of cultures to colchicine at a final concentration of o.ooi p. 100 , for 4 hours prior to terminating the cultures. Aceto-orcein stained chromosome preparations were made according to the procedure described previously (B ASRUR and G ILMAN , 19 66) and metaphase plates with well spread chromosomes were selected and photographed with phase optics on a Zeiss Photomicroscope. From each culture over 50 cells were examined and the cytogenetic make up of the embryos were recorded on the basis of their macrochromosomes including the sex complements. RESULTS AND DISCUSSION A majority of the embryos used in this study ( 25 out of 34 ) were males on the basis of gonadal histology although in three of these males a well developed gonad was detected only on the left side while the right gonad was vestigeal. Nine of the embryos were recorded as females on the basis of the histologic feature of their gonads. Karyotype analysis confirmed the hybrid constitution of 29 embryos all of which carried a set each of chicken and pheasant chromosomes (Plate I). The histologic males (table i) among the embryos exhibited the ZZ sex complement (Plate II a) and the females carried the ZW complement (Plate II b). Three male embryos belonging to chicken female-pheasant male crosses had the cytogenetic make up of domestic chicken whereas two male embryos (table 2 ) had varying percentages of chicken and « hybrid » cells. Tetraploid cells were frequently noted in cultures of all hybrid embryos examined although the proportion of tetraploid cells in the two chimeric hybrids ( 1 8 out of 6 2 and 19 out of 66) was greater than in normal chicken embryo or « normal hybrids (table 3 ). dibulum (V AN DxiMM!!,!rr, rg5i) or that some of the hen's eggs are fertilized parthenogenetically. The fact that two of the hens used for hybridization were previously mated with domestic chicken roosters supports the possibility of fertilization of some of the eggs by rooster spermatozoa which have retained their viability in the hens reproductive tract. It has been shown previously that the spermatozoa stored in the infundibular sperm nests could be released into the lumen of the oviduct by experimentally distending the oviduct (G RIGG , ig 57 ). It is possible that the stimulus provided by the process of insemination with pheasant semen causes the release of the stored rooster sperms for fertilization. Alternatively, it is equally possible that the chromosomally confirmed chicken embryos of the chicken pheasant crosses are those resulting from parthenogenesis through polar body fusion. Parthenogenesis has been reported frequently in turkey (Or,s!N,ig6o ;P OOLE ,195 6) and may be also prevelant in chicken. Since the chicken embryos detected in this investigation are all males, the possibility of their origin through parthenogenesis cannot be ruled out. The presence of chimeric embryos with hybrid cells and chicken cells are difficult to explain. One possibility is that the haploid chicken elements segregated at one of the early cleavage stages and that these haploid elements subsequently « doubled » to give the diploid chicken complement noted in approximately 33 p. 100 and 1 8 p. 100 of the cells in these embryos. It is also possible that the segregation of chicken chromosomes occured in one of the tetraploid « hybrid » cells during the course of embryonic development. Tetraploid cells have been noted frequently in some of the tissues of mammalian and avian hybrids (B ASRUR , rg6g ; O HNO , ig68). Since the proportions of tetraploid hybrid cells noted in the chimeric embryos were similar to those detected in other hybrid and chicken embryos (table 3 ), it is unlikely that the original zygotes in these cases were of tetraploid constitution. It is conceivable that the chimeric embryos are the result of double fertilization involving a pheasant and a chicken spermatozoa. If such an event had occurred in an ovum and its retained polar body, the failure of subsequent separation of these fertilized cells could give rise to an embryo which is composed of chicken and hybrid cells. The occurrence of these chimeric embryos may be interpreted as cytogenetic evidences of premature fertilization and/or impaired polar body separation in the females employed in the present crosses although the number of such embryos detected during this investigation is small. It is possible that in these instances, the polar body separation which coincides with ovulation in domestic chicken has been interrupted by the premature fertilization of the oocytes by a chicken and a pheasant spermatozoa.

v3-fos

2018-04-03T06:02:05.313Z

1972-04-01T00:00:00.000Z

195693273

s2

Chemistry and the two organic kingdoms of nature in the nineteenth century. which, we repeat, are only applicable to inorganic machines. We could cite many physiological errors which have resulted from this indirect mode of procedure, while, on the contrary, the experimental study of the phenomena of nutrition, conducted directly in the organs, tissues and even in the elements oftissues, have led to fruitful discoveries. The formation of sugar in the liver would never have been discovered if one had been restricted to the comparison of analyses of materials entering and leaving the organism. The physiologist must rely on these general chemical results, but he must not be content with them; he has to descend, with the aid of the direct experiment, into the intimacy of the organs, into the tissue, into the living cell whose function is identical in animals and plants. It is by this study alone that he will be able to grasp the mystery of intimate nutrition and succeed in mastering these phenomena of life, which is his supreme goal." Their symmetrical division into two segments; the beautiful disciform, finely-cut and toothed Micrasterias, the lobed Euastrum, the Cosmarium glittering as if it were with gems, the Xanthidium armed with spines, the scimitar-shaped Closterium embellished with striae, the Desmidium resembling a tape-worm, and the strangely insect-like Staurastrum sometimes furnished with arms, as if for the purpose of seizing its prey, all these characters seem indeed to pertain more to the lower animals than to vegetables.8' D. C. Goodman original properties. They formed a heterogenous collection of sugars, fats, albuminous substances, acids and pigments. It was above all in the study of these, in the discovery of their origin and physiological function, that fundamental physiological problems were solved with chemical assistance in the nineteenth century, though, as will be seen, the path was not a smooth one. Most responsible for directing inquiries along these lines was Chevreul, a pupil of Vauquelin. He said the immediate principles were compounds which had been formed in life, and that an exact determination of their nature was an essential preliminary in physiology.4 They had to be isolated by weak solvents, such as water and alcohol, working at moderate temperatures to preserve their nature, and finally characterized by precise properties like their melting points. In this way Chevreul first demonstrated that fats were compounds of glycerol with various fatty acids. Since a number of immediate principles were common to the two organic kingdoms, Chevreul preferred not to classify them as products of vegetation and animalization, but to put them under the mixed heading of 'products of organized bodies'.5 Albumen was one of the common immediate principles, existing both in the organs of herbivores and in their vegetable diet." He looked to chemistry to explain how food was altered in the body. Another important step was to investigate the immediate principles in situ in the tissues. Raspail argued that this was the correct method to adopt. He complained that chemistry, as traditionally practised, had told us nothing about the tissues, the seat of vital reactions, because analysis mixed up substances which nature kept apart in separate organs.7 Chemistry on the large scale, and alone, could give no indication of the original nature of the various organs. But, in association with anatomy, and particularly in conjunction with the microscope, he said, chemical tests would become valuable. As an example of his new method, Raspail described a test which is still in use for proteins. When the ovaries of barley were treated with a drop of concentrated sulphuric acid, on the slide of a microscope, an intense purple colour resulted.8 Further experiments showed that this was due to the combined presence of sugar and albumen. This test produced the same colouration in the membranes of the uterus during gestation. Raspail concluded that there was a remarkable analogy between embryonic animals and plants, and that this was perhaps the stage of their development when they were most alike.9 He could find no basis for dividing organic chemistry into vegetable and animal chemistry, since this separated immediate * F. V. Raspail, 'Nouveau r6actif destin6, dans les analyses microscopiques, A distinguer des quantit6s minimes de sucre, d'albumine, d'huile et de r6sine; et l'analogie que l'on d6couvre, par ce moyen, entre les ovules des plantes et les organes femelles de la g6n6ration des animaux pendant le temps de la gestation', Bull. Sci. math. phys. chim., 1828, 10, 267-72. 9 F. V. Raspail, Nouveau Systeme (n. 7), p. 261. 114 Chemistry and the two Organic Kingdoms of Nature in the 19th Century principles common to the two kingdoms. No distinctive definitions could be given for animal and vegetable substances, so, like Chevreul, he classified them together as organic or organized. If a judgment had to be made on the kingdom of origin of an organic material, he said, chemistry would be useless, and only zoology or botany could decide. 10 But the recognition of analogous immediate principles in animals and plants led to consequences which Raspail had wanted to avoid. It was possible to argue that the nutrition of animals occurred directly through the incorporation of essential principles which already existed in vegetable foods. This highly simplified account was in fact adopted by the leading chemists of the time. In so doing, they abandoned the organism and set up false barriers between animals and plants. VITAL DUALISM Besides albumen, which was known to exist in both organic kingdoms in the eighteenth century, the discovery of principles resembling milk or cheese in plants was another source of this simplification. Einhof, professor of chemistry at the agricultural institute at M6glin, announced the discovery of a white immediate principle, having the odour of cheese, in peas, lentils and other leguminous plants." He said its similarity to the animal substance explained the nutritional value of these plants. It was called legumin by Braconnot who later said it was really no different from the casein of milk.'2 Braconnot even supposed lactose existed in plants. He applauded the anatomical comparison of cotyledons and mammals, and thought there was a development of milk in both.13 Attention also turned to milk of almonds,'4 which appeared to have an astonishing resemblance to cow's milk. On standing it turned sour, a white deposit formed on the surface and a cheese-like smell was given out. Certain fractions were compared to whey and butter. The most striking example of all came in Humboldt's description of a tree, which he was amazed to find during his South American travels.'5 He had heard stories of a tree growing in the mountains of Venezuela, which the natives called the cow-tree on account of the milk it provided. Humboldt was sceptical, but soon found that the reports were true. He saw the thick, milky juice pour from the incisions in the trunk. It became sour on standing and formed a clot, which the natives called 'cheese'. Humboldt could not carry out chemical tests, since he said he was almost without 10 Ibid., pp. 84-85 and 90-91. 115 D. C. Goodman reagents, but he was convinced of the similarity with mammal's milk. He said he had drunk much of the vegetable milk without bad effects. The opportunity of a detailed analysis came with the departure of Boussingault for the same region. Humboldt particularly asked him to study the juice. Boussingault reported that it was physically like cow's milk and had the same taste, but he thought it differed chemically in containing fibrine instead of casein.'6 Many years later he carried out a further analysis on the vegetable milk he had first mixed with his coffee in Venezuela. Some $ottles of the milk had been sent by the Venezuelan government to the International Exhibition in Paris. This time Boussingault found casein, and he compared the juice of the cow-tree to cream."7 It was tempting to suppose that casein, albumen and other principles originated in plants and served as the sole source of the same principles in animals. Comparative analyses finally persuaded the chemists that this was how nutrition occurred. Mulder reported that there were identical percentages of carbon, hydrogen, oxygen and nitrogen in vegetable and animal albumen, fibrine of the blood, and the casein of animal milk. The differences in phosphorus and sulphur content were small and seemed unimportant. He believed these animal and vegetable principles consisted essentially of the same quaternary, nitrogenous compound, which he called 'protein'. He stated that protein originated in plants and then entered the animal kingdom through ingested food. 18 In his laboratory at Giessen, Liebig supervised research along the same lines. It was concluded that vegetable casein, vegetable albumen and vegetable fibrine (gluten) were isomeric, and individually identical with their animal counterparts. 20 Liebig wrote: How beautifully and admirably simple, with the aid of these discoveries, appears the process of nutrition in animals, the formation of their organs, in which vitality chiefly resides! Those vegetable principles, which in animals are used to form blood, contain the chief constituents of blood, fibrine and albumen, ready formed, as far as regards their composition. All plants, besides, contain a certain quantity of iron, which re-appears in the colouring matter of the blood.... Vegetables produce in their organism the blood of all animals, for the cvora, in consuming the blood and flesh of the graminivora, consume, strictly speaking, only the vegetable principles which have served for the nutrition of the latter.'1 The milk with which the mother fed her young also came from plants. It derived either from the casein in the peas and lentils she had eaten, or chiefly from the supposedly simple conversion to casein of the isomeric albumen and fibrine, the con-stituents of her blood, which were of vegetable origin. In the young the ingested casein was converted back to blood.22 The constituents of the blood, which Liebig believed only plants could form, were the starting-point for animal syntheses, resulting in the production of their tissues, membranes, nerves and brains, materials which no vegetable could supply.23 During the incubation of the chick's egg, albumen, in the presence of atmospheric oxygen, was somehow elaborated into membranes, veins, arteries, feathers and claws. 24 Liebig therefore described the animal organism as 'a higher kind of vegetable'.25 The syntheses initiated in the vegetable kingdom from simple starting materials (carbon dioxide, water and ammonia) and producing protein compounds, were continued in the animal kingdom, to form the complex substances of the nerves and brain, the seat of the distinctive animal functions of sensation and thought. The separation of the two organic kingdoms, based on their ability to perform particular syntheses, was taken much further by Dumas. It was he who was most responsible for the false vital dualism which attributed different physiologies to animals and plants. He wrote: 'We have found, in fact, by results beyond the reach of question, that animals do not create any of the truly organic substances, that they consume or destroy them; that vegetables, on the contrary, habitually create these substances, and that they destroy but few.... It is in the vegetable kingdom therefore, that the great elaboratory of organic life is found.'26 Dumas argued that the inability of animals to synthesize protein was established by his analyses of foods and excreta. Through this technique, which he and Boussingault employed in collaboration, Dumas made the grave mistake of ignoring the changes taking place inside the organism. He compared the nitrogen content of vegetable foods with that of urea, the waste product of the animal's destructive action on proteins. The two quantities were about the same. He made a physiological deduction, in which his neglect of the organism was explicit: 'So, abstracting from all the phenomena occurring in the organs and only considering the balance of entry and exit, one finds that man converts nearly all the nitrogen he receives into urea.... Is it not easy to conclude that the nitrogenous material in our food produces this urea, and that the entire activity of the animal organism is confined to assimilate the nitrogenous material, when it needs to, or to convert it to urea?'27 Like the protein, all the fats and sugars in animals came from the plants, which alone could synthesize them. Their fate in the body was the same, either to be retained unchanged or to be destroyed. The process of destruction was revealed by the nature of the products eliminated in the excreta. Carbon dioxide and water, discharged from the lungs, and urea in the urine were all products of oxidation. Dumas concluded that oxidation was the characteristic feature of animal physiology. Employing the oxygen of respired air, the animals performed acts of combustion with the fuel D. C. Goodman provided by the vegetable kingdom. In this way the animal restored the heat, which it was continually losing through radiation and other ways, and received the energy for locomotion.28 Inevitably he was drawn into comparisons between the animal body and the steam-engine. The approach, which Dumas had recommended as the best way in which chemistry could serve physiology, resulted in a drastic reduction of all animal functions to the chemical process of combustion. The extent to which he insisted on this as the criterion of animality was clear from his discussion of various phenomena exhibited by plants. Referring to the production of carbon dioxide by plants at night and by their flowers and ripening fruit during the day, and to the evolution of heat in plants, he remarked: 'In a word, in all circumstances in which the plant needs heat, and when it does not receive this from outside, it behaves like an animal ... it becomes an apparatus of combustion, and one can say, without being metaphorical, that at this time the plant becomes animal and really forms a part of the animal kingdom, from the point of view of the general physics of the globe.'29 But he said the true nature of the vegetable kingdom was displayed in the other activities of plants, in which they behaved in the opposite way from animals. The simple oxides excreted by animals were absorbed by plants, which then synthesized them into the complicated proteins, sugars and fats, which animals consumed. Concerned with the eternal circle of this global exchange, and not with the individual organism, Dumas, assisted by Boussingault, summarized his conclusions in tabular form-'* The table consisted of two contrasting columns which displayed the opposition of the two organic kingdoms. It rested on the false antithesis of plant synthesis and animal destruction. This theory was first challenged by Liebig, who had denied only the animal synthesis of protein. He was convinced, from analyses of ingesta and excreta, that animals were able to synthesize fats from sugars and starch. He wrote: 'There is no butter in the cow's grass, nor goose-fat in potatoes or barley. They do contain substances like wax, but in such small quantities that I do not attribute the formation of fat to them.'3' Solubility in ether was the test employed for fat. Experiments at Giessen showed that only minute portions of potatoes and fodder behaved in this way, yet pigs fattened remarkably and cows yielded much butter. Liebig denied that the chlorophyll of ingested green vegetables was converted to fat, since the excreta of cattle was green. He found that the excreta contained the same small quantity of fat as the ingesta.32 Liebig also asked how the origin of all the fish-oil and spermaceti could be I' J. B. A. Dumas, Traite de Chimie appliquee aux Arts, Paris, 1828-1846, 8 vols., vol. 8, p. 417f. explained, since none was present in the marine plant food of cetacea and fish.8" He could only assume what Dumas had denied: 'Can animals perform acts of the same nature as plants relative to the formation of their principles? One can scarcely doubt it.'34 Dumas replied: 'The hay eaten by Liebig's cow was richer in fat than he thinks.'" Dumas, Boussingault and Payen, who had worked on the problem together, still maintained that any fat in animals was due to an accumulation from vegetable sources.86 Attention then turned to the production of beeswax, which Liebig had also referred to. This had continued to puzzle investigators, since the eighteenth century, and agreement had not yet been reached on whether bees synthesized wax or simply collected it from plants."7 Dumas supposed the source was vegetable wax,88 but decided to investigate the claim, based on Huber's experiments,8' that bees fed on a diet of sugar could make wax. Dumas and Milne Edwards40 isolated a number of bees, and estimated the average quantity of fat already existing in their bodies, before feeding, to see if they held reserves taken from plants. The bees were then fed with honey, which was also examined for fat content, and left to construct a comb. The total quantity of wax which this contained was then determined, as was the average amount of wax left in the bees. The arithmetic, which involved minute quantities, seemed to show that there was not enough wax in the food or in the bees' reserves to explain the quantity produced. It was concluded that bees really made wax. In the discussion of the results, the strength of the resistance to the idea of animal synthesis was apparent. Payen wondered if the fat content of honey had been understated, and speculated on other possible causes for the wax produced in the bees' prison: 'Perhaps the wood of the box, the mastic of the windowpanes, the paints, cements, or some cryptogamic plant developing in the humid conditions provided the elements of wax?'4" Payen added that even if the wax originated in the bees, this was a special act, unrelated to the formation of fat in the tissues of all other animals. He was far more impressed by the rapid fattening of cattle by fodder, and so retained his opinion of the vegetable source of animal fats. Milne Edwards agreed that, in view of the peculiarity of bees' glands and of the wax produced, no general conclusions could be drawn from the experiments on the *s J. von Liebig, 'Note sur la formation de la graisse chez les animaux', C. r. hebd. Sianc D. C. Goodman origin of fats. But he preferred the simple explanation that animal fats resulted from an accumulated deposition from vegetable foods. He said the theory one adopted depended on what limits were placed on the animal's ability to modify foods. He approved of Dumas' restrictions on this. Edwards would allow animals to convert one protein to another, or to modify vegetable oil to animal fat, but he excluded what he regarded as greater transformations: the synthesis of fats from proteins, involving immediate principles of different families.42 Dumas commented that if animals made fat from sugars, as bees made wax, the process was to be regarded as one of fermentation, intermediate in nature between plant synthesis and animal destruction.43 Doubts remained on the production of beeswax, the formation of animal fat, and the possibilities of synthesis performed by animals. As Magendie said, the whole question of animal nutrition remained obscure. He warned that, while it was of interest to demonstrate the existence of analogous immediate principles in animals and plants, it was 'a great leap' to draw conclusions from this on the origin of substances in the animal body." The inadequacy of the current methods of investigation was the chief obstacle, as Lehmann pointed out: We cannot, it is true, arrive at any conclusion regarding the working of the process itself by a mere juxtaposition and quantitative comparison of the ingesta and excreta of the animal ornism.... It need scarcely be observed that science should not rest satisfied with a knowledge of the final results of chemical processes in the animal body. . . but should be made to enter more deeply into the course of the separate processes, and into the causal relations of phenomena. Here the statistical method cannot of course afford any satisfactory solution to our enquiries; for when we have ascertained by this experimental method that fat is formed in the animal body, we must learn from other methods the manner in which this substance is formed."6 A biological chemistry required far more attention to the living animal than Liebig, Boussingault and Dumas had shown. It was through the application of an improved experimental method that discoveries of fundamental physiological importance were made with the assistance of chemistry. This occurred, not in the context of fat or protein synthesis, but in the solution to the problem of the origin of carbohydrates in animals.46 ANIMAL CARBOHYDRATES The related compounds of starch, cellulose and the sugars were regarded as the most characteristic products of the vegetable kingdom, because of their abundance there. They were ternary compounds of carbon, hydrogen and oxygen, created by green plants from water and the carbon dioxide exhaled by animals. "9 Ibid., pp. 542-45. vol 1, pp. 14-15. 4' The complicated details of the metabolism of fats, proteins and carbohydrates were not worked out until the twentieth century. This awaited the discovery of a technique which could for example distinguish an ingested fat from one that was already present in the body. A tracer in the form of a chlorinated fat was employed by Bernard and Berthelot: C. Bernard, Le!ons sur les Phgnomines de la Vie communs aux Animaux et aux Vegetaux, Paris, 1878-1879, 2 vols., vol. 2., pp. 31-32. But a satisfactory labelling method was not available until the recent application of radioactive isotopes. 120 Chemistry and the two Organic Kingdoms of Nature in the 19th Century The presence of sugars in animals was attributed to their vegetable diet, or to a pathological condition, diabetes. The existence of starch and cellulose in animals was hardly ever considered.47 Indeed their supposed confinement to the plant world served as a basis for separating the two kingdoms, at a time when they had been brought together morphologically by the cell theory. Payen maintained that the cells of plants were bounded by cellulose, while the exterior of animal cells consisted of a quaternary, nitrogenous principle. He drew up a table contrasting the chemical behaviour of these two types of cell.'8 Cellulose was generally resistant to the reagents which attacked animal cells. Above all it was detected by the blue colour which appeared when sulphuric acid, followed by iodine, were applied. Payen said this was never observed in animal cells. This test for cellulose resembled the important test for starch,'9 which gave a blue or red colour with iodine. But they differed in that cellulose had first to be swelled by sulphuric acid, before the iodine was introduced. The same cellular distinction was made by Nageli,50 who saw in it the material cause for the functional differences of animals and plants. The nitrogenous boundary of the animal cell was the underlying cause of sensation and motion; plants, whose cell-walls were non-nitrogenous, lacked these faculties. He also believed that plant cells were peculiar in containing starch, which he said was never found in animal cells. The presence of nitrogen, which the eighteenth century had proposed as a criterion for distinguishing animal and vegetable substances, had reappeared in this new version of qualitatively distinct cell-membranes. But this was soon invalidated by Schmidt's surprising discovery of cellulose in tunicates.51 The chemically resistant covering of these animals was found by analysis to have the same composition as the cell-walls of plants. Schmidt remarked that tunicates lived within a plant-like exterior. He concluded that there was no chemical distinction between animals and plants, which could only be separated on psychological grounds. Schmidt's results were checked, and reluctantly it had to be conceded that cellulose could no longer serve as a distinguishing sign of a vegetable nature.52 The discovery '7 In 1821 Odier had reported the discovery of a resistant material, which he called chitin, in the elyptera of insects. He supposed it was identical with the structural material of plants. A. Odier, 'M6moire sur la composition chimique des parties corn6es des insectes', Meim. Soc. Hist. nat., Paris, 1823, 1, 29-42. This was falsified by Lassaigne, who showed the compound to contain nitrogen. Nevertheless chitin is a derivative of cellulose, and the physiological function of both is to provide structural rigidity. i' A. Payen, 'Memoire sur les developpements des v6g6taux', Mdm. div. savants Acad. Roy.Sci., 1846, 9, 1-42. See also his paper on 'Propri6t6s distinctives entre les membranes v6g6tales et les enveloppes des insectes et des crustac6s', C.r. hebd. Sdanc. Acad. Sci., Paris, 1843, 17, 227-31. The detection of cellulose in the Corallina officinalis was decisive in his judgment that it was a plant: A. Payen, 'Note relative aux caracteres distinctifs qui s6parent les v6g6taux des animaux, et aux s6cr6tions min6rales dans les plantes', ibid., [16][17][18][19]. ' D. C. Goodman also implied that animals were capable of synthesis. Loewig and Kolliker speculated on how this might occur. They ruled out a protein origin in animal food, since this seemed too unlike cellulose to be capable of conversion to it. But they remarked that tunicates also fed on vegetables, and the cellulose of these might be decomposed to sugar by their gastric juice. The sugar would then enter the blood, where it would somehow be converted to cellulose for circulation to the envelopes. They suggested an analysis of tunicate blood to study the process. They also looked to a chemical analysis of embryonic ascidia to explain the further difficulty of cellulose formation in the foetal stage.53 Amid the speculations, animal synthesis was finally demonstrated with Claude Bernard's momentous discovery of hepatic glycogenesis.54 This destroyed the foundation of Dumas' vital dualism and provided the experimental method which all previous attempts had failed to find. The way in which Bernard combined chemistry with physiology constituted the beginnings of a true biochemistry. His doctoral thesis on gastric digestion had convinced him of the primary importance of the organism itself in nutrition. Gelatine taken into the stomach reappeared in the urine, but glucose and cane sugar disappeared in the organism. It was clear to him that the organism was active in assimilating certain substances and eliminating others. He set out to trace the fate of ingested sugars, studying the interior of the organism. Chemical techniques would be required, but chemistry alone could not solve problems relating to animal functions.55 Nor could investigations be restricted, as the chemists had done, to the study of ingesta and excreta, since these were merely the beginning and end of a whole chain of events which constituted nutrition. In a remarkably clear and eloquent statement of the differences in the chemical and physiological approaches to the living organism, Bernard later wrote: We recognize the great importance of chemical statics, since it provides the preliminary data, which form the basis of the physiologist's study of the intimate phenomena of nutrition in our tissues. But experimental physiology teaches us that these intermediary problems ofnutrition must then be investigated step by step with the aid of delicate experiments, instead of being deduced by hypothetical explanations based on the comparison of materials in entry and exit. The phenomena of nutrition are too complicated to lend themselves to this type of investigation, Ann. Sci. Nat. Zool., 1846, 5, 193-238. Nevertheless they argued that chemistry could still separate the two kingdoms quantitatively. If over three-quarters of some tunicates consisted of a cellulose exterior, their other animal parts inside the envelope consisted of cells with the usual nitrogenous membrane. They said that no animal was yet known in which every cell-membrane consisted of cellulose, as was the case in plants. Besides only animals had nitrogenous cell-membranes. The latter statement was invalidated with the discovery ofchitin in fungi, towards the end of the nineteenth century. Is Ibid., p. 224f. Berthelot was not satisfied with an elementary analysis of the tunicate envelope. This merely showed it to be isomeric with cellulose. A more signifcant comparison required the demonstration of identical transformations, since this would imply the same physiological role. He showed that like cellulose, the tunicate envelope could be hydrolyzed to glucose, but with much greater difficulty. Another form of cellulose, he called it 'tunicine'. M. Berthelot, 'Recherches sur la transformation en sucre de divers principes imm6diats contenus dans les tissus des animaux invert6br6s, C. r. Soc. BioL, 1857, 4, 77-80, and 'Sur la transformation en sucre de la chitine et de la tunicine, principes imm6diats contenus dans les tissus des animaux invert6br6s', Ann. Chim. "T The most recent study of Claude Bemard is J. Schiller, Claude Bernard et les Problemes scientifiques de son Temps, Paris, 1967. This is a profound study, which has been invaluable in the preparation of this paper. 'C. Bernard, 'De l'origine du sucre dans l'economie animale', C. r. Soc. Biol., 1849, 1, 132. 122 which, we repeat, are only applicable to inorganic machines. We could cite many physiological errors which have resulted from this indirect mode of procedure, while, on the contrary, the experimental study of the phenomena of nutrition, conducted directly in the organs, tissues and even in the elements of tissues, have led to fruitful discoveries. The formation of sugar in the liver would never have been discovered if one had been restricted to the comparison of analyses of materials entering and leaving the organism. The physiologist must rely on these general chemical results, but he must not be content with them; he has to descend, with the aid of the direct experiment, into the intimacy of the organs, into the tissue, into the living cell whose function is identical in animals and plants. It is by this study alone that he will be able to grasp the mystery of intimate nutrition and succeed in mastering these phenomena of life, which is his supreme goal." Employing vivisection, Bernard tested various parts of the organism for sugars, using cupropotassium tartrate (reduced to red copper oxide) and yeast (alcohol and carbon dioxide were produced by fermentation). The experiments were conducted on dogs, fed on a sugar-free diet of meat, or starved. The tests showed an abundance of sugar in the blood leaving the liver by the ligatured hepatic veins; no sugar was found elsewhere. This indicated that sugar existed in the liver, and the tests confirmed it. The control experiments had established that the sugar could not have come from ingested vegetable sources. The prolongation of the experiments excluded the possibility that the sugar had been merely deposited in the liver by an earlier vegetable diet. It was undeniable that sugar was produced in animals by a process that was independent of the diet. Bernard drew the important conclusion: 'Therefore the law that animals create no immediate principles, but only destroy those provided by plants must cease to be true, since, like plants, animals can create and destroy sugar physiologically.'57 This was the contradiction of Dumas' fundamental premise. This line of separation between the two organic kingdoms had been erased. Bernard proceeded to isolate the precursor of hepatic sugar, glycogen.58 At first he had thought it was a protein, since cooking the liver inhibited the production of sugar. But it turned out to have the same properties as starch. It gave the characteristic reaction with iodine, and could be converted to dextrine and glucose by acid hydrolysis or fermentation. He called the new substance 'animal starch'.59 Just as in plants, a starch formed in animals, and was subsequently converted to glucose. Bernard wondered if starch was synthesized in the same way in both. He said 'the most perfect parallelism' between the kingdoms was established by the conversion of the starch to sugar by ferments.60 THE FERMENTS The ferments or enzymes (as they were later called by Bernard's pupil Kuhne, from their presence in yeast) were unknown in a pure state, because of the technical difficulties which their isolation presented. Nevertheless enough was known of their properties to recognize their existence, and to establish their analogous functions in the digestive processes of animals and plants. " 123 D. C. Goodman The first to be described was the agent in malt. This was called diastase,6' from its ability to separate the contents of starch granules from their supposed teguments, converting the starch to dextrine and glucose. Payen and Persoz crushed germinated barley in cold water, added alcohol, and collected a nitrogenous, white precipitate. They were astonished by its powerful effects, since in a few minutes it could alter two thousand times its weight of starch. They also discovered that the activity of a solution of diastase was destroyed by boiling, a characteristic property of enzymes which was repeatedly observed. They found diastase in cereals and potatoes only after germination, and in those parts where starch was consumed. The physiological role of diastase in vegetation was already apparent. A ferment with the same properties was then found in the human saliva by Mialhe,62 who called it 'animal diastase'. He carried out comparative tests on vegetable diastase and the white substance which alcohol had separated from the saliva. It seemed that equal weights ofthe two decomposed the same quantities of starch. The two agents behaved identically, but he wondered if the substance in the saliva was simply vegetable diastase introduced with the food. He later denied that the origin was external, when he failed to detect the ferment in the saliva of herbivores. A starch-digesting ferment was also discovered in the pancreas, though the investigators would not commit themselves to its identity with diastase, since they were unsure if they had studied the pure substance.63 A clue to the existence of this type of ferment in the liver was given by the temperature dependence of the process of sugar production there. Bernard found that the process came to a halt when the liver was placed in boiling water." He washed the liver to remove the sugar and glycogen, and then treated it with glycerol. The solution was precipitated by alcohol, the same technique, he said, for obtaining vegetable diastase. Like the latter, the liver ferment decomposed starch and glycogen to sugars. Bernard concluded that sugar was formed identically in animals and plants. In each case starch was created and converted to glucose by the same ferments. Wherever starch was digested, in the germinating potato, in the liver of animals, in their saliva and pancreas, the diastasic ferment was present. As Bernard explained, the complicated starch was decomposed to the simpler sugars which were soluble, and so could be circulated and assimilated.A Bernard could draw similar parallels in the digestion of other foodstuffs. The growing beetroot consumed reserves of cane sugar and produced a mixture of laevulose and glucose. This was caused by a ferment which Bernard extracted. He found a similar substance in the intestines of dogs, rabbits and birds. In each case he followed its action in the inversion of cane sugar with a polarimeter." The digestion of fats involved the production of an emulsion, as Bernard had seen 61 A. Payen and J. F. Persoz, 'M6moire sur la diastase, les principaux produits de ses r6actions, et in his early experiments on dogs. He discovered that the pancreatic juice would emulsify fats, dividing them into minute globules, and also saponify them.67 He said these changes were not due to the alkalinity of the pancreatic juice, but to the presence of ferments, since the action was suspended by boiling. He argued that the same ferment occurred in oleaginous grains. For example, crushed almonds also gave a milky emulsion, which was similarly precipitated by alcohol. 68 Bernard said there must in addition be a protein-digesting ferment in plants similar to pepsin, which Schwann had found in the stomach of an ox. In this way germinating plants could convert their protein reserves to soluble peptones.69 The prediction was confirmed in experiments on carnivorous plants. Lumps of meat placed on Drosera, Dionaea and Nepenthes were rapidly gelatinized and finally consumed, in a way that could only be compared to animal digestion. It was concluded that these plants produced a ferment like pepsin.70 The study of the ferments had led to profound physiological results. A remarkable organic unity was revealed in the acts of digestion of animals and plants. In both kingdoms, as Bernard said, the same reserves of carbohydrates, fats and proteins were decomposed by the same processes, and by identical, or at least similar ferments, to provide soluble substances for assimilation. He said the absence of a digestive apparatus in plants was unimportant, and had led to their false separation from animals. What really mattered was that the purely chemical processes of digestion were identical in both.7' In addition, the ferments clearly exhibited the peculiarity of the processes occurring in the organism. If digestion was entirely chemical, and if the reactions were of the same types as those occurring outside the body, the agents and the conditions were different.72 In the laboratory the chemist had to employ mineral acids to hydrolyze starch to glucose, and caustic potash to saponify fats. The ferments produced by the organism allowed the same changes to occur in conditions of mild acidity or alkalinity, and at moderate temperatures. The drastic procedures of industrial chemistry, impossible in life, were avoided in the organism, but the results were the same. THE UNITY OF RESPIRATION It was through his general physiological approach, searching for the common phenomena of life, that Bernard was able to remove a further barrier between the kingdoms, which the chemical study of respiration had erected. The eighteenthcentury contrast between the behaviour of green plants in sunlight, absorbing carbon D. C. Goodman dioxide and emitting oxygen, and the respiration of animals, had been continued by Dumas and others. Bernard would show that this too was a false contrast. The green substance in plants had been characterized as a distinct immediate principle. Soluble in alcohol, non-nitrogenous, and bleached by chlorine, it had been called 'chlorophyll', from its presence in green leaves.73 Nageli had stated that it was exclusive to the plant kingdom, and therefore another basis for distinguishing animal and plant cells.7' This was undermined by the discovery of its existence in infusoria, microscopic organisms of controversial status, although many were regarded as animals. Ferdinand Cohn studied the green spherules in the euglena and stentor, and declared them to be chlorophyll, from their reaction with concentrated sulphuric acid, turning blue. He considered these infusoria to be green animals, breathing like plants.7" Schultze referred to numerous green animals in ditches and ponds, such as the hydra viridis and volvox viridis. Their green substance behaved just like chlorophyll with acids and alkalis, and also faded in the dark. He kept the volvox viridis for a month in a dark room and found that the intense green had changed to a yellow. 76 A spectroscopic examination of the green matter in the euglena was undertaken by Angstrdm. He found the spectrum to be similar to that given by chlorophyll in the leaf of a Trifolium plant, and identical with the spectral bands of chlorophyll in the conferva and other algae. He concluded that chlorophyll was not peculiar to plants, since it existed in the euglena, and that his experiments, far from supporting the separation of animals and plants, confirmed an old law: natura non facit saltus. 77 It could be argued that the chlorophyll in infusoria had come from ingested vegetables, but this seemed unlikely from its presence within the parenchyma, instead of in the digestive system.7" A different problem which had not yet been elucidated was whether the green colour in certain animals was due to symbiotic algae. This was certainly the case with the green planaria studied at Lacaze-Duthiers' marine zoological laboratory at Roscoff.7" The green worms in the aquaria moved towards the light, and generated gas bubbles rapidly. These were collected in test-tubes and observed to rekindle a glowing match. There was no suspicion that the chlorophyll inside the worms actually belonged to smaller algae within them. The green colour of the freshwater sponge was misleading for the same reason."' Claude Bernard said that a classification might be attempted of a kingdom of organisms with chorophyll and one without, but this would not correspond with the Chemistry and the two Organic Kingdoms of Nature in the 19th Century division into animals and plants. 81 The euglena would have to be put with the plants on this view, and the whole class of mushrooms separated from the vegetables. Bernard distinguished between plant and chlorophyll, which he said should not be confused. More important he distinguished the chlorophyll function of certain organisms from the respiration which was universal in life. Green plants absorbed oxygen and exhaled carbon dioxide, by the destructive act of respiration which they performed in common with animals. But this was obscured by the simultaneous evolution of oxygen, performed by the chlorophyll function. Bernard found that he could separate the two processes by the use of anaesthetics.82 He placed two plants under bell-jars in sunlight, and near one he put a sponge with chloroform. The chlorophyllic function of this plant was suspended, and only carbon dioxide was emitted, like a breathing animal; the other plant exhaled oxygen as usual. In another experiment he passed air, freed from carbon dioxide, into vessels containing a rat and a cabbage, and collected the gases which were given off. In both cases barytes was turned cloudy by carbon dioxide.83 Far from establishing a system of separate physiologies for animals and plants, Bernard The decline of vital dualism can be traced in the classification of the infusoria. The study of these minute forms of life was facilitated by the nineteenth-century improvements in the microscope. Lamarck had declared them animals because of their irritability, a property which he said was caused by the distinctive chemical compounds which Nature had employed in the fabrication of animals.84 But this chemical difference could not be found. Richard Owen later wrote that chemistry 'will not serve as a rigorous basis for definition in the lower forms where the aid of'the chemist has been most wanted for that purpose.'85 The simultaneous presence of animal and vegetable characteristics in infusoria defied attempts to put them in either of the organic kingdoms. The enigmatic euglena moved like an animal and exhaled oxygen like a green plant. Ehrenberg8 had taken its red spots to be eyes. He had observed its ingestion of indigo, and searched for an intestinal tube. Without hesitation he accepted the euglena as an animal. As for the evolution of oxygen, he simply said the euglena falsified the view that this faculty was restricted to plants. Similarly Ehrenberg classified other moving and feeding microscopic organisms as animals, and described imaginary anatomical analogies with the higher animals. He said these characters were 'more determinate' than chemistry in deciding which kingdom the infusoria belonged to.87 61 C. Bernard (n. 56), vol. 1, p. 146 and p. 208f. D. C. Goodman Protests came from the botanists. They complained that Ehrenberg had taken some plants into the animal kingdom, particularly the desmids and diatoms, which he had classified as infusorial animals of the bacillaria family.88 Ralfs said Ehrenberg had been led astray through the neglect of chemical evidence. At first he had also regarded the desmids as animals, from their forms: Their symmetrical division into two segments; the beautiful disciform, finely-cut and toothed Micrasterias, the lobed Euastrum, the Cosmarium glittering as if it were with gems, the Xanthidium armed with spines, the scimitar-shaped Closterium embellished with striae, the Desmidium resembling a tape-worm, and the strangely insect-like Staurastrum sometimes furnished with arms, as if for the purpose of seizing its prey, all these characters seem indeed to pertain more to the lower animals than to vegetables.8' But forms were misleading, and besides the desmids were of a herbaceous green colour. Above all they contained starch. Ralfs had tested the desmids with iodine solution, and observed the characteristic blue colour. He wrote: 'Of all the facts which indicate the vegetable nature of the Desmidieae, this is undoubtedly the most important, since it is the most easily subjected to the test of experiment."'0 He denied that the starch had come from ingested aquatic plants, deposited in the supposed stomachs of desmids. This would not explain the gradual increase of starch as the desmid seed formed, nor its absence in the earliest stages of development."' He appealed to impartial observers to repeat his tests for starch, which had been overlooked by Ehrenberg and his followers. He argued: 'Again, it has been seen that starch is abundantly produced in this family. Can a single example be referred to where it is an animal product? . . . Until these facts have been denied, or the arguments deduced from them refuted, I shall presume that the claim of the Desmidieae to be considered vegetables is firmly established'.'2 The same arguments were used to reclaim the diatoms for botany.93 They were rendered useless by Bernard's discovery of glycogen in the animal kingdom. In a treatment of the infusoria which appeared soon after, Clapar6de and Lachmann said chemistry was unable to guide their classification, since the claims based on the exclusive presence of starch, cellulose or chlorophyll in the vegetable kingdom had been disproved." Similarly, referring to the significance of the discovery of cellulose in tunicates for the classification of unicellular organisms, von Siebold wrote: 'I must here remark that we can scarcely expect chemistry to decide what is animal and what plant, having several times been deceived in our hopes in this respect.'95 In a paper read to the British Association in 1860, John Hogg described the difficulties he had found in classifying ambiguous organisms. He said the discovery of starch in animals had greatly weakened the determination of vegetability.96 No more successful than other criteria, chemistry had failed to establish a sharp dividingline. He therefore proposed that desmids, diatoms, infusoria and all other disputed creatures should be put into a fourth kingdom, which he called Regnum Primigenum. He said: 'Since, indeed, the vegetable and animal kingdoms have been well compared to two lofty pyramids, which diverge from each other as they ascend, but are placed on, or united in, a common base; this base, then, might fairly represent the Primigenal kingdom, which includes the lower creatures or organisms of both the former, but which are of a doubtful nature, and can in some instances only be considered as having become blended or mingled together. '97 Huxley98 remarked that the failure of the chemical distinctions had caused fundamental changes in the naturalist's conception of animals and plants. The former clear divisions had gone. He despaired of finding a single character for the border territory between the kingdoms, which he called 'no-man's land'. He thought the last hope lay in protein synthesis, which perhaps only plants could achieve. The ambiguous bacteria multiplied in a solution of tartrates and phosphates, a process involving the synthesis of their proteins. Huxley therefore took them to be plants, but other infusoria left him in a state of indecision. When Frankland, in a lecture to the Chemical Society, announced that all microorganisms were animals, because of their destructive acts of oxidation, his firm tone shocked the biologists present."9 Burdon-Sanderson commented that it was for the chemist to consult the biologist on this subject, and that in any case it was 'of little practical consequence' to decide whether the organisms were animals or plants. Foster added that the behaviour of the micro-organisms mattered far more than their classification. These remarks were significant. The insistence on a classification into two organic kingdoms was receding, as the impossibility of this separation on any grounds became increasingly apparent. As one writer of the late nineteenth century put it: 'The entire fabric of living nature is, in truth, a great tree, the branches of which diverge most widely in their highest levels, but which in its lowest parts, unites and blends all diversities in a common and inseparable unity.'1" In looking to chemistry to divide this unity, he said the dilemma of the biologist becomes 'confusion worse confounded'.'01 CONCLUSION In this paper, and the previous one (Med. Hist., 1971, 15, 23-44), we have considered the relation between chemistry and physiology in the eighteenth and nineteenth D. C. Goodman centuries. The problem of nutrition was approached by the analytical techniques which characterized organic chemistry up to the middle of the nineteenth century. In experiments which were conducted outside the living organism, vegetable foods and animal materials were analysed, and from the results, deductions were made on the action of the animal economy. In the eighteenth century, the process of animalization was attributed principally to nitrogenation, while in the next century, Dumas explained the entire animal economy by the single act of combustion. The neglect of the living organism had resulted in a reduction of physiology to chemistry. This procedure left in obscurity the very facts which were being sought: the intimate events of creation and destruction occurring within the organism. It was through Bernard's insistence on the study of these, involving the detection of intermediates, such as glycogen, in living processes, that the way was opened to an understanding of nutrition. The acts of digestion were then shown to be purely chemical processes, but involving enzymes, a class of compounds peculiar to living organisms. Nutrition, previously assumed to occur directly, was found to be indirect. Food taken into the body was subject to destruction, synthesis and storage. The same processes were observed in animals and plants. Bernard had shown how chemistry could be combined with physiology, so constructing a basis for biochemistry. Both sciences acquired benefits: physiology adopted chemical tests and received explanations for the phenomena of digestion; organic chemistry became synthetic, imitating, as Berthelot said, the synthesis of immediate principles and their metamorphoses in life. We have also considered the part played by chemistry in classification. Its growing use in this way, since the eighteenth century, was apparent from the chemical analyses of ambiguous organisms, and from the chemical content of the definitions which were given to animal and plant by Dumas, Nigeli, and Charles Robin. But the chemical distinctions, on which the separation of the two organic kingdoms had increasingly: relied, were found to be baseless. One by one, the chemical barriers between animals and plants fell. The claims for a monopoly in one kingdom of nitrogen, chlorophyll, sugar, cellulose and starch were all discredited by the middle of the nineteenth century. The fundamental antithesis of plant synthesis and animal destruction fell with them. Chemistry in the end provided strong arguments for the inseparability of the two kingdoms, from its demonstration of the identity of nutrition in both.

v3-fos

2018-04-03T00:05:51.600Z

1972-05-01T00:00:00.000Z

8629169

s2

Storage of conidia of Penicillium chrysogenum in liquid nitrogen. Conidiated slope cultures of derivative of Penicillium chrysogenum Wis 54-1255 were stored at -196 or +4 C for a period of 3.5 years. After this time, the viability fell to 68% in the former case and to 4% in the latter. At the end of the experiment, 65 single conidial isolates from each series were tested for penicillin yield. Among those from conidia stored at -196 C, the spread of penicillin yields did not differ markedly from that of 65 single conidial isolates made as controls prior to storage. However, 18% of those from conidia stored at +4 C formed a subpopulation with substantially lower penicillin titers than those of control isolates. Storage at -196 C may reduce or prevent a possible source of penicillin yield decay, namely, the selection of spontaneous mutants of low titer present in small numbers in the original culture and selected, as viability decreased, by virtue of their increased longevity relative to that of the parental culture. Conidiated slope cultures of a derivative of Penicillium chrysogenum Wis 54-1255 were stored at -196 or +4 C for a period of 3.5 years. After this time, the viability fell to 68% in the former case and to 4% in the latter. At the end of the experiment, 65 single conidial isolates from each series were tested for penicillin yield. Among those from conidia stored at -196 C, the spread of penicillin yields did not differ markedly from that of 65 single conidial isolates made as controls prior to storage. However, 18% of those from conidia stored at +4 C formed a subpopulation with substantially lower penicillin titers than those of control isolates. Storage at -196 C may reduce or prevent a possible source of penicillin yield decay, namely, the selection of spontaneous mutants of low titer present in small numbers in the original culture and selected, as viability decreased, by virtue of their increased longevity relative to that of the parental culture. Microbial mutants giving increased amounts of commercially useful products are of obvious interest, and it is clearly important that their genetic integrity be maintained in culture. Whether or not this can be done will depend on their spontaneous mutability and the environmental pressures to which they are subjected. For example, such mutants can be susceptible to yield decay during replication so that serial subculturing leads to a reduction in productivity (1,2,4,14). Even the preservation of a culture in the quiescent state does not necessarily avoid a drop in product yield if viability decreases during storage (4). It is normal industrial practice to avoid serial subculturing, and, from a master culture, subcultures are made in parallel to establish separate fermentations. The preservation of a culture under conditions where there is little or no loss of viability might be a way of preventing yield decay during storage (4, 6, 10). Previous work indicated that when a strain of Penicillium chrysogenum producing a relatively high yield of penicillin was stored at +4 C, mutants with low titers accumulated as viability fell (4). Heterokaryon tests (3) showed that these were nuclear rather than cytoplasmic in origin. There were two possibilities, neither being mutually exclusive: the selection of pre-existing mutants with low penicillin productivity because of their increased longevity relative to the parent or the induction of such mutants by the storage conditions. The present paper reports results with the same strain of P. chrysogenum when a comparison was made of preservation at temperatures of + 4 and -196 C. Studies by Wellman (13) indicated that when conidiated slope cultures of two strains of P. chrysogenum were stored for 38 months in liquid nitrogen, conidial survival approached that of control cultures, when estimated as per cent germination. Fermentations derived from mass conidial inocula of material stored in liquid nitrogen yielded similar amounts of penicillin to controls. The work reported here is an extension of Wellman's studies in that a strain with a higher penicillin titer was used and also tests were made of single conidial isolates after storage to discover whether there were population changes in the pattern of penicillin yield not detectable by mass conidial transfer. MATERIALS AND METHODS Organism. From P. chrysogenum Wis 54-1255 (11), a mutant was produced after three serial ultraviolet-light treatments (5) which had brown conidia and requirements for biotin and nicotinamide. This mutant was used in the work reported here, and conidia from a lyophilized culture were dispersed on CM (see below) so that separate colonies grew after incubation. A single colony was then sown on to 10 CM slopes. After incubation, one was set aside as master culture and the remaining nine when tested 990 STORAGE OF P. CHRYSOGENUM CONIDIA for penicillin yield, as described later, averaged about 3,000 units/ml, which was similar to that of the parental culture Wis 54-1255 tested under the same conditions. Penicillin yield testing. Each isolate for test was grown on a slope of CM prepared in a 1-oz (ca. 31.1 g) universal container. Conidia from a slope were inoculated into a 100-ml Erlenmeyer flask containing FM which was placed on a rotary shaker [2-inch (ca. 5.08 cm) throw, 220 rev/min] at 24.5 C for 6 days before assaying the filtered broth. The assay method in its essentials was the arsenomolybdate technique as described by Pan (8). Methods of storage. Cultures were stored on slopes of CM either in a refrigerator at +4 C or in liquid nitrogen at -196 C in a Union Carbide LR-1OA-6 unit. Those stored in liquid nitrogen were on slopes in 0.5-dram (ca. 0.58 g) vials (Johnson and Jorgensen Ltd., London) measuring approximately 3.5 by 1 cm with screw caps. The latter were tightened before immersion in liquid nitrogen. Care had to be taken when removing slopes from liquid nitrogen because of the possibility of leakage of the liquid into slopes and the danger of explosion when they were thawed. No trouble was experienced, but as a precaution slopes removed from liquid nitrogen were placed in a large metal container and allowed to rise to room temperature before use. Estimation of viability. Conidia were dispersed in a wetting agent consisting of 0.02% (v/v) calsolene (ICI Ltd) in distilled water and plated on at least 10 plates of CM at dilutions to give approximately 100 colonies per plate. RESULTS Conidia from the master culture of the auxotroph of P. chrysogenum were inoculated on to several CM slopes for storage, after incubation, at +4 and -196 C. Conidial viabilities of slope cultures were estimated immediately before preservation, after one week at +4 and -196 C and then at intervals throughout a storage period of 42 months. There was a drop in viability after 1 week at -196 C which could be attributed to the effects of freezing and thawing ( Table 1). Slope cultures were simply immersed in liquid nitrogen and removed to room temperature as required. No attempts were made to establish controlled conditions of cooling or heating. The results in Table 1 showed that, after 42 months, viability was much better preserved at -196 than at +4 C. At the beginning of the experiment when slope cultures were inoculated from the master culture for preservation, conidia from this master culture were also plated on CM, and 65 single conidial isolates were tested for penicillin yield with the results shown in Fig. 1. When viabilities were estimated after 4 and 21 months, 10 single conidial isolates from each series at both periods retained the penicillin yield of the parental auxotrophic culture. After 30 and 42 months, 65 single conidial isolates were made from each series and tested for penicillin yield. At 30 months, single conidial isolates from both series included a small proportion with low penicillin yields (Fig. 1). This was substantiated after 42 months among isolates from material stored at +4 C but not from isolates grown from conidia held at -196 C (Fig. 2). After 42 months, 100 single conidial isolates of each series were also tested for growth requirements. With one exception, all 200 retained the three genetic markers carried by the parental strain determining brown spore color and requirements for biotin and nicotinamide. The exception was a single conidial isolate from material stored at +4 C which had lost its requirement for nicotinamide and had a penicillin titer of less than 500 units/ml. None of the three genetic markers had a substantial effect on penicillin yield, so, unless this isolate also bore an independent mutation which reduced penicillin titer, the loss of its requirement for nicotinamide was probably due to a suppressor mutation having a pleiotropic effect which lowered penicillin yield rather than a reversion at the original site of the mutation determining the vitamin requirement. The'evidence was minimal but suggested storage at -196 rather than at +4 C to prevent loss of auxotrophy. VOL. 23, 1972 FIG. 1. Penicillin yields of 65 single conidial isolates from the master culture immediately before storage (top) and from slopes stored for 30 months at +4 C (middle) and -196 C (bottom). DISCUSSION Some mutants yielding more of a useful metabolite than their parent may not be stable and range from those so unstable as to escape discovery, because of poor viability or an extreme liability to yield decay on replication, to those whose instability only becomes evident after their operational use on an industrial plant. The latter sort may be of the kind which show yield decay on storage, a characteristic not amenable to test immediately after a mu- tant's isolation. As yields continue to be raised by serial mutagenic treatments, it may become increasingly necessary to use unstable mutants in industry. If so, methods will have to be sought to minimize the effects of instability both on replication and during storage. Obviously an attempt could be made to reduce or prevent any decreases in productivity during replication by using fewer seed stages in industrial fermentations. However studies with Streptomyces griseus indicated that when an iron salt was omitted from the culture me- (7). Presumably sufficient Fe2+ was available as a contaminant in other medium ingredients to allow growth. It was suggested that the presence of porphyrincontaining enzymes, involved in the biosynthesis of streptomycin, was dependent on a genetically labile step inactive in the absence of sufficient Fe2+ and that possibly the chances of deleterious mutations were then limited (7). Perhaps further investigations in the field of nutrition may indicate other ways of yield stabilization during replication. Attention to the effects of media ingredients may also be important in preserving viability during storage. For example, raising the level of Fe2+ above that required for growth increased the longevity of Pseudomonas cultures (12). It was proposed that when cells have ceased to divide pools of primary metabolites may have to be converted to innocuous secondary metabolites to prevent distorted growth and loss of viability, and that the metal was required for the operation of syntheses in these conversion processes (12). The present work investigated the possibility of precluding or reducing instability by preserving cultures of P. chrysogenum at -196 C. When conidia were stored at this temperature over a period of 42 months, they retained good viability. Furthermore, 65 single conidal isolates had penicillin yields similar to that of the same number made prior to storage. That a reduction in viability can be correlated with loss of penicillin yield (4) has been supported by the demonstration that storage of conidia at +4 C for 42 months resulted in poor survival and a decrease in penicillin titer in a proportion of 65 isolates grown from individual surviving conidia. It has not been possible to decide whether mutants of low titer were selected as viability fell or storage per se induced mutation to low titer. Proof of the latter event would require a demonstration of an absolute increase in the number of low-yielding mutants during storage at +4 C. When after storage for 42 months at +4 C, the viability had dropped to 4%, then among 65 conidial isolates 18% had low penicillin titers (Table 1, Fig. 2). Assuming that mutants with low yields survived preservation, this represented a level of 0.72% in the original sample prior to storage. No single conidial isolates of low yield were present among a sample of 65 made before storage (Fig. 1 and 2). The results were not incompatible with a selection hypothesis, although they did not disprove that preservation at +4 C could induce mutations to low titer, particularly if such mutants died off during storage, albeit at a slower rate than the parent. However, even if storage at +4 C did induce mutation to low titer, storage at -196 C, as was pointed out previously (4), should tend to inhibit metabolic reactions including those involved in mutation. Whether, in the future, precautions will become necessary in mutational screening programs with highly developed industrial strains to preserve the viability of putative mutants, immediately after mutagenic treatment, will be a matter for the individual experimenter to decide in the light of his experience and rate of success in producing mutants with increased yields. ACKNOWLEDGMENT I am indebted to Teresa M. Tessier for expert technical assistance.

v3-fos

2020-12-10T09:04:12.762Z

1972-01-01T00:00:00.000Z

237229312

s2

Growth of Salmonella typhimurium in Skim Milk Concentrates The influence of various levels of skim milk solids and temperature on the duration of lag phase, growth rate, and extent of growth of Salmonella typhimurium was investigated. The effect on growth of salmonellae (and a strain of Escherichia coli) of reduced pressure at a constant solids level and under conditions simulating vacuum condensation of skim milk was also studied. S. typhimurium grew when inoculated into skim milk solutions ranging from 10 to 60% solids and over a temperature range of 23 to 44 C. At 10 to 12 C, growth was evident only in the 10% skim milk. As the total solids level was increased or incubation temperature was deviated from the optimum, or both, there was an increase in the lag phase and generation time of salmonellae. A lower cell population also resulted. The generation time at 37 C of S. typhimurium incubated at atmospheric pressure was approximately one-half that in skim milk concentrates held under reduced pressure. In addition, a slightly longer lag phase and lower cell yield characterized the growth under reduced pressure. Concentration of skim milk had little or no effect on viability of salmonellae or E. coli when the vapor temperature in the vacuum pan was below the maximum growth temperature for salmonellae. Increasing the vapor temperature to 48 C caused a two-log reduction in viable organisms during the concentrating period (65 min). The influence of various levels of skim milk solids and temperature on the duration of lag phase, growth rate, and extent of growth of Salmonella typhimurium was investigated. The effect on growth of salmonellae (and a strain of Escherichia coli) of reduced pressure at a constant solids level and under conditions simulating vacuum condensation of skim milk was also studied. S. typhimurium grew when inoculated into skim milk solutions ranging from 10 to 60% solids and over a temperature range of 23 to 44 C. At 10 to 12 C, growth was evident only in the 10% skim milk. As the total solids level was increased or incubation temperature was deviated from the optimum, or both, there was an increase in the lag phase and generation time of salmonellae. A lower cell population also resulted. The generation time at 37 C of S. typhimurium incubated at atmospheric pressure was approximately one-half that in skim milk concentrates held under reduced pressure. In addition, a slightly longer lag phase and lower cell yield characterized the growth under reduced pressure. Concentration of skim milk had little or no effect on viability of salmonellae or E. coli when the vapor temperature in the vacuum pan was below the maximum growth temperature for salmonellae. Increasing the vapor temperature to 48 C caused a two-log reduction in viable organisms during the concentrating period (65 min). The genus Salmonella has received increased notoriety in recent years, prompted in part by recovery of these organisms from dried milk implicated in a 1965 outbreak of Salmonella food poisoning (3). Subsequent investigation disclosed that various dried dairy products were contaminated with salmonellae. Although various constructive opinions (4, 5, 7) have been given regarding probable sources of salmonellae and suitable conditions conducive for growth of these organisms in processing plants, the literature contains little information on the actual behavior of salmonellae during the manufacture of nonfat dry milk (NDM). It is becoming increasingly evident that the behavior exhibited by salmonellae (and other microorganisms) in laboratory media is not necessarily characteristic of the organisms in a food environment. The basis for this discrepancy is in part attributable to the physicochemical properties of the given food substrate and in part to the influence of other microor-ganisms constituting the normal flora of the product. Consequently, to be certain how salmonellae will behave in a given product or process environment, experimentation must be conducted using the food in question and simulating the processing this food may receive. This study was conducted to generate data on the behavior of salmonellae during the manufacture of dried milk products. MATERIAS ANe MEJHODS Bacterial cultures. The strains of S. typhimurium and Escherichia coli used in this study were obtained from the Food Research Institute culture collection. Stock cultures were maintained on nutrient agar slants at room temperature. Working cultures were transferred daily in Trypticase soy broth (TSB) and incubated without agitation at 32 C unless otherwise noted. Skim milk. The milk was prepared from a single lot of antibiotic-free skim milk, which was spraydried in the dairy plant facilities of the University of Wisconsin. The NDM met the following specifications: no detectable coliform or Salmonella microor-82 ganisms when tested by the procedures advocated by the Food and Drug Administration (1), a standard aerobic plate count of less than 300 per g, and a moisture content of approximately 2.5%. Growth at atmospheric pressure. A 12-hr culture of S. typhimurium was added at a 0.5 to 1.0% (v/w) level to milk solutions, which were at 10, 30, 40, 50, and 60% (w/w) total solids (TS) levels. Concentrates were prepared in Nalgene containers by adding the appropriate amount of NDM to sterile distilled water which had been tempered to the test incubation temperature, i.e., 10 to 12, 23, 32, 37, and 44 C. A mechanical blending apparatus was used to mix thoroughly the inoculum into the milk concentrate. The inoculated concentrates were then incubated at the test temperature to which they have been pretempered. Growth under reduced pressure. Reduced pressure growth studies were conducted at 35 to 37 C with a 40% (w/w) milk concentrate, which was contained in a laboratory vacuum pan that had been modified for bacterial quantitation by the incorporation of a sampling port into the system (3a). To simulate vacuum condensing conditions, the laboratory vacuum pan including an external heating source (steam) was employed. Concentration of 10% skim milk to higher TS levels was carried out by matching the rate of inflow of 10% skim milk to the outflow rate of condensate. Material balance calculations were used to determine the TS level attained per volume of 10% skim milk added (volume of fluid in the system constant) at the desired vapor temperature. The inoculum was added into and samples were taken from the vacuum pan as described previously (3a). A manometer and a thermometer were employed to monitor the pressure and vapor temperature during the concentration process. Enumeration of salmonellae. Quantitation of S. typhimurium in the growth experiments was accomplished by periodically transferring a 10-g sample to a sterile, chilled Waring Blendor containing 90 ml of sterile distilled water. After blending for approximately 1 min at low speed, 0.1-ml samples of the appropriate dilutions were surface-plated on Salmonella-Shigella agar (BBL). These plates were examined for typical Salmonella colonies after incubation at 35 to 37 C for 48 hr. Enumeration of salmonellae during the vacuum concentration of milk was performed by periodically removing 1-ml samples to 9 ml of 0.1% peptonewater. A 0.1-ml amount of the appropriate subsequent dilutions were surface-plated on Trypticase soy agar (BBL) fortified with 0.2% yeast extract (TSAYE). The plates were examined after incubation at 35 to 37 C for 48 hr. Enumeration of E. coli. Enumeration of E. coli in the growth experiments was accomplished by making pour plates with violet-red bile-agar of the appropriate dilutions of concentrate. Plates were examined after 18 to 24 hr at 35 to 37 C. Enumeration of E. coli during the vacuum-condensing operation was performed in an identical manner as that described above for salmonellae. RESULTS AND DISCUSSION Growth at atmospheric pressure. The initial approach was to study the effect of various incubation temperatures and TS levels on the growth of S. typhimurium at atmospheric pressure. Although the NDM used in the concentrate preparation was high-quality powder, it was not a sterile product. This necessitated the employment of a selective 'and differential) medium for the quantitation of viable salmonellae throughout the incubation period. Salmonella-Shigella agar was chosen as the recovery medium after preliminary experiments disclosed that the interfering organisms were primarily Bacillus spp. which grew well and masked typical Salmonella colonies on Brilliant Green-agar. The inoculum was at a level to insure detection of salmonellae by the surface-plating procedures throughout the experimental period. The growth curves of S. typhimurium in milk solutions (10 to 60% TS) incubated at several temperatures are shown in Fig. 1 (a-e). Figure 2 depicts the growth pattern of S. typhimurium at a single TS level (40%) as the growth was influenced by incubation temperature. In all trials, there was a loss of recoverable salmonellae upon introduction into the milk solutions. In solutions containing higher TS levels, most of the loss was manifested during the time (5 min) that elapsed between introducing the inoculum and taking the initial sample. At the lower TS levels, the loss was not as great during this time, but there was a continued gradual loss of recoverable cells for several hours thereafter, the rate being dependent on the temperature of the concentrate. It is quite probable that the major adverse effect on the cells was the abrupt change in osmotic pressure. The more drastic the change, the more rapid the loss of recoverable salmonellae. The simultaneous change in temperature could also be seen to contribute to the demise of salmonellae as is indicated when the curves obtained at a single TS level but different incubation temperatures are examined (Fig. 2). However, this effect was slight in comparison to the osmotic pressure influence. Since the data depicted in these figures were derived from counts made on Salmonella-Shigella agar, these points represent the number of salmonellae recoverable on this agar. Parallel experiments in which TSAYE was used as a recovery medium (until overgrowth by the normal flora made enumeration of salmonellae impossible) were run. The same pattern of behavior was obtained, i.e., a die-off of salmonellae in the concentrates followed by an "adjustment period" before the number of replicating cells exceeded the number of cells that were dying. However, the loss in recoverable salmonellae upon inoculation as measured on TSAYE did not exceed 0.5 log in any of the milk concentrates. Moreover, the adjustment period was shorter when TSAYE agar was used. It is thus apparent that the osmotic stress imposed by inoculation into concentrated milk rendered the cells less able to cope with the rather adverse environment of a selective agar medium. This is not unexpected since the work of others (2,8) has repeatedly demonstrated that injured or stressed cells are physiologically debilitated and are more susceptible to the harsh environment of selective recovery media. The loss of recoverable salmonellae upon inoculation into concentrated milk solutions underscores the importance of performing counts on the inoculated material rather than basing the zero time cell count on an enumeration of the organisms in the cell suspension used as the source of inoculum. Unless this initial count is made, the time at which actively growing cells constitute the major portion of the population and the curve begins to approach a logarithmic nature may be missed by several hours. For example, the data of Mc-Donough and Hargrove (9) would indicate that a lag phase of slightly more than 24 hr occurred before salmonellae were able to prolif- erate in a 60% TS concentrate at 37 C. Our results show that, although the most rapid growth began at 15 hr, the population of S. typhimurium began to increase 3 hr after inoculation into 60% TS at 37 C. Thus, these cells would manifest the characteristics (e.g., sensitivity to chemical and physical agents) of logphase cells rather than lag-phase organisms. Whether this difference in sensitivity is sufficient to have a bearing on the survival by salmonellae of in-plant processing is questionable. The mean generation times for S. typhimurium in milk concentrates are given in Table 1. Increasing TS concentrations and lower incubation temperatures both resulted in slower growth by salmonellae. Similar results were reported by Wodzinski and Frazier (10) in their study of the effect of solute concentration and incubation temperatures on three species of bacteria. It should also be noted that increased concentrations of solids also resulted in a lower cell yield of salmonellae. This effect was particularly in evidence for the 50 and 60% TS solutions incubated at and above 32 C. Although this may be of interest to the researcher, it is rather unimportant to the proc- essor since significant levels of salmonellae were achieved in all concentrates stored at or above 23 C. At 10 to 12 C, salmonellae were able to proliferate only in the 10% TS solution, and a reduction in viable salmonellae was noted in all other solutions. Most of the die-off was completed within 72 hr, and the population remained relatively stable thereafter. Thus, refrigeration of milk concentrates will not only prevent growth of salmonellae but also result in a reduction in viable cells. However, it would not be prudent to assume that such practices would ensure a Salmonella-free concentrate. Growth under reduced pressure. Experiments designed to generate data on the behavior of salmonellae under reduced pressure were undertaken to determine the conditions that would permit the proliferation of salmonellae during the vacuum concentration of skim milk. For comparative purposes, a strain of E. coli was also included. Both organisms were inoculated (in separate experiments) into 40% TS milk solutions and incubated at 35 to 37 C and 55 i 5 mm of Hg pressure. Under these conditions, the mean generation times were 0.8 and 1.0 hr for E. coli and S. typhimurium, respectively. The more rapid growth of E. coli was most probably due to its ability to ferment lactose, a characteristic lacking in S. typhimurium. It should be noted that the generation time at 37 C of S. typhimurium in the 40% TS solution at atmospheric pressure was approximately one-half as long as under reduced pressure. Anaerobic growth of salmonellae in skim milk is possible by the energygenerating arginine dihydrolase system that these organisms have. However, this system would not be as efficient as an oxidative amino acid metabolism. This would be a possible explanation for the difference in growth rates of salmonellae in the aerobic and anaerobic environments. In addition to a slower growth rate, 85 VOL. 23, 1972 r-v--* the culture grown under reduced pressure achieved a lower final population than the culture grown at atmospheric pressure. This latter observation is in accord with that of George et al. (6) on the growth of S. aureus in skim milk concentrates incubated under reduced pressure. These investigators concluded that the subatmospheric pressures used in vacuum concentration would not afford sufficient retardation of growth to be of practical significance. Based on our observations, we would agree that this premise is also true for control of salmonellae during vacuum concentration of milk. The effect of vacuum concentration of skim milk from 10 to 42% TS on the behavior of E. coli and S. typhimurium was investigated. Multiple trials employing the pressure-temperature parameters summarized in Table 2 were conducted. The results of a typical single trial with each organism are shown in Fig. 3 and Fig. 4. It is evident that no significant decrease in cell number occurred when the vapor temperature was below the maximum growth temperature for salmonellae. At temperatures slightly above 46 C, there was approximately a 2-log decrease in viable cells during the concentration process. It is probable that the short time (47 to 56 min) necessary to accomplish the concentration precluded growth of the organisms when the temperatures were below the maximum growth temperature. It is quite probable that given sufficient time to adjust to the environment, these organisms would multiply in the concentrate as was described above. This work and that of McDonough and Hargrove (9) have amply demonstrated that salmonellae will grow quite readily in milk solutions (up to 60% TS) if the temperature is appropriate. The application of reduced pressure to milk solutions to effect a concentration of , , N solids does reduce the growth rate of salmonellae, but the process will not kill the cells if the temperature is below the maximum growth temperature for salmonellae. The processor should now realize that if salmonellae do gain entry to pasteurized milk, the surest method of preventing increases in cell number during further processing, i.e., vacuum concentration, is strict temperature control. Product cannot be left within the growth temperature range for salmonellae for any significant period of time if the population of the contaminant is to be maintained at a level low enough to be destroyed during subsequent drying treatments.

v3-fos

2020-12-10T09:04:17.004Z

1972-02-01T00:00:00.000Z

237230998

s2

Effect of Four Nematocides on Activities of Microorganisms in Soil Tests were conducted to determine the effects of four nematocides, Dasanit, carbofuran, D-D, and Vorlex on microbial activities in a loamy sand. The results indicated that bacterial and fungal populations initially decreased with some nematocide treatments but recovered rapidly to levels similar to those in the controls. In some instances, ammonium production from added peptone increased in the nematocide-treated soils, whereas mineralization of soil organic nitrogen and nitrification and oxidation of elemental sulfur were depressed. Oxygen consumption generally increased in proportion to the concentration of nematocide in the soil. However, with Vorlex, an increase in respiration was evident at the lower concentration, whereas an inhibitory effect occurred at the higher concentration. The study indicated that indigenous soil microorganisms can tolerate these chemicals used for control of nematodes in soil. Tests were conducted to determine the effects of four nematocides, Dasanit, carbofuran, D-D, and Vorlex on microbial activities in a loamy sand. The results indicated that bacterial and fungal populations initially decreased with some nematocide treatments but recovered rapidly to levels similar to those in the controls. In some instances, ammonium production from added peptone increased in the nematocide-treated soils, whereas mineralization of soil organic nitrogen and nitrification and oxidation of elemental sulfur were depressed. Oxygen consumption generally increased in proportion to the concentration of nematocide in the soil. However, with Vorlex, an increase in respiration was evident at the lower concentration, whereas an inhibitory effect occurred at the higher concentration. The study indicated that indigenous soil microorganisms can tolerate these chemicals used for control of nematodes in soil. Pesticides are often applied directly to the soil for pest control. Some of the organophosphorus and carbamate pesticides have been shown to be moderately persistent (4) and to have some effect on microbiological activities in the soil (1,8). For many years, fumigants such as D-D and Vorlex have been widely used for control of nematodes. Other compounds such as Dasanit and carbofuran have also shown promise when applied at relatively high rates of application. Little is known about the effects of these chemicals on the beneficial soil microbes that are important in soil fertility. This paper reports the effects of four nematocides on the activities of microorganisms in the soil. MATERIALS AND METHODS The experiments were conducted with Delhi loamy sand, a typical agricultural soil in southwestern Ontario. Composite soil samples were taken to a depth of 6 inches and analyzed for chemical, mechanical, and physical characteristics. The soii contained 0.81% organic matter and 0.03% total nitrogen. The moisture holding capacity was 27%, and the pH was 8. hydrocarbon mixture) at rates of 30 and 180 ug/g (14 and 84 GPA) were mixed into the soil. Chemical purities of nematocides were at least 94.5%. Reagent grade peptone and elemental sulfur powder were added to each soil sample at 1,000 jzg of nitrogen or sulfur per g for ammonification and sulfur oxidation, respectively. Oxidation of ammonium from soil organic nitrogen was studied by nitrification. The additives were thoroughly mixed with the soil. The mixtures and controls were transferred to 0.236-liter (0.5 pint) milk bottles, which were closed with 0.0381-mm (1.5 mil) thick polyethylene film. Soil moisture was maintained at 60% of the moistureholding capacity. The treatments, in duplicate, were incubated in the laboratory at 28 C for appropriate periods, i.e., 1 week for ammonification, 1 and 2 weeks for nitrification, and 4 weeks for sulfur oxidation. Changes in the population of soil microorganisms were determined after 1, 2, 4, 8, and 12 weeks. Rose bengal-streptomycin-agar was used for determination of changes in fungi in the soil, and sodium albuminate-agar was used for bacteria and actinomycetes. Procedures for chemical, physical, microbial, and statistical analyses of the soil samples have been described elsewhere (8). Respiratory studies were conducted with Warburg reaction flasks containing 0.15 ml of 20% KOH solution in the center wells to absorb CO2. An 8-g sample (oven-dry weight) of each soil and nematocide mixture was placed in a flask with 0.70 ml of distilled water to bring the soil to 60% of its moisture holding capacity. One hundred micrograms of glucose-C per g was added and mixed into each soil sample. Oxygen consumption was measured at 30 C for 80-hr periods with a Gilson differential respirometer. RESULTS AND DISCUSSION The plate count data obtained indicated that none of the materials tested affected the fungal population drastically, although statistically significant differences were observed in many cases ( Table 1). Because of the limitations of the plate technique for determining populations of soil fungi, the results can be taken only as an indication of what is likely to occur in the field. There was a slight depression in population after the first week with 30 jug of Vorlex per g, 600 ug of D-D per g, 5 ug of carbofuran per g, and Dasanit at both concentrations; after the second week, there was a slight depression at both concentrations of the two fumigants and with carbofuran at 5 gg/g (Table 1). An inhibitory effect was also observed in the samples treated with 5 ug of Dasanit per g and both concentrations of carbofuran after 4 weeks of incubation. All populations subsequently recovered to the level of those in the controls. The plate counts also indicated that the bacterial populations decreased significantly during the first week of incubation (Table 1) with samples treated with 5 ,ug of carbofuran per g, 120 Mg of D-D per g, 180 ug of Vorlex per g, and at both concentrations of Dansanit. Populations subsequently recovered to levels at or above those found in the controls. During the 12-week incubation period, plate counts in the controls showed a decrease in the populations of fungi and bacteria (Table 1) in the soil. These declines probably resulted from the fact that over a long incubation period aeration was inadequate, the sources of available nutrients were depleted, and waste metabolites had accumulated (2,7). In general, organisms capable of forming spores or of existing at low metabolic levels would survive (7). In the later part of the incubation period, populations in the nematocide-treated soils exceeded those found in the controls (Table 1). Trichoderma species became dominant. These species may be more tolerant to the toxic pesticides and thus, after partial sterilization of the soil, may be reestablished rapidly in the less competitive condition. It has been shown that Trichoderma species can tolerate and degrade pesticides (5). The results indicated that, in some instances, ammonium production from added peptone-N increased by 1 to 10% in the nematocide treatments ( Table 2). Similar responses with herbicides and organophosphorus insecticides have been reported (8,9). The fumigants temporarily depressed ammonification of soil organic nitrogen. Vorlex depressed nitrification of ammonium from soil organic nitrogen slightly during the first week after treatment (Table 2). However, nitrate production was equal to or better than the control after two weeks of incubation with all four nematocides. Similar responses have been demonstrated with other organophosphorus and carbamate pesticides and fumigants (1,3,10). Oxidation of added elemental sulfur ranged from 20 to 40% (Table 2). With the exception of Dasanit, the nematocides caused a slight but significant decrease in sulfur oxidation. Soil pH was not significantly altered by any of the nematocide treatments. Sulfur oxidation lowered the pH by 1.6 units, whereas ammonification of the added peptone increased pH to 9.2, thus preventing nitrification which is inhibited above pH 8 (6). The effect of the nematocides on the soil microbial population as a whole is illustrated by changes induced in respiration as a result of the nematocide treatments (Fig. 1). With the exception of Vorlex at the higher concentration, oxygen consumption from the decomposition of native organic matter was greater in the treated soils than in the controls. In addition, oxygen consumption increased with increasing concentration of Dansanit and carbofuran, both in soils with and without supplemented glucose-C. Similar results were obtained with some organophosphorus insecticides (8). Respiration was depressed with both D-D treatments and with the higher level of Vorlex in the glucose-treated soils (Fig. 1). Soil that received the lower concentration of fumigants consumed a greater amount of oxygen. The temporary depression of soil respiration by the fumigants was apparently the result of partial sterilization, as indicated by the plate counts (Table 1). This study has pointed out that the indigenous soil microorganisms can tolerate these chemicals used for control of soil nematodes. Temporary inhibition of mineralization of soil organic nitrogen, nitrification, and oxidation of elemental sulfur in soil occurred, but the microorganisms recovered rapidly. Increased respiration associated with the higher levels of Dasanit and carbofuran might result from the degradation of these chemicals by the soil microorganisms. ACKNOWLEDGMENTS I gratefully acknowledge the advice and encouragement given to me by C. R. Harris, the critical review of this manuscript by L. T. Richardson, and the technical assistance provided by G. Hietkamp.

v3-fos

2020-12-10T09:04:12.438Z

1972-10-01T00:00:00.000Z

237231252

s2

Assessment of the Sanitary Effectiveness of Holding Temperatures on Beef Cooked at Low Temperature Beef cubes cooked at low temperature were surface inoculated and incubated at 43.3 through 53.3 C to establish temperature limits for growth of staphylococci, salmonellae, and Clostridium perfringens. A greater than 99% reduction in staphylococci was achieved after 6 hr at 48.8 C, of salmonellae at 51.1 C, and of C. perfringens at 53.3 C. There were no survivors of a mixed inoculum of Staphylococcus aureus, Salmonella enteritidis, and C. perfringens after 12 hr at 51.1 C. The prevention of microbial development in prepared food has most often depended upon control of time and temperature. Among recent innovations in the food industry is the precooked product served to order by carry-out vendors. Temperature control in these operations is frequently based upon quality considerations rather than public health. One such precooked product is the sliced beef sandwich, prepared at the customer's request from beef cooked at low temperature (60 C). The cooked beef is maintained at approximately 48.8 C by commercial heat lamps for as long as 12 hr. A similar situation exists in the case of beef roasts held on conventional cafeteria serving lines. The practice of holding cooked beef for extended periods in the operations mentioned poses the question of whether enteropathogens would survive and multiply at the prevailing temperatures. Angelotti et al. (1) studied the growth of strains of salmonellae and staphylococci in custard, ham salad, and chicken a la king. They found that both groups of organisms decreased at temperatures of 46.6 C and above. Bryan and Kilpatrick (2) made a bacteriological survey of raw and cooked meat and the kitchen environment in a fast-food service operation. They isolated Clostridium perfringens from 11 of 36 samples of sliced roast beef, cooked to an internal temperature of 68.4 C or higher and held under heat lamps. They demonstrated that beef roasts are held at optimum temperatures for the multiplication of C. per-fringens for many hours in such an operation. The purpose of this study was to obtain data on the growth of staphylococci, salmonellae, and C. perfringens on roast beef when held at various temperatures. MATERIALS AND METHODS Test organisms. Test cultures, selected on the basis of incrimination in food poisoning outbreaks, included Salmonella typhimurium; S. anatum; S. enteritidis; Staphylococcus aureus MS149, 196E, and 161; and C. perfringens E-3, A86, and A91. The salmonellae and staphylococci were maintained on Trypticase soy agar (BBL), and the clostridia were maintained in cooked meat medium (Difco). Inocula for a given experiment were prepared by transferring the stock cultures of staphylococci and salmonellae to Trypticase soy broth (BBL) and the clostridia to fluid thioglycolate medium (Difco). All cultures were incubated 24 hr at 35 C. Preparation of beef. Commercial grade topround beef roasts, approximately 15 lb each, were purchased locally over-the-counter. The meat, freshly cut at the time of purchase, was brought to the laboratory, where all fat and sinew were removed. The roasts were cooked to an internal temperature of 60 C in a standard electric oven, modified to provide a maximum cooking temperature of 98.8 C. The accuracy of the oven temperature was determined by using thermocouples distributed at the bottom, top, sides, and center of an empty oven and inserted similarly into the beef roast during control and alternate experimental runs. Two "thermo-pins" (5) were placed in parallel positions in the roast in such a manner that during cooking they would direct the heat from the bottom 599 upward into the center of the roast. Cooking oil (Mazola) was poured over the surface of heavy parchment paper that lined the bottom of the cooking pan. The roast was placed on the paper, covered with an oil-soaked cloth towel, and an ordinary meat thermometer was inserted into the center of the roast. The pan containing the roast was put into the oven, which had been preheated to 98.8 C, and the roast was cooked for 3.5 hr, after which the thermometer in the center of the roast registered the desired internal temperature of 60 C. This procedure duplicates commercial practice (5), and the resulting roasts are well done externally and rare internally. Temperature studies. Each roast was removed from the oven, cooled at room temperature for 15 min, and the thermo-pins were removed. The center, rare portion of beef was cubed into 50-g samples (2inch cubes), and each cube was placed in a 9-oz wide-mouth screw-capped jar that had been equilibrated to the appropriate incubation temperature. Four jars and a control were prepared for each temperature and each organism. The upper surface of each experimental beef cube was covered with 0.1 ml of 24-hr broth inoculum of the organism to be tested. This volume yielded an initial population of approximately 4 x 104 to 4 x 105 clostridia, 3 x 106 salmonellae, or 5 x 105 staphylococci, determined by viable plate counts of broth cultures as well as of the 0-hr blended sample. Each organism was tested three times. Each jar, tightly capped, was heat-sealed inside a plastic bag and was submerged in one of a series of carefully controlled water baths set at selected temperatures between 43.3 and 53.3 C. Each strain was incubated at all temperatures in a given experiment. At 6-hr intervals for 24 hr, an inoculated jar was removed from each water bath, and the contents were transferred to a sterile Waring blendor cup to which 400 ml of sterile, cold (7.2 C) 0.1% peptone water had been added. Each jar was rinsed with an additional 50 ml of peptone water, which was then added to the blendor cup. The mixture was blended for 2 min at low speed, and appropriate tenfold dilutions were prepared from the blend in sterile 0.1% peptone water. Duplicate plates were poured from these dilutions using plate count agar (Difco) for the total aerobic counts and sodium sulfite polymyxin sulfadiazine (SPS, BBL) for the anaerobic counts. Egg-yolk medium 110 was used as a selective medium for staphylococci, and Brilliant Green sulfadiazine was used as the selective medium for salmonellae. Preliminary studies showed that plate count agar was the most effective recovery medium for staphylococci and salmonellae, and anaerobic SPS was the most effective medium for recovery of C. perfringens. Verification. Three typical colonies from each medium were confirmed biochemically. The control, or uninoculated, meat sample was also tested for any growth or contamination, in the same manner as the inoculated samples. None of the uninoculated meat samples yielded any significant bacterial growth. In one experiment, 0.1 ml of inoculum of a representative strain from each bacterial group (S. aureus 161, S. enteritidis, and C. perfringens E3) was added to a 50-g cube of beef to approximate natural mixed contamination. Only temperatures between 51.1 and 53.3 C were used, because the individual strains of salmonellae and C. perfringens had survived the 48.8 and 50-C exposure temperatures in earlier experiments. RESULTS The results of this study are compiled in Table 1. Each value tabulated represents the mean of three separate analyses. The resistance of individual strains of each of the three groups to the temperatures tested was quite similar. The growth of S. aureus MS149, the most resistant of the staphylococci, is shown in Fig. 1. The organisms grew well at 43.3, 44.4, and 45.5 C. At 46.6 C, a slow decrease in numbers took place during 24 hr. A greater than 99% reduction in staphylococci occurred after 6 hr at 48.8 C. Some survivors were detected after 12 hr, but none after 18 hr. Salmonellae grew well at all temperatures below 48.8 C, requiring that this species be tested at higher temperatures. The growth of S. typhimurium at temperatures between 48.8 and 53.3 C is shown in Fig. 2. At 48.8 C, a slight increase occurred after 6 hr, a decrease of greater than 90% after 18 hr, and greater than 99% after 24 hr. A greater than 99% reduction in viable organisms occurred in 18 hr at 50 C, in approximately 6 hr at 51.1 C, and in less than 6 hr at 53.3 C. The growth of C. perfringens A86 at the higher temperatures is shown in Fig. 3. The organisms grew extremely well during the first 12 hr at 48.8 C, and declined sharply after 18 and 24 hr. The number of viable organisms increased 90% or more by 6 hr at 50 C and by 12 hr at 51.1 C. A subsequent reduction of approximately 90% or more was exhibited by 18 hr at both temperatures. A greater than 99% reduction was achieved in less than 6 hr at 53.3 C. The growth responses of a representative strain from each of the genera inoculated together onto beef cubes and held at 51.1 C are shown in Fig. 4. No S. enteritidis or S. aureus 161 survived after 6 hr at 51.1 C; a few C. perfringens E-3 cells survived 6 hr at this temperature, but none remained after 12 hr. No survivors of the three genera were found after 6 hr at 52.2 and 53.3 C. 13,000,000 450,000 <300 <300 a Total counts for staphylococci and salmonellae in plate count agar, and Clostridium in sulfadiazine polymyxin sulfite agar. DISCUSSION The results of this study show that the strains of staphylococci, salmonellae, and C. perfringens used in this study are capable of growing on the surface of roast beef at temperatures as high as 43.3 to 45.5 C. Death of staphylococci took place in 18 hr at 48.8 C. The population of salmonellae decreased rapidly above 48.8 C. Destruction of C. perfringens occurred above 51.1 C. Strains representing all three groups in a mixed inoculum were destroyed in less than 12 hr at 51.1 C. It is apparent that the growth curves which resulted when all three genera were added to beef cubes simultaneously (Fig. 4) represent a more dramatic decline than when the organisms were tested alone. No Salmonella or Staphylococcus, and only a. few Clostridium, survived 6 hr at 51.1 C in a mixed inoculum. When tested individually, the genera showed sharp decreases at 6 hr, but significant numbers of survivors were still present at this temperature. One explanation that could be offered for this difference in resistance is competitive inhibition. The metabolic activities of a mixed flora inhibit the growth of individual species in the population. Recently, Speck (6) offered another illustration of inhibitory competition when he studied the interactions among commercial lactic streptococci and different foodborne pathogens. When inoculated alone into sterile, reconstituted, nonfat milk solids, Salmonella gallinarum grew rapidly. However, when a mixed-strain starter culture was present, growth was depressed remarkably. The cooking process eliminated all significant contamination on the surface of the roast beef. The general distribution of the three groups of organisms in the environment, however, would facilitate recontamination. Furthermore, the continued handling of a vended product such as roast beef would transfer enteropathogens from human sources to the food. When conditions of incubation, time, and temperature are proper, these cells multiply and produce toxins. The recommended temperature for holding potentially hazardous foods in food-service operations, in vending machines, and aboard aircraft is 60 C (3,4). Our data on C. perfringens, staphylococci, and salmonellae substantiate the earlier studies of Angelotti et al. (1) and Bryan and Kilpatrick (2) and suggest that reducing the holding temperature of 60 C for beef served to order is possible without endangering the public health from growth of staphylococci, salmonellae, or C. perfringens.

v3-fos

2018-04-03T02:32:02.521Z

1972-09-01T00:00:00.000Z

33941198

s2

Enterobacteria in feedlot waste and runoff. Samples of beef cattle feedlot waste (FLW), runoff from the pens, and water from a large drainage ditch at the feedlot were examined for Enterobacteriaceae. The drainage ditch receives the runoff but contains primarily subsurface drainage from fields on which FLW is spread for disposal. Planting and enrichment techniques with seven different media were used to isolate 553 cultures of enterobacteria. FLW contains about 50 million enterobacteria/g dry weight. More than 90% of these were Escherichia coli, none of which were enteropathogenic types as determined with multivalent sera. Citrobacter and Enterobacter cloacae were other organisms present in moderate numbers. Application of enrichment techniques broadened the spectrum of enterobacteria isolates to include the four Proteus spp., both Providencia spp., Klebsiella, Enterobacter aerogenes, Arizona, and a single isolate of Salmonella (serological group C(2)). Shigella was not isolated. The wide spectrum of enterobacteria in FLW may be a hazard if unsterilized waste is refed. Fewer enterobacteria occurred in the runoff and in the drainage ditch; the most numerous species in FLW also were most numerous at these sites. However, neither Salmonella nor Arizona was isolated from runoff or drainage-ditch waters. Samples of beef cattle feedlot waste (FLW), runoff from the pens, and water from a large drainage ditch at the feedlot were examined for Enterobacteriaceae. The drainage ditch receives the runoff but contains primarily subsurface drainage from fields on which FLW is spread for disposal. Plating and enrichment techniques with seven different media were used to isolate 553 cultures of enterobacteria. FLW contains about 50 million enterobacteria/g dry weight. More than 90% of these were Escherichia coli, none of which were enteropathogenic types as determined with multivalent sera. Citrobacter and Enterobacter cloacae were other organisms present in moderate numbers. Application of enrichment techniques broadened the spectrum of enterobacteria isolates to include the four Proteus spp., both Providencia spp., Klebsiella, Enterobacter aerogenes, Arizona, and a single isolate of Salmonella (serological group C2). Shigella was not isolated. The wide spectrum of enterobacteria in FLW may be a hazard if unsterilized waste is refed. Fewer enterobacteria occurred in the runoff and in the drainage ditch; the most numerous species in FLW also were most numerous at these sites. However, neither Salmonella nor Arizona was isolated from runoff or drainage-ditch waters. Cattle feedlots represent a serious, but largely undocumented, pollution hazard. Most studies on feedlot waste (FLW) concern disposal methods and movement of nutrients into surface and subsurface waters through runoff and percolation. Currently, refeeding is being investigated as a means for combating the increasing accumulation of waste from intensive animal production. Surprisingly little attention has been paid to the microbiological aspects of either FLW or runoff. We have enumerated and categorized the microflora of FLW and associated sites (10). Gram-negative bacteria were the third most numerous group of organisms encountered; coliform counts were approximately 1 x 107/g dry weight of FLW and slightly less than 1 x 105/ml of runoff. These counts varied only slightly during 1 year. A brief report by Witzel et al. (12) gave coliform counts of 5 x 105/g wet weight in cattle manure, of which more than 95% were "typical" Escherichia coli. In an overall examination of coliforms in warmblooded animals, Geldreich et al. (6) found a similar percentage of E. coli present when they examined strains from cattle manure by the IMViC tests. Miner et al. (9) isolated Salmonella infantis from litter and runoff at two feedlots, and the investigation by Bromel et al. (1) demonstrated transfer of antibiotic resistance from enteric bacteria of farm animals to those from human sources. The problem of identifying pollution from FLW in surface waters has led to the suggestion that Streptococcus bovis might be a better indicator than coliform organisms (8). We evaluated the Enterobacteriaceae in FLW and runoff because methods for dealing with animal wastes must include consideration of potential health hazards. MATERIALS AND METHODS Samples. Samples were collected in July from a cattle feedlot in central Illinois capable of sustaining 5,000 to 10,000 animals at a time. A detailed description of this commercial feedlot is given by Rhodes and Hrubant (10). Four types of samples were collected. (i) Composite FLW: 12 specimens of 3 to 5 g each were taken from scattered sites in two adjacent animal pens and combined to yield a 50to 60-g composite sample. (ii) Individual FLW: 10 fresh manure deposits in seven different pens were individually sampled with paired sterile swabs for enrichment procedures. (iii) Runoff: multiple dips from a small drainage ditch adjacent to the pens were combined to yield a single 100-ml sample. (iv) Field ditch: a combined sample of 500 ml taken by multiple dip from the intersection of two drainage VOL. 24,1972 ENTEROBACTERIA IN FEED ditches located about .5 mile from the pens. FLW had been spread on adjacent cornfields for several years. Runoff from the pens also emptied into this field ditch at the sample site via the drainage ditch of sample iii. All samples except sample ii were stored in cracked ice until analyzed in the laboratory within 4 hr of collection. Plate counts and FLW isolates. The composite FLW sample i was diluted 1:3 (w/v) with sterile water and blended for 30 sec in a Waring Blendor. A 40-ml portion was then diluted with 60 ml of sterile 0.1% tryptone (Difco) to give a 1:10 dilution. Subsequent 10-fold dilutions were made with 0.1% tryptone; the 90-ml dilution blanks contained glass beads to aid dispersion. Colony counts were made from triplicate spread plates inoculated with 0.3 ml/plate; four dilutions were spread on each medium. Eosin methylene blue agar (EMB), deoxycholate lactose agar (DCL), sorbitol agar (Sorb), bismuth sulfite agar (BS), and Salmonella-Shigella agar (SS) were the plating media. All colonies on plates of the proper dilution were counted after incubation for 18 to 24 hr at 37 C. All colonies from one, two, or three plates of the countable dilution of each plating medium except EMB, selected to total 75 to 100 colonies, were transferred to Kliger's iron agar (KIA) slants. Of 250 colonies on EMB, 105 were transferred to KIA; the remaining 145 were pinpoint colonies and were not transferred to KIA. A total of 452 colonies were transferred to KIA from the count plates; from these, 352 isolates were obtained. Isolates from enrichment cultures. Portions (10 ml) of composite FLW (1:10 dilution of sample i), runoff liquid (sample iii), and ditch water (sample iv) were added to 10 ml of double-strength brilliant green bile broth (BGB) and to selenite cystine broth (SC). Swabs of the individual FLW samples ii were added to BGB and to SC at the sampling sites. After incubation for 18 to 24 hr at 37 C, loopfuls of the BGB enrichment cultures were streaked on Sorb while the SC enrichments were streaked on BS and SS. The streak plates were incubated for 24 hr at 37 )LOT WASTE AND RUNOFF 379 C, and colonies from each plate then were transferred to KIA. Ten colonies from each streak plate medium were transferred to represent the enrichment cultures of composite FLW, runoff, and ditch water (90 isolates). Each of the 10 individual FLW swab samples was represented by six subcultures on KIA from each streak plate medium (180 isolates). KIA and primary screen. After the KIA slants were incubated for 18 to 24 hr, the isolates were grouped by their acid, gas, and H2S reactions. The media and tests used for subsequent differentiation of the Enterobacteriaceae were selected from those of Ewing and Davis (5). Cultures on KIA that were alkaline or exhibited no growth in the stab were transferred to pigment-enhancing media, glucose broth, and a repeat KIA test. These organisms were characterized no further. Presumed enterobacteria were all subjected to the following primary screen: indole production and motility on SIM medium; methyl red; Voges-Proskauer (VP) using the Barritt method for acetyl methyl carbinol; citrate utilization (Simmon's); urease production by the liquid method of Stuart et al. as cited by Ewing and Davis (5); mannitol fermentation; and growth in potassium cyanide broth (Difco). Secondary tests. The isolates were regrouped after results of the primary screen were collated. Additional tests were selected for these groups from among the following: lysine and ornithine decarboxylase; arginine dehydrolase; phenylalanine deaminase; fermentation of sucrose, dulcitol, salicin, inositol, sorbitol, arabinose, rhamnose, arabinose plus dulcitol, and adonitol plus inositol plus sorbitol. All cultures were also checked for nitrate reduction in fluid medium with inverted insert vials. In a few instances, tests from the primary screen were repeated. Production of gas from glucose and the lactose fermentation were confirmed in carbohydrate fermentation media; urease was checked on Christensen's urea agar; and the VP, citrate, and potassium cyanide tests were repeated by the original techniques. Cultures were identified by their bio- aIn Tables 2-6, a blank space means that the specific organism was not isolated. For abbreviations, see footnote to Table 1. chemical reactions and serotyping in accordance with the schema of Ewing (3,4). The carbohydrates used were from Sigma (St. Louis) or Difco (Detroit); all other media and media components were from BBL (Division of BioQuest, Cockeysville, Md.), except where noted. RESULTS AND DISCUSSION FLW contains between 4.4 and 6.8 x 107 enterobacteria/g dry weight (Table 1). These values represent only those cultures shown to be enterobacteria by biochemical tests. Total plate counts on BS and SS were about 100-fold lower than counts obtained on EMB, DCL, or Sorb. On these three media, total counts varied from 1.1 x 101 organisms/g on EMB to 3.6 x 108 on DCL. Pinpoint colonies and those that did not grow on transfer to KIA accounted for about 60% of colonies on EMB and DCL but only about 16% of those on Sorb. About 50% of the isolates from DCL and Sorb were demonstrably not enterobacteria by their reaction on KIA; in contrast, enterobacteria represented more than 90% of the isolates from EMB. The numbers of enterobacteria per gram of FLW, as calculated from plate counts on EMB, DCL, and Sorb, correspond to undifferentiated counts on EMB of cecal contents in cattle fed high-roughage diets (7). More than 90% of the enterobacteria in FLW were E. coli (Table 2). Citrobacter and Enterobacter cloacae were also isolated from EMB. Single isolates of Proteus mirabilis and E. cloacae were obtained from DCL and Sorb, respectively. The inability of E. coli to grow on BS and SS is largely responsible for the difference between the total plate count on these media and that on EMB, DCL, and Sorb. Inhibition of most of the E. coli permitted isolation of Enterobacter aerogenes, Klebsiella, Providencia stuartii, and the four species of Proteus from BS and SS count plates. Between 105 and 106 of these organisms occur per gram of FLW. Enterobacter species are the a Swabs from 10 individual feedlot waste samples incubated for 18 to 24 hr at 37 C in first designated medium; a loopful of this growth streaked on second medium and incubated for 24 hr at 37 C. Six colonies from each streak plate transferred to KIA as primary isolates subsequently characterized (180 total isolates). For abbreviations, see footnote to Table 3. O of 60 isolates picked from streak plates, number characterized as indicated. most numerous of the enterobacteria outside of the E. coli and Citrobacter groups; Proteus species are somewhat less abundant than are Enterobacter. EMB appears to be the best single medium for enumeration of coliform organisms in FLW and related sources. Both BS and SS agar are also required for determination of those enterobacteria present in small numbers compared to E. coli. The results of enrichment culture techniques applied to the composite FLW (sample i) and to 10 individual FLW samples (sample ii) from seven pens are given in Tables 3 and 4, respectively. BGB enrichments, plated on Sorb, were used in attempting to isolate pathogenic E. coli. None were found although all sorbitolnegative isolates were tested with polyvalent OB sera which detect the 10 serotypes of E. coli most often implicated in infantile diarrhea. Salmonella and Shigella were sought in SC enrichments plated on BS and SS. One Salmonella group C2 was isolated from FLW. Polyvalent 0 and group C2 antisera confirmed the generic biochemical identification. S. typhimurium (group B) and S. newport (group C2) appear to be the Salmonella most frequently isolated from cattle (2,11). S. infantis (group C1) was isolated from the litter and runoff at two experimental feedlots by Miner et al. (9). Several isolates from FLW were biochemically identified as Arizona strains. Neither Shigella spp. nor members of the alcalescens-dispar group were isolated. Although no numerical evaluation can be made from the enrichment procedure, frequency of isolation confirms the overall abundance of the types of enterobacteria found by plating. Enrichment cultures of runoff and of field ditch water were similarly checked for enteric pathogens (Table 5). None were isolated. The infrequency of isolation indicates that E. coli does not survive well in these waters. Counts done on the runoff and ditch water show few coliforms compared to the numbers encountered in FLW (10). Table 6 summarizes the classification of all isolates studied. The percentage of coliforms that are E. coli is similar to that reported previously in bovine feces (6,12). Although E. coli constitutes more than 90% of the total enterobacteria in FLW, its poor survival in related waters indicates that it may have limited value as an indicator of pollution from feedlots. Middaugh (8) has suggested S. bovis as an indicator of pollution from bovine sources. The presence of a broad spectrum of other enterobacteria in lesser numbers was demonstrated by enrichment culture techniques. Since these organisms, particularly the Proteus species, have poor assimilative capacity, they probably have a subordinate role in the degradation of FLW. However, coliforms and other enterics including Proteus and Klebsiella isolated from animal waste and a waste treatment lagoon were shown to be a potentially hazardous source of transferable R-factors carrying multiple antibiotic resistance (1). The isolation of a single Salmonella supports the position that agricultural wastes do have public health implications (2,9,11). The occurrence of a wide spectrum of enterobacteria also should give pause to proposals for indiscriminate refeeding of unsterilized FLW as a method of utilizing this waste. a Swabs incubated for 18 to 24 hr at 37 C in first designated medium; a loopful of this growth streaked on second medium and incubated for 24 hr at 37 C. Ten colonies from these streak plates transferred to KIA as primary isolates subsequently characterized. For abbreviations, see footnote to Table 3. O of 10 isolates picked from streak plate, number characterized as indicated.

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2018-04-03T05:57:49.753Z

1972-06-01T00:00:00.000Z

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Recognition of group D streptococcal species of human origin by biochemical and physiological tests. The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species. The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species. Shattock (19) in 1962 and Deibel (2) in 1964 presented classification schemes for group D streptococci that included the two nonenterococcal group D streptococci, Streptococcus bovis and S. equinus. The inclusion of S. bovis and S. equinus into the group D classification is based on the demonstration of the group D antigen in extracts of cells of these two species (17,20). Shattock (19), Deibel (2), and Hartman et al. (9) have also included S. faecium in their classification schemes. The results of studies by Barnes (1), Deibel et al. (3), and Shattock (17) clearly show that S. faecalis and S. faecium are distinct species. The terms "fecal streptococci," "enterococci," and "group D streptococci" are discussed by Hartman et al. (9). It is now evident that these terms are not synonymous. The term fecal streptococci has no definitive meaning and should not be used. Various investigators use the term to describe many different streptococcal species of fecal origin (9). The term enterococci may be defined as including S. faecalis and its varieties (zymogenes and liquefaciens), S. faecium, and S. durans. Group D streptococci may be defined as all those streptococci possessing the group D antigen. This includes all the enterococcal species plus S. bovis and S. equinus. It is probably advisable to retain the use of the term enterococcus be-cause of the difference in antibiotic therapy for patients with enterococcal infections and those with nonenterococcal infections. Clinical studies which have demonstrated multiple antibiotic resistance have been done on enterococci and not on the entire group D streptococci. Even though the term has no real taxonomic meaning, it does have clinical significance. A review of the literature on speciation of group D streptococci isolated from human sources will not be attempted but the reader is referred to early reviews by Thomson and Thomson (21), Dible (4), Graham and Bartley (8), and an excellent review by Evans and Chinn (6). Earlier classification schemes (before 1962) did not include the species S. faecium, S. bovis, or S. equinus. Recently, during a study of antibiotic resistance of human isolates, Toala et al. (22,23) speciated the enterococci, and included S. faecium in their identification procedures. Although they did not include the two nonenterococcal group D species (S. bovis and S. equinus) in their identification scheme and although the number of differentiating tests was somewhat limited, their interpretations appear sound. Pleceas (16) advocated the use of a limited number of differential tests to recognize all the group D species. She included phage typing as a means of dif-ferentiating between species and recommended that atypically reacting strains be recognized as separate entities. Duma et al. (5) did not feel that a limited number of tests would sufficiently speciate the group D streptococci, but they did feel that a few tests would adequately place them into the division set forth by Deibel (2). Isenberg et al. (10) attempted speciation of group D streptococci, but failed to incorporate sufficient tests into their scheme to differentiate the species accurately. Food and water microbiologists have been using the identification procedures summarized by Shattock (19) and Deibel (2) for several years, and many publications have appeared showing the successful speciation of food, water, and fecal isolates. It is the purpose of this investigation to demonstrate that speciation of group D streptococci isolated from clinical material can be accomplished if the recommendations of Deibel (2) are heeded. Deibel emphasized that, in all identification procedures, dependence must be placed on a spectrum of characteristics possessed by the strain in question, and its failure to comply in a few specific tests does not constitute sufficient grounds to negate speciation. Deibel also cautioned against the attempt to establish species on the basis of a few differing characteristics and emphasized the need to recognize transitional strains as types. Accurate speciation procedures are necessary to determine the species distribution of group D streptococci in human infection and to determine differences among species in regard to their susceptibility to various antibiotics. MATERIALS AND METHODS Cultures. A total of 262 strains of group D streptococci isolated from the clinical sources listed in Table 1 were tested. These strains were received at the Center for Disease Control (CDC) between July 1968 and September 1971 and constituted all the group D streptococcal isolates from human sources excluding feces. An additional 6 S. faecalis, 39 S. faecalis var. zymogenes, 5 S. faecalis var. liquefaciens, 9 S. faecium, 14 S. faecium var. casseliflavus, 12 S. durans, 16 S. bovis, 18 S. equinus, and 6 S. avium (group Q streptococci) stock strains were speciated to determine test proficiency. The reactions of these stock strains are not included in the following tables. The majority of stock strains of the group D streptococci were obtained from R. L. Lancefield, S. D. Elliot, and J. 0. Mundt. Preparation of media. The procedures for serological grouping; Gram staining; determining hemolytic and catalase activities; and testing for reactions on bile-esculin medium (BEM), growth in 6.5% NaCl broth, acid reaction in Streptococcus faecalis broth (SF, Difco), and growth in 0.1% methylene blue milk (MBM) have been previously described (17). All of the following media were sterilized by autoclaving for 15 min at 15 psi (121 C) unless otherwise stated. All pH measurements were conducted with a pH meter. Growth at 10 and 45 C was determined by inoculating 5 ml of Todd-Hewitt broth (THB, Difco); after incubation the tubes were examined for growth by rotating them in front of an incandescent lamp. Blood-bile-agar was prepared by adding 40 g of oxgall (Difco) per liter of HIA. A 50-ml amount of defibrinated rabbit blood was added to 1 liter of the sterilized cooled medium just before the plates were poured. Tolerance to 40% bile was determined by examining the plates for growth daily for 3 days. The tetrazolium plates were prepared by adding 5 g of dextrose per liter of HIA. The mixture was adjusted to pH 6.0 by adding 1 N HCL. Ten milliliters of a 1% solution of 2,3,5-triphenyl tetrazolium chloride, sterilized by filtration, was added to the sterilized, cooled medium (50 C) just before the plates were poured. A positive reduction of the tetrazolium was recorded when examination of the plates revealed brick-red colonies at any time interval within 3 days. The base tellurite medium was prepared by adding 1 N HCl to 1 liter of HIA to pH 6.0. Fifty milliliters of defibrinated rabbit blood was added to the sterilized medium at 90 C. The potassium tellurite solution (0.5 g of K-tellurite per 150 ml of distilled water sterilized by filtration) was added to the cooled medium (less than 50 C) just before the plates were poured. Tolerance to tellurite was recorded as positive when examination of the plates revealed black colonies after any time interval up to 3 days. The starch agar was prepared by adding 20 g of soluble starch (Merck) per liter of HIA. The medium was sterilized and poured when cooled. Hydrolysis of starch was determined by flooding the surface of the plate with Gram's iodine 48 hr after inoculation and incubation. A zone of hydrolysis appeared colorless, and a dark blue to purple color indicated that the starch had not been hydrolyzed. The sucrose plates were prepared by adding 50 g of sucrose per liter of HIA. The medium was sterilized and poured when cooled. Plates were examined daily for 3 days for large gummy colonies and colonial adherence to the media characteristic of extracellular polysaccharide production. The medium for determining pH 9.6 tolerance was prepared by adding NaOH pellets to Heart Infusion Broth (HIB, Difco) until a pH of 9.6 was reached as indicated by a pH meter. The medium was dispensed in 3-ml amounts into screwcap tubes (13 by 100 mm) and sterilized. Tolerance was determined by observing the medium for growth daily for 3 days by rotating the tube in front of an incandescent lamp. The medium for determining hydrolysis of gelatin was prepared by adding 120 g of gelatin to 1 liter of HIB (11). The medium was dispensed in 5-ml amounts into 15 by 125 mm screwcap tubes and sterilized. Hydrolysis of the gelatin was determined after incubation for 3 days by refrigerating (10 C) the growth tube and an uninoculated control tube. When the control tube had solidified, the tubes were removed and inverted. Tubes that remained unsolidifled were recorded as positive, all others as negative. Litmus milk (Difco) was prepared and dispensed in 10-ml amounts into cotton-stoppered tubes (15 by 150 mm) and sterilized. The reactions of acid production and clotting of litmus milk were noted daily for 3 days. Heart infusion broth (HIB) was used as the base medium for 1% broths of sucrose, raffinose, mannitol, inulin, lactose, esculin, sorbitol, glycerol, and arabinose. One hundred milliliters of a 10% solution of each carbohydrate and 1 ml of indicator solution (1.6 g of bromcresol purple in 100 ml of 95% ethanol) were added to 900 ml of HIB. The medium was dispensed in 3-ml amounts into screwcap tubes (13 by 100 mm). The medium was sterilized by autoclaving for 10 min at 15 psi. A positive reaction was recorded when the indicator changed from purple to yellow, which indicated that acid had been produced. Recordings were made daily for 3 days. The 5% sucrose broth was prepared by adding 28.5 g of dehydrated thio broth (Difco), 10.0 g of K2HPO4, and 12.0 g of sodium acetate to 500 ml of distilled water. This solution was autoclaved separately as was 50 g of sucrose in 500 ml of distilled water. After sterilization, the solutions were mixed and dispensed into screwcap tubes (16 by 125 mm). The medium was checked daily for 3 days for an increase in viscosity. All media were inoculated with a Pasteur pipette that dispensed one to two drops of a 24-hr THB culture of the specimen being tested. Media were incubated at 35 C aerobically unless otherwise indicated. RESULTS Speciation by a spectrum of physiological tests. Table 2 shows the reactions used to speciate the group D isolates. This table is compiled from various publications (1,2,3,7,9,14,15,18,19) and from our own results with 125 stock strains representing all species of group D and Q streptococci. All strains studied were gram-positive cocci and varied in chain length. All strains studied failed to release 02 from H202 with the exception of half of the S. faecium var. casseliflavus strains. Very weak reactions were observed with most of these strains, and at least two of them, upon several laboratory transfers, lost the ability to release 02 from H202. Thus this was not considered a stable characteristic of the species. For a strain to fit the speciation scheme perfectly, all the reactions had to agree with those listed in Table 2. However, there are many variants within a species, and, to best place the organism into a particular species, a spectrum of reactions was used. VOL. 23,1972 S. faecalis (division I) and its varieties, zy-flavus and S. avium in division II because of mogenes and liquefaciens, are differentiated by certain similarities to S. faecium. The variety hemolytic activity and gelatinase production. casseliflavus is a relatively poorly definable All other characteristics of these taxons are species in our system. The species shares charsimilar in our system. There is very little difacteristics of both division I and II. The posiference between these three taxons, and it is tive reactions on tellurite and tetrazolium doubtful that they need to be treated as sepa-media indicate that the organisms belong to rate entities. Other investigators have noted division I, but the negative reaction in sorbitol that the hemolytic action of S. faecalis var. and positive reaction in arabinose broths are zymogenes on blood-agar depends on the kind characteristics of S. faecium (division II). The of blood used in the pour plates (6,12,24). We casseliflavus strains produced a characteristic have observed this same phenomenon with yellow-pigmented, mucoidal colony on 5% susome of our isolates. By definition, S. faecalis crose agar, whereas all other members of diviis not beta-hemolytic and does not liquify gel-sion I and II were white (nonpigmented) and atin; S. faecalis var. liquefaciens is not beta-mucoidal on the same media. We failed to hemolytic but liquefies gelatin; and S. faecalis identify any strains from our collection which var. zymogenes is beta-hemolytic and may or fit this particular spectrum of reactions. may not liquefy gelatin. We also failed to identify strains resembling We arbitrarily placed S. faecium var. casseli-S. avium in our collection from human sources although they are easily recognized in our system. All of our division II species grew in MBM and all but three grew at 10 C (Table 3). None of our stock strains of S. avium was able to grow in MBM or at 10 C. The failure to initiate growth in litmus milk and to form acid in lactose broth are characteristics unique to S. equinus in the group D streptococci. We feel that these characteristics are distinctive and make the species easily recognizable. We failed to recognize any strains resembling S. equinus in our collection from human sources. Recognition of streptococci as members of group D. Table 3 shows the percentages of positive reactions of the various species of group D streptococci in our collection. Extracts of four S. durans, two S. bovis, and one S. faecium failed to react with CDC group D antisera. All of these strains showed typical reaction patterns of their respective species described in Table 2. One strain failed to blacken BEM. This strain failed to form acid in any carbohydrate (CH) broth tested, including esculin, but was still classified as a S. faecalis var. liquefaciens by the remaining tests. Growth on 40% bile, growth at 45 C, and acid from esculin are not properties unique to group D streptococci but are characteristics shared by the majority of strains of all group D streptococcal species. The results of the MBM-tolerance test support our previous contention (7) that MBM should not be used as a differential test for enterococci or group D streptococci. Although nearly all of the enterococcal strains (divisions I and II) reduced MBM, 60% of the nonenterococcal strains (division Ill) were able to reduce MBM. This variability of division III strains to reduce MBM limits the usefulness of the test to differentiate accurately either enterococci or group D streptococci from other streptococci. It does serve as a useful test in recognizing S. avium strains as previously discussed. Placement of strains into divisions and species. The ability of enterococci (divisions I and II) to grow in SF broth and in 6.5% NaCl broth and to initiate growth at 10 C differentiates enterococci from nonenterococcal (division III) group D streptococci. Table 3 shows that very few strains of division III give positive reactions in these three tests. Table 3 also shows that pH 9.6 broth did not clearly differentiate between enterococcal and nonenterococcal group D species; however, the pH of this medium was adjusted before rather than after autoclaving, and this may have affected the results. We did not rely on this test to establish placement of the organisms into division categories. The reduction of tetrazolium, resistance to tellurite, and acid production in sorbitol and glycerol broths are characteristics shared by division I organisms. Division II species (faecium and durans) ordinarily fail to give positive reactions on these tests, but exceptions do occur ( Table 3). The same exception occurs with acid from arabinose by S. faecium: S. faecium routinely forms acid in arabinose, whereas most other group D species do not. Table 3 shows that only 78% of division I specimens formed acid in glycerol broth within 3 days. This was not totally unexpected since the test was designed as an anaerobic test and we ran all our tests aerobically. We did not rely on acid from glycerol to place the organisms into division I. Acid production from mannitol, arabinose, and sucrose and failure to clot litmus milk are tests generally considered to differentiate S. faecium from S. durans. Acid from mannitol and arabinose clearly demonstrated this capacity, but 10 of 13 strains that were mannitoland arabinose-negative were sucrose-positive. By our system, these strains were closer to S. durans than S. faecium. We do not feel that they should be recognized as distinct entities but rather that they should be considered as a biotype within S. durans. Clot formation in litmus milk was not used to differentiate between S. faecium and other group D species. The test was unreliable because many strains of faecium clot litmus milk and some strains of faecalis and durans do not ( Table 3). The division III organisms are recognized by the failure to grow in SF broth at 10 C and in 6.5% NaCl broth. Table 3 shows that the hydrolysis of starch and slime formation in 5% sucrose broth and agar are unique characteristics of S. bovis among the group D isolates. We recognized a variant in division III organisms that did not hydrolyze starch, form slime in 5% sucrose broth or agar, or form acid in mannitol as did nearly all the S. bovis strains. These seven variant strains were all very similar to one another and were easily distinguished from typical S. bovis. We feel that they may be a separate species or a biotype of S. bovis. These isolates are unlike S. equinus which fails to grow in litmus milk or form acid in lactose broth. All seven strains were positive in both tests. Table 4 shows the distribution of the 262 strains according to the speciation scheme in Table 2 and the number of exceptional reactions that occurred. The reactions listed as variable were not counted for this data. The formation of a clot in litmus milk, the formation of acid from glycerol, and growth in pH 9.6 broth were considered unreliable tests for speciation under our conditions and were not used for speciation or counted in the exceptional reactions. Table 4 shows that nearly 73% of the specimens fit the spectrum of reactions for the species perfectly, whereas 20% had one exceptional reaction. Therefore, of the specimens tested, almost 93% had one or no exceptional reactions according to our spectrum of reactions in Table 2. Fourteen specimens had two exceptional reactions, one had three, three had five, and one had six. The correct identity of the specimen with six exceptional reactions may be questioned, but we believe that the other 261 strains have been correctly identified. The three strains with five exceptional reactions were similar to one another. Most of the exceptional reactions of these three strains were the result of the failure to form acid in CH broths. DISCUSSION Significance of the isolates. Clinical information on some of these isolates is not available, but a significant portion of the clinical information was provided with many of the isolates. Although these specimens are not a random sample of isolates from the United States, we believe that they represent a distribution of species of group D streptococci that is found in human infections. This supposition is supported by the fact that 54% of the specimens were blood isolates; 39% of these were from subacute bacterial endocarditis. It is also significant that in our series of subacute bacterial endocarditis (SBE) isolates from the Mayo Clinic (J. Washington), 9 of 48 (19%) group D isolates were S. bovis or S. bovis variants. In our series from the Cleveland VA Hospital (P. I. Lerner), 6 of 25 (24%) group D isolates were S. bovis. This high percentage of nonenterococcal group D isolates demonstrates the necessity of correct identification of enterococci for the best management of these infections. Recognition of group D and enterococcal streptococci. Group D streptococci are defined as those streptococci possessing the group D antigen. Other investigators (17,20) have demonstrated that S. bovis and S. equinus possess the group D antigen and recommended that these two species be included in the group D classification. We have previously shown that the BEM reaction correlates with the group D serological reaction (7). Identification of group D streptococci can be presumptively made by the BEM reaction and definitively made by demonstrating a serological group D reaction. The inclusion of S. bovis and S. equinus into the group D classification (2,9,19) and the occurrence of S. bovis strains in human infections (Table 1) indicates that the clinical laboratories cannot use the serological or BEM reactions to establish enterococcal identification. Twenty percent of the group D isolates in our collection were S. bovis, all were BEM positive, and 96% reacted with group D antisera. A significant number of misidentifications would result if either the BEM or group D reactions were used as indicators of enterococci. Only limited evidence is available but apparently S. bovis strains do not resemble enterococci in their susceptibility to antibiotics (25). S. bovis infections probably do not require the intensive treatment required by enterococcal infections. Mundt and Graham (14) described a new streptococcal species, S. faecium var. casseliflavus. We were able to demonstrate a satisfactory group D precipitin reaction with Lancefield extracts of all 14 of their strains. Although Isenberg et al. (10) felt that several of their isolates resembled S. faecium var. casseliflavus, they failed to perform a sufficient number of tests to identify accurately any of their group D isolates. Although S. faecium var. casseliflavus physiologically resembles S. faecalis in some respects and S. faecium in others, we feel that the species can be recognized by the set of physiological characteristics listed in Table 2: pigment production and release of 02 from H202 by some of the strains. This spectrum of characteristics was not observed among any of the human isolates reported here. Nowlan and Deibel (15) described the physiological and serological characteristics of S. avium (group Q streptococci). We were able to demonstrate weak group D reactions in Lancefield extracts with five of six strains of S. avium (7). This serological reaction was very weak and required more than the usual 30-min time limit to develop. Lancefield extracts of S. equinus stock strains reacted in the same manner. We would not have accepted these reactions as bona fide group D reactions in extracts of diagnostic strains of streptococcal isolates. Increased group D precipitin reactions (both tube and gel diffusion) were reported by Nowlan and Deibel (15), who used a method of concentrating Lancefield extracts of S. avium. The physiological characteristics of S. avium are very similar to those of S. faecium. However, failure of S. avium to tolerate MBM and the formation of acid in sorbitol broth differentiates the species from S. faecium. All of our strains of S. faecium grew in MBM and only two fermented sorbitol. The three strains of S. faecalis that failed to gr6w in MBM did not resemble S. avium. These three S. faecalis isolates tolerated tellurite. S. avium does not tolerate tellurite. Recognizing S. equinus is not difficult. The species has two very distinctive physiological characteristics-failure to grow in litmus milk and failure to form acid in lactose broth. Like S. faecium var. casseliflavus and S. avium, strains resembling S. equinus were not encountered among our 262 human group D isolates. Thus, to differentiate between enterococcal and nonenterococcal streptococci, laboratory personnel need to perform additional tests after they have established presumptive or confirmatory identification of a streptococcal isolate as a group D streptococcus. The results show that growth at 10 C, in 6.5% NaCl broth, or production of acid in SF broth will provide an acceptable differentiation between members of division I and II (enterococci) and those of division III (nonenterococci).

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Dry Heat Inactivation of Bacillus subtilis var. niger Spores as a Function of Relative Humidity Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Dry heat is the principal process being considered for spacecraft sterilization by the National Aeronautics and Space Administration. It is known that, at a given temperature, Dvalue (time to sterilize 90% of a population) varies with air moisture content (3). However, there is a paucity of data available for setting spacecraft sterilization cycles as a function of relative humidity. This is a report on dry heat sterilization of Bacillus subtilis var. niger spores at relative humidities (RH) from 0.0033 to 1.67% at oven temperature. 105 C D-values were determined for 20 RH values in this range. These values were used to set parameters in a kinetic model which incorporates relative humidity as an environmental parameter. The model is then used to predict D-values at 115 C and 125 C and compare them to experiments at these temperatures. MATERIALS AND METHODS Humidity determination. The standard formula for RH is (5): % RH = (e/e,.t) x 100 (1) where e is the pressure of water vapor present and esat is the pressure of saturated water vapor at the same temperature. Since, for air of a given moisture content, saturation temperature and dewpoint are the same, the RH inside an oven during dry heat sterilization (RH,) is given by % RH. = (ed/eo) x 100 (2) where ed is the saturated vapor pressure at dewpoint and e. is the saturated vapor pressure at oven temperature. The saturated vapor pressures are found in handbook tables (4). If the RH is known at a given temperature (T), the relative humidity of the same air at oven temperature is given by % RH, = (% RHTeT)/eo (3) where RHT is the relative humidity at T and eT is the pressure of saturated water vapor at T. Accuracy of RH determinations. RHO was determined either by use of equation 2 and direct dewpoint readings or by use of equation 3 and RHT readings from LiCl sensor-strip chart recordings. It was assumed that the direct dewpoint readings were accurate to 40.5 C. If ed is the saturated vapor pressure at dewpoint, ed+0.0 -ed > ed -edO0.5. For this reason we took the accuracy range for dewpoint RH0 determinations to be (ed/e0) + I(ed+0.Ied)/eol x 100. The LiCl sensor-strip recorder units were calibrated as a system by the Primary Standards Laboratory of Sandia Laboratories and gave % RH + 1% at ambient temperature. The temperature at which these readings were taken was 26 C. It can be seen from equation 3 that the error in RHO which results from a 1% error in RHT is the ratio eT/eo. The accuracy range for LiCl RH0 determinations were calculated as % RHO 4 (eT/eG). Humidity control systems. The humidity control used for a given experiment depended on the desired air moisture content. This determined the dewpoint for the oven input air. The initial step in the humidity control system assured an excess air moisture content. The ambient RH at our location is frequently as low as 10%. Thus for dewpoints above -10 C the input air was first passed through fritted glass tubes submerged in deionized water at 26 C. For dewpoints below -10 C, the moisture content of the incoming air was adequate. After assurance of a sufficient moisture content, the air was then either cooled to the desired dewpoint and the excess moisture was removed by condensation, or, if the dewpoint was below 2 C, the air was cooled to 2 C and excess moisture was removed by condensation under pressure. Condensation under pressure enabled us to attain dewpoints as low as -18 C. For dewpoints below -18 C, air having this dewpoint was passed through a desiccant bed having a bypass arrangement for dewpoint regulations. For dewpoints above 2 C, control was maintained by regulation of condensation temperature. Condensation occurred in cooling coils submerged in a tem-perature bath controlled to ± 1 C. For dewpoints in the range -18 to 2 C, dewpoint was controlled by regulation of the pressure under which condensation took place. If condensation takes place under the pressure P. absolute, RH % = f[e(P./P.)I/e..dt x 100 (4) where P. is ambient pressure absolute. Thus the dewpoints covered by this system configuration were easily controlled by pressure regulation. This pressurized air was expanded to ambient pressure prior to entry to the subsequent phase. A dewpoint of -52 C was attainable by passage of -18 C dewpoint air through a desiccant bed. Control was achieved through a bypass arrangement which allowed the mixing of the desiccated and -18 C dewpoint air. Air having the proper moisture content was warmed to 26 C, the RH determination was made either by dewpoint or LiCl sensor measurement, and the air was then fed into the oven. Air flow was metered at a level which maintained an oven overpressure of 0.03 ± 0.001 inch H20 and provided a 1-min replacement of oven air. The equipment configuration shown in Fig. 1 is for a test using input air with a dewpoint above -18 C. The desiccant bed, the only item not shown, was constructed from a sealable cylinder of approximately 3 ft3 volume. A 6-inch layer of the desiccant, CaSO4, was supported at the center of the chamber by a porous mesh. Air entered at the bottom and exited at the top. Spores. B. subtilis var. niger spores acquired from Fort Detrick were cleaned of vegetative material by multiple centrifugation and suspended in 95% ethanol at a concentration of approximately 107 spores/ml. The suspension was stored at 4 C. Sample preparation. The spore suspension was insonated for 2 min to distribute the spores uniformly. For each sample, 0.1 ml of the insonated spore suspension was pipetted onto the surface of an aluminum disc 1.25 inches in diameter. These discs were cut from 0.0015-inch biological grade aluminum foil. After the ethanol had evaporated, four of the inoculated discs were placed on an aluminum strip 1.5 by 7 by 0.020 inch. A single clean foil disc was placed over each inoculated disc, and the entire unit was covered by another aluminum strip. The assembly was held together by wire clamps. One such assembly was prepared for each sampling period. The sample strips were then placed in an evacuated, 23-inch Hg vacuum, dessicator over CaSO4 for approximately 16 hr before exposure to the dry heat environment. Exposure method. The spores were exposed to the RH-controlled dry heat environment in the temperature chamber shown in Fig. 1. The strips were placed on a perforated metal cage within the chamber. The chamber door was modified by the addition of small plugs at its center. Thus, individual strip assemblies could be inserted or removed quickly with negligible effect on temperature and RH. The slight overpressure, 0.03 inch H2O, in the temperature chamber was necessary for the maintenance of RH stability. This overpressure prevented the diffusion of outside air into the temperature chamber. Recovery methods. Each sample strip of -four inoculated discs represented a single sampling period. After exposure, the spores were removed from the foil discs by 2-min insonation at an energy level of 11 watts/inch2 in 10 ml of sterile 0.1% Tween 80water. Tenfold serial dilutions were made as required and plated out in duplicate on Trypticase Soy Agar. Plate counts were made after 72 hr of incubation at 35 C. All inoculation, assembly, and recovery operations were carried out in a class 100 vertical laminar airflow clean room. Data analysis. Each experiment covered from four to six sampling periods. Data for each sample time came from eight plate counts for each of the serial dilutions. All data for a given experiment were input to a computer program which (i) computed the coefficient of variation for the eight plate counts for each dilution; (ii) selected from the data for a given sampling time that dilution which had the smallest coefficient of variation; (iii) computed a best linear least squares fit in the semilog plane to those data points from the dilution values with smallest coefficient of variation; (iv) computed the D-value from this least squares fit; (v) computed a 95% confidence interval about the D-value as follows: 0.95 CI = D 4 1.96 DE/S where D is the D-value, E is the standard error in the estimate of the slope of the least squares linear fit, and S is the slope of that line; and (vi) computed the average coefficient of variation for the data used for the least squares fit. The coefficient of variation provides a measure of the tightness of the data for a given dilution. The 95% confidence would be 0 if the standard error in the estimate of the slope were 0. This provides a measure of the linearity of the data and indicates the extremes of the D-value range for that experiment. Since there are inherent errors in both the D-value and RH determination, the data were smoothed by taking a moving average of each three consecutive points in the D-value-RH plane, consecutive in the sense of increasing RH. RESULTS The test series consisted of 27 experiments. Twenty-two were carried out over an RH range of 0.0033 to 1.6% at a sterilization temperature of 105 C. Twenty of these experiments were for determining 105 C D-values as a function of RH and two were designed for checking possible diffusion effects resulting from our use of covered discs. The environmental conditions, D-values, average coefficients of variation, and 95% confidence intervals on the D-values for the 20 D-value determination experiments are shown in Table 1. Figure 2 shows the D-values and 95% confidence intervals as a function of RH at sterilizing temperature. The moving averages discussed above are also shown in Fig. 2. Figure 3 shows a comparison of survivors from covered foil disc assemblies and open planchetts. The solid line is the least squares fit to the covered disc data, and the dotted line is that for the planchetts. The simultaneous exposure to 105 C of the covered discs and planchetts was at an RH of 0.0033 ± 0.00014%. To set sterilization parameters, it was necessary to have D-values for a fixed RH at two temperatures. D-values other than 105 C were desired for predictive checks. To this end, three experiments were carried out at 125 C and two at 115 C. The results of these experiments are shown in Table 2. Kinetic analysis. A model for dry heat sterilization which gives D-value as a function of temperature and relative humidity is useful for spacecraft sterilization applications. It was suggested in (1) that if AH# were constant over the RH range of interest, D-value could be modeled by expressing AS# as a function of RH. Here AHI and AS$ are the thermody- where WH# is the enthalpy of activation and AS# is the entropy of activation. The assumption that sterilization can be expressed in terms of D-values is equivalent to the assumption that sterilization is logarithmic. Analogously, first-order kinetics is logarithmic. We will assume that both temperature and RH can be modeled as environmental sterilization parameters by first-order kinetics. The relationship between D-value and the reaction rate constant of equation 5 is given by r = (loge 10)/D (7) where D is D-value. From equations 5 and 6 we see that TAS# -AHf = RT loge (rh/kt). namic parameters of Eyring kinetics in which reaction rate (r) is expressed by (2) r = (kT/h) exp ( -AF/RT) (5) where k is Boltzmann's constant, T is the temperature in degrees Kelvin, h is Planck's constant, R is the gas constant, and AF# is the energy of activation. Table 3 shows the moving averages of Fig. 2 and AS as a function of RH under the assumption that AH# = 25 kcal/mole. Figure 4 shows these AS# values as a function of RH. Finally, the utility of a modeling technique depends on its predictive qualities. Figure 5 shows D-values with 95% confidence intervals for the 115 and 125 C~sterilization experiments given in Table 2 DISCUSSION The technique of holding AH# constant and expressing ALSa as a function of RH appears to be a useful supplement to the kinetic modeling of dry heat sterilization. This allows for the inclusion of both RH and temperature as envi- ronmental parameters. Equation 3 shows how one can convert the RH scale of Fig. 4 to a temperature other than 105 C and equation 10 shows how to construct a AS4 curve for a different AH# base. Considering the number of experiments and lengths of confidence intervals, the drop in Dvalue through the oven temperature RH range of 0.02 to 0.2% must be real. An analysis which predicted this drop was presented in (1).

v3-fos

2020-12-10T09:04:12.759Z

1972-04-01T00:00:00.000Z

237229172

s2

Antimicrobial Protection of Moisturized Deglet Noor Dates The growth of Saccharomyces rouxii and Saccharomyces mellis, which are two of the main spoilage organisms of dates, can be inhibited by various treatments. The most effective treatment found in this study that did not affect flavor consisted of a predip of the dates in 2% potassium sorbate solution followed by injection of methyl bromide into the sealed package. The growth of Saccharomyces rouxii and Saccharomyces mellis, which are two of the main spoilage organisms of dates, can be inhibited by various treatments. The most effective treatment found in this study that did not affect flavor consisted of a predip of the dates in 2% potassium sorbate solution foltowed by injection of methyl bromide into the sealed package. Dates have long been recognized as an energy-rich food throughout many countries of the world. About 22,000 tons of dates are harvested annually in the United States, giving a return to the grower of more than $3,000,000. Normally, these dates are quite dry when harvested and seldom create a microbial spoilage problem. However, if they are marketed at this moisture level, which is usually around 21%, the consumer complains about their hardness. The texture of dates can be softened by hydrating them to 24% moisture or higher, depending upon the amount of sugar inversion that has occurred. At this moisture level, yeast spoilage of the dates can be a problem (10). Microbial spoilage in dates, as well as prunes and figs, has generally been controlled in the past by an epoxide treatment. The epoxides have a high degree of killing power and produce essentially a sterile product (3,9,12). However, in the past few years the use of epoxides on dried fruits has been prohibited. Since then, the major prune and fig processors have used potassium sorbate for treating high-moisture fruits (7). The present study was undertaken to determine the effectiveness of potassium sorbate and its derivatives (11) as well as other potential antimicrobial agents in retarding microbial spoilage of processed dates. MATERIALS AND METHODS Untreated Deglet Noor dates, pitted and unpitted, packaged in 9-kg bulk cartons, were obtained from Indio, Calif. The dates were divided into two batches and their moisture levels were adjusted in a steam chamber to 22% and 27 to 30%, respectively. The 22% moisture dates were used in dip treatments, which resulted in a final moisture after treating of 28 to 30%. The dates were hydrated to a high moisture level for this study to provide conditions in which spoilage was certain to occur without treatment. Dates were inoculated after hydration with approximately equal numbers of the osmophilic yeasts, Saccharomyces rouxii (Zygosaccharomyces japonicus) and Saccharomyces mellis, which are two of the main spoilage organisms of dates (5). The yeasts were washed from 48-hr potato-dextrose-agar slants with 0.1% peptone water, and the optical density of the resulting suspension was adjusted by dilution. A calculated inoculum of 3 x 10' yeast cells/g was added to the dates. The dates with yeast inoculum were then incubated for 24 hr at 25 C before antimicrobial treatment. The 22% moisture dates were dipped into the following solutions for 30 sec: 2 and 5% potassium sorbate; 1 and 2% propyl-p-hydroxy benzoate; 2% heptyl-2-hydroxybenzoate; 2% 3-acetyl-4-hydroxy coumarin; 2% octylgallate; 2% obtusastyrene (p-cinnamylphenol); and 2% calcium propionate. These inoculated and treated dates were drained for 1 min, after which 160-g portions were sealed in polyesterpolyethylene laminated pouches (Scotchpak). Duplicates were prepared of samples from each treatment. The inoculated 27 to 30% moisture dates were packaged as above, and the respective antimicrobial agents, peracetic acid, ethyl formate, diethyl pyrocarbonate, propylene oxide, and ethyl sorbate, were added before sealing. The peracetic acid was dispersed over the inside surface of the pouch before the dates were added. The bags of dates treated with ethyl sorbate were heated for 2 min in a 93 C oven to vaporize the material. For isomaltol vapor treatment, a 20% solution of isomaltol in methanol was prepared. Three milliliters of this solution was absorbed on absorbent paper (3 by 12 cm). Alcohol was allowed to evaporate and the paper was put into the bag of inoculated dates. For gaseous treatment, the bags of dates were sealed and the gas was injected through a needle into the bag; then the hole was sealed over. These packages of treated dates were all stored in a 25 C incubator. Samples were removed periodi-cally during the 90-day testing period to determine whether any microbial growth had occurred. Microbial growth was determined in two ways: by visual examination and by measuring the changes in bag volume by water displacement. Potassium sorbate was determined by the method of Nury and Bolin (6) and moisture was determined by the AOAC (Association of Official Agricultural Chemists) method (1). RESULTS AND DISCUSSION Propylene oxide provided good microbial protection at the 0.3% level (v/w). However, an objectionable odor was noticed when the packages treated with this chemical were opened. This off-odor was present regardless of the purity of the commercial propylene oxide. Taste was not affected and the odor disappeared when the dates were aerated. Potassium sorbate has been used successfully and extensively for treating other highmoisture dried fruits, such as prunes and figs, by spraying or dipping in a 2% solution (7). However, this same dip did not keep yeast from growing on dates during extended storage (Table 1). This difference in effectiveness is probably because dates have a higher pH than either prunes or figs. Sorbic acid imparts antimicrobial activity in the undissociated form, and dissociation becomes greater at higher pH. In prunes and figs having a pH between 4 and 5, 50 to 80% of the sorbic acid is present in the undissociated form. Dates have a pH of about 5.9, so that only a small percentage of the sorbate is present as undissociated sorbic acid. Therefore, a greater amount of sorbate is required in dates to provide microbial protection. Taste threshold for sorbate varies with the dried fruit to which it is added. In prunes about 600 to 700 Mg/g can be detected. A taste panel evaluation of date paste indicated that a significant percentage of panel members could not detect potassium sorbate added at 4,800 ,Mg/g. However, sensitivity to sorbate varied considerably, with a few panel members consistently able to detect sorbate in dates at 1,000 ug/g. At higher levels, those who could taste it indicated that it produced a slight burning effect or a reduction in flavor. Moreover, the sorbate was not distributed uniformly throughout dates. Pitted dates, dipped in 2% potassium sorbate solution at 23 C and analyzed after 2 weeks, contained 900 gg/g sorbate in the whole date, but concentration in the skin was 1,250 Isg/g while the flesh contained 690 ,ug/g. Therefore, sorbate can be used to give antimicrobial control for dates if the concentration is kept in the range where no taste difference occurs and yet the needed protection is obtained. This would be applicable only to dates having a low moisture level and a low level of contamination. Methyl and propyl p-hydroxybenzoates are members of a group of antimicrobial agents known to retard mold and yeast growth in certain commodities (4). Dates were dipped in propyl p-hydroxybenzoate solutions of various concentrations ( Table 2). Even a 0.5% dip provided protection for about a month, and a 2% dip inhibited microbial growth for the full 90day storage period. However, off-odor and flavor were imparted to the dates at this higher treatment level. Therefore, it appears that these hydroxybenzoates cannot by themselves be useful in inhibiting microbial growth on high-moisture dates. Numerous other treatments were tried with varying success (Table 3). When 20 ml of nitrous oxide and 4 ml of diethyl pyrocarbonate were added to 160 g of dates, no protection was obtained; also, 2% dips in 3-acetyl-4-hydroxy coumarin and octylgallate were ineffective. However, dips in 2% solutions of obtusastyrene or calcium propionate, or isomaltol vapor treatment extended storage life up to a maximum of 2 weeks. Heating was also investigated, but excessive heat caused the dates to darken noticeably. Darkening also seemed to occur more readily as the moisture level of the dates increased. Ethyl sorbate, a derivative of sorbic acid, when added to the bags of packaged dates, did not provide microbial protection. However, if the sealed packages were heated for a few minutes to vaporize the ethyl sorbate, protection was provided depending upon concentration (Fig. 1). Full protection was realized at a 0.3% treatment level; however, dates treated at this level also had a possibly objectionable odor which would limit the usefulness of this treatment. Methyl bromide gas is widely used for the ++, +++ = '3, 33, and all fumigation of dried fruits to prevent insect infestation. This gas was investigated for its effectiveness against microorganisms on fruits. When dates were treated at levels of 1 to 10 ml of gas per package, varying degrees of protection were obtained (Fig. 2). Only the 10-ml gas injection gave complete protection. Yet, this effective treatment imparted an off-odor to the dates. At this point, studies were undertaken to determine whether combination treatments would result in an increased antimicrobial activity at chemical concentrations below the odor and flavor thresholds. Potassium sorbate (2% dip) was used as the first primary treatment, because it did not impart a noticeable flavor change at this level, and various other chemicals were added as secondary treatments. When methyl bromide was added to inoculated sorbate-treated dates, microbial protection was obtained with as little as 0.5 ml of gas (Table 4). At this low treatment level, taste panel evaluation indicated that there were no detectable odor or flavor changes in the fruit. Therefore, this appears to be a practical method of microbial control for high-moisture dates. Methyl bromide has been cleared by the Food and Drug Administration for use on dates if the residual bromide is below 100 ppm, calculated as inorganic bromide. If all of the methyl bromide injected (using 1 ml of the gas) were absorbed by the dates, the maximum possible would be 21 ppm. However, methyl bromide passes readily through packaging materials, so the amount absorbed by the dates should only be a fraction of the amount injected into the headspace (8). No methyl bromide could be detected by gas chromatographic analysis when bag headspace was analyzed 2 weeks after addition of 2 ml of methyl bromide. This loss of methyl bromide also illustrates the advantage of using a combination treatment. The methyl bromide reduces the original microbial count and is then dissipated while the potassium sorbate remains to provide lasting protection. Peracetic acid alone also exhibited antimicrobial properties, but its effectiveness was greatly increased when the dates were first dipped in a 2% potassium sorbate solution (Table 5). However, even at the 0.2% peracetic acid treatment level, a certain tartness and vinegar odor were given to the dates. Ethyl formate was found to inhibit microbial growth when added to dates at 3 ml per lb (ca. 453.6 g) ( Table 6). However, at this concentration it also imparted an objectionable odor. When the dates were first dipped in a sorbate solution, ethyl formate treatment could be reduced to 1.5 ml per lb, but even at this level it could be detected in the treated dates. The usefulness of this combination treatment is questionable. Possibly the effectiveness of the formate could be increased by a short heating in the sealed package to vaporize it quickly. The best overall method for prevention of yeast spoilage in this series of experiments was the combination sorbate-methyl bromide treatment. Other procedures provided protection but imparted a flavor or odor change to the moisturized dates. The sorbate or sorbate-ethyl formate treatment could have some application possibilities if either the microbial level or the moisture level of the dates is sufficiently low. The effectiveness of antimicrobial agents is increased as the moisture content of the fruit is decreased (2). This knowledge, plus the information on the effectiveness of various chemical or chemical treatment combinations, can be used to produce packaged succulent dates free from microbial spoilage.

v3-fos

2018-04-03T00:11:01.820Z

1972-06-01T00:00:00.000Z

9483684

s2

Loss of lactose metabolism in lactic streptococci. Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C(2)F. Instability of lactose metabolism was noted in other strains of S. lactis, in strains of S. cremoris, and in S. diacetilactis. Colonies of S. lactis C(2)F grown with lactose as the carbohydrate source also possessed lac(-) cells. Treatment of lactic streptococci with the mutagen acriflavine (AF) increased the number of non-lactose-fermenting variants. The effect of AF on growth and on loss of lactose-fermenting ability in S. lactis C(2)F was consequently further examined. The presence of AF appears to favor competitively the growth of spontaneously occurring lactose-negative cells and appears to act in the conversion of lactose-positive to non-lactose-fermenting cells. The lactose-negative mutants partially revert to lactose-positive variants which remain defective in lactose metabolism and remain unable to coagulate milk. The lactose-negative cells become dominant in continuous culture growth and provide evidence that alterations in the characteristics of starter strains can be produced by continuous culture, in this case, the complete loss in ability to ferment lactose. During studies of lactose metabolism in lactic streptococci (11,12), it was observed that acriflavine (AF) treatment of Streptococcus lactis C2F resulted in the appearance of lactose-negative (lac) cells. In addition, spontaneous lacmutants were isolated from stock cultures of the same organism. This variation in lactose metabolism by certain lactic streptococci was noted by earlier workers. In 1937, Yawger and Sherman (22) isolated four variants of S. lactis from milk which did not ferment lactose. Okulitch and Eagles (16) observed that the successive transferring of S. cremoris 142 in a glucose, mannose, fructose, or salicin medium caused the organism to lose the ability to ferment lactose. Galactose and lactose were the only fermentable carbohydrates studied which failed to induce a complete loss of lactose fermentation. These authors suggested that the inhibitory power of glucose or one of its metabolic products was the cause for the sudden or gradual loss of lactose metabolism in starters. In 1939, Okulitch (15) described the microbic dissociation of lactic acid streptococci. He suggested that the organism must be in a susceptible condition I Journal article no. 7759, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, Minn. before dissociation could be induced. The dissociation was accompanied by loss of lactose metabolism. His attempts to repeat the experiments at a later date were unsuccessful. Hunter (8) isolated variants of S. cremoris which failed to ferment lactose and which were defective in galactose metabolism. The occurrence of these variants was spasmodic, and all attempts to define the precise conditions leading to the change were unsuccessful. Many attempts resulted in the production of 100% of the lace cells, but even then the actual factor(s) responsible for the change could not be determined. Hirsch (6), in 1951, observed that S. lactis 354 lost the ability to ferment lactose after repeated subculturing in glucose broth. The question remains, however, as to how the loss of lactose metabolism is induced in cultures of lactic streptococci. Lactose metabolism and its possible instability seem particularly important at the present time since there is widespread interest in production of concentrated starter cultures in which the cells are grown by continuous culture techniques (9). It is only assumed that in the continuous culture there is no selection of cells which might be lacking in enzymes essential for acid production when the cells are grown in milk. In fact, little or no evidence is available concerning possible mutagenic changes in starter strains produced by continuous culture. It was the purpose of this investigation to establish whether lactose metabolism is unstable in lactic streptococci and to examine the possible cause(s) for this instability. MATERIALS Detection of lac-mutants. Lactic agar (4), containing 1% lactose as the primary carbon source and supplemented with 0.004% bromocresol purple, served as the indicator medium. Plates were spread with 0.1 ml of the appropriate cell dilution and incubated at 32 C for about 48 hr. On this medium, lactose-fermenting colonies were yellow in contrast to the white non-lactose-fermenting variants. To avoid the selection of contaminants, only catalase-negative lac mutants were quantitated, and, for S. kactis CYF, the lacmutants were examined for lysis by the host phage. Treatment of lactic streptococci with AF. Two loopsful of a 24-hr-old culture in lactic broth of each organism were inoculated into 2.0 ml of lactic broth containing various concentrations of AF. The AF was separately sterilized at 121 C for 15 min. After 24 hr at 21, 32, or 37 C, each culture was diluted and spread over the surface of the indicator agar for total bacterial count and for scoring of the lacmutants. For the determination of the direct effect of AF on lactose-fermenting cells of S. lactis C2F, a culture was diluted to contain 50 to 100 colony-forming units (CFU) per 0.1 ml of the test solution. The test solution consisted of 0.2 ml of lactic broth containing 1.2 Ag of AF. The tubes were incubated at 32 C for 3 hr, and the 0.2-ml samples were spread over the surface of the indicator agar. Control tubes, plated after 1.5 hr at 32 C, lacked AF and initially contained 10 to 20 CFU per 0.1 ml. Comparative growth rates of lac+ S. kactis C.F and three lacvariants isolated from this strain were determined in lactic broth containing 6 jg of AF per ml. Cultures were incubated at 32 C after receiving an inoculum to give 103 CFU per ml. Samples were removed at periodic intervals and diluted. and the total count was determined by using pour plates of lactic agar. The plates were incubated at 32 C for 48 hr. Occurrence of lac-cells within lactose-fermenting colonies of S. lactis CF. To obtain evidence for instability of lactose metabolism in lactic streptococci, S. lactis C2F was cloned several times on the lactose indicator agar. A typical colony was placed into a tube of lactic broth and incubated at 21 C for about 16 hr. The culture was diluted and spread over the surface of the indicator agar to obtain individual colonies. The plates were incubated at 32 C for 65 hr after which 10 lac+ colonies were separately picked into individual tubes containing 1.0 ml of 0.85% saline. The tubes were mixed thoroughly and scored for the presence of any lac-cells by diluting and spreading over the surface of the lactose indicator agar. Reversion of lac-mutants. For each of the various isolates, 10 cultures, each containing about 10 organisms per ml, were prepared and incubated at 21 C for 16 hr. A 0.1-ml sample from each culture was then spread onto the lactose indicator agar and incubated for 2 days at 32 C. The number of colonies utilizing lactose was then counted. At the time of sampling, equal portions from each of the 10 cultures were pooled, and the resultant bacterial suspension was plated to give the basis for a reversion frequency. Continuous culture growth of S. lactis C2F. Continuous culture studies were made in a culture vessel designed by H. M. Tsuchiya, Department of Chemical Engineering, University of Minnesota. In this system, hyponeedle wire (gauge 21) transferred nutrient from the nutrient reservoir to the growth vessel. The height of the nutrient reservoir was adjusted to deliver the nutrient at a continuous feed rate of approximately 0.17 ml per min. The vessel with a culture volume of 100 ml was water-jacketed for constant temperature control. The circulating water temperature was maintained by using a Beckman Thermocirculator model 1818. Mixing of the cells was accomplished by introduction of air from the bottom of the culture vessel. Air was sterilized by passing through field monitor disposable plastic membrane filter holders (Millipore Corp., Bedford, Mass.) and then by bubbling through sterile water before entering the culture vessel. Lactic broth containing defoamer (Marschall, Division of Miles Laboratories, Inc., Madison, Wis.) at 10 gg/ml to control foaming served as the nutrient medium. Continuous cultures were initiated by inoculating the culture vessel with 2 to 5 ml of S. lactis C2F. Samples were periodically removed from the culture vessel for determination of the presence of lacmutants. RESULTS Effect of AF. The effect of AF concentration at several temperatures on appearance of lacmutants from S. lactis C2F is shown in Table 1. No lacmutants were observed in the absence of AF; yet, in the presence of AF, lacmutants were readily isolated at 21, 32, and 37 C. The highest frequency was noted at 32 C in the presence of 6 Mg of AF per ml. In some experiments, spontaneous lacmutants were observed in the control tubes; however, AF always increased their number. Whether AF increased the frequency of conversion from lac+ to lacdirectly without appreciable selective growth of any naturally occurring lac-cells was unknown. Figure 1 illustrates that the number of viable S. lactis C2F cells decreased when exposed to AF. No cells could be recovered after 24 hr of incubation. On the other hand, the lacmutants derived from S. lactis C2F were resistant to the mutagen. The lac-culture isolated in the presence of AF exhibited a lag period of 4 hr before growth occurred. The other lac-cultures also grew after an extended lag and reached maximum populations in 24 to 48 hr. This shortened lag period of the AF mutant could be due to the cells prior exposure to AF. Figure 2 indicates the spontaneous lacmutants did exhibit a shortened lag if they were first grown in the presence of AF. It therefore appears that the lacmutants are more resistant to AF than are the lac+ cells and that the lacvariants can mutate more readily to AF resistance, thus increasing their selective advantage in the presence of AF. Conversion of lac+ cells of S. lactis C2F to lac-cells by treatment with AF. The results obtained in Fig. 1 indicate that AF selectively allows growth of lac-cells; however, it does not rule out the possibility that AF is involved in the direct conversion of lac+ cells to lacvariants. To obtain evidence for this possibility, a small number of cells were inoculated into lactic broth containing AF, and 0.2-ml samples were dispensed into 100 tubes. After 3 hr of incubation at 32 C, the contents were spread over the surface of the indicator agar. In the presence of AF, 56 tubes contained evidence of lac-cells. Some were pure lacclones, but many lac+ colonies were found to contain outbursts or papilliae consisting en-I nL. *"0 2 tirely of lac-cells which formed at the edge of the colony. In the absence of AF, only 10 tubes of the 100 inoculated were found to contain laccells, suggesting that the mutagen was involved in the conversion of lac+ to lac-. AF treatment of S. lactis C2F, as shown above, increased the occurrence of lack variants. Table 2 Growth of AF treated and untreated spontaneous lacmutants of S. lactis C2F in lactic broth supplemented with 6 pg of AF/ml. A spontaneous lacmutant of S. lactis CF was grown with 6 pg of AF/ml. These AF-treated cells were subcultured once in lactic broth without AF and then inoculated into the experimental broth (0). The control curve (0) represents growth of untreated laccells in the presence of AF. by AF treatment in S. lactis C2F, S. cremoris B1, S. cremoris Wg2, and S. diacetilactis 18-16. Spontaneous lacmutants were also isolated from S. lactis b, S. cremoris C,, and S. cremoris W, as well as S. lactis C2F, S. cremoris B1, and S. diacetilactis 18-16. Thus, it appears that the loss of lactose metabolism is a general phenomenon among the lactic streptococci. In addition to certain chemical agents, elevated temperature is also known to increase the loss of metabolic functions from bacteria (14). When cultures of S. lactis C2F were incubated at 37 C for 72 hr and subsequently held at 25 C, a high proportion of the cells which grew out were lac-. The percent of laccells in the total population ranged from 19 to 64 depending upon the experiment. Occurrence of lacvariants within lactose-fermenting colonies of S. lactis CF. To obtain further evidence for the instability of lactose metabolism in lactic streptococci, we examined lactose-fermenting colonies of S. lactis C2F to determine whether any laccells were naturally present. Table 3 indicates that laccells were obtained from presumably pure lac+ colonies. From the 10 lac+ colonies examined, five were found to contain laccells. Even the others may have contained lac-cells if a larger number of colonies had been examined. It has subsequently been observed that on occasion, when abnormally light yellow colonies are picked from a plate and restreaked, they contain a mixture of lac+ and lac-cells. It should be emphasized that this spontaneous conversion of lac+ to lac-cells must occur at a fairly high rate or it would be difficult to detect any cells by the direct plating techniques used. Reversion of lacmutants. Table 4 illustrates the reversion of the lacmutants to lac+ variants. Mutants selected from the three treatments were examined. Although partial revertants were observed, they did not possess the original S. lactis C2F phenotype. They remained defective in lactose metabolism and never regained the ability to coagulate milk. These revertants appeared on the plate only after extended periods of incubation. When S. lactis C2F cells were mixed with an excess of a lac-culture and were spread over the surface of the indicator agar, the S. lactis C2F cells developed into colonies which produced acid within 24 hr. Thus, the procedure did not inhibit any true lac+ revertants. Continuous culture growth of S. lactis C2F. The spontaneous occurrence of lac-cells in cultures of lactic streptococci suggested that continuous culture growth of starters could present a problem for mass culturing if conditions were selective for dominance by lacvariants. Table 5 shows the continuous culture growth of S. lactis C2F and the subsequent occurrence of lacmutants. After prolonged continuous growth, the lacmutants slowly became dominant. In one experiment at 37 to 39 C, about 82% of the population consisted of lacmutants after 85 hr of continuous growth. With continued incubation, the lacmutants eventually became the only surviving cells. DISCUSSION During studies on lactose metabolism in lactic streptococci, a peculiar loss of lactose metabolism in S. Iactis C2F was observed. In the isolation of lac-strains from S. lactis C2F, over 70% of the survivors were lac-under certain conditions of mutagenesis. In addition, on December 10, 2020 by guest http://aem.asm.org/ Downloaded from spontaneous lacmutants were isolated from stock cultures of this strain after an extended period of daily subculturing in lactic broth at 21 C. Since these variants grew as well as the wild type on glucose, maltose, mannose, and fructose, it was argued that the defect did not involve the glycolytic enzymes but was specific in the metabolism of lactose (11). Their relationship to the parent strain was assured since these mutants retained sensitivity to the bacteriophage specific for its parent. Thus, lactose metabolism in S. lactis C2F, previously believed to be a stable trait, was unstable under certain conditions of cultivation. The question that arises is how the loss of lactose-fermenting ability is induced in lactic streptococci. Okulitch and Eagles (16) observed that successive transferring in any fermentable carbohydrate medium resulted in the loss of lactose metabolism in S. cremoris 142. Lactose and galactose were the only fermentable sugars which did not result in a complete loss of lactose metabolism. They suggested that the inhibitory activity of glucose or one of its metabolic products may cause this sudden loss. A prime candidate could be lactic acid or H202 since the latter compound is mutagenic. They also suggested that the specific configuration of the carbohydrate in the medium was important as well as the physiological state of the microorganism. The data presented here suggest that lactic streptococci could be carrying a genetic element which is responsible for the cells ability to ferment lactose. The loss of this element would cause the cell to become lac-. If this were the case, then lactic streptococci would be expected to throw off extrachromosomalnegative variants as a result of occasional errors in its replication (14). This spontaneous loss of lactose metabolism by lactic streptococci was observed. In the present study it was noted in S. lactis C2F, S. lactis b, S. cremoris B1, S. cremoris Ct, S. cremoris W, and S. diacetilactis 18-16. In earlier work, it was observed in S. cremoris 142 by Okulitch and Eagles (16), in S. cremoris HP by Hunter (8), and in S. lactis 354 by Hirsch (6). The frequency of such variants can often be increased by treatment with certain chemical agents such as AF which selectively inactivates extrachromosomal elements (5). The appearance of lacmutants was increased by AF treatment in S. lactis C2F, S. cremoris B1, S. cremoris Wg2, and in S. diacetilactis 18-16. In S. lactis C2F, it was shown that the lacvariants are selected by the presence of AF, but, in addition, it was shown that AF may also be involved in the direct conversion of lac+ to lac-cells. The high incidence of the spontaneous loss of lactose metabolism is presumptive evidence for an extrachromosomal particle's being responsible for lactose metabolism. The effect of AF and elevated temperatures on the occurrence of lac-variants strengthens this hypothesis. However, other sources of genetic alterations such as point mutations, phase variations, and deletions were not ruled out. Point mutations and phase variations are revertible, but deletions or loss of extrachromosomal material are nonrevertible. Unlike the earlier workers who considered the change in S. cremoris and S. lactis to be stable (6,8,15,16,22), we observed a reversion from lacto lac+ to occur. Whether these mutants represent reversions in chromosomal-linked lactose genes (ruling out deletions or loss of genetic material) or represent the ability of the lac-culture to utilize lactose by mutation in alternate genes is not known. Any change in metabolism could mean that a reversion had occurred by changes in a cryptic alternate pathway similar to that reported for mannitol utilization in Aerobacter aerogenes (19). Vakil and Shahani (20) demonstrated that S. lactis UN can utilize lactose by first converting it to lactobionic acid. This could be one alternate pathway. These revertants were considered partial because they lacked the parental lactose-fermenting phenotype and were unable to coagulate milk. They appeared only after prolonged incubation of plates spread with a lawn of laccells. Thus, although partial lactose-fermenting variants were observed by the strain of S. lactis C2F, the evidence gained from studies on their spontaneous occurrence and the findings observed with AF strongly suggest the loss of genetic material as being responsible for the instability of lactose metabolism observed in lactic streptococci. It was previously noted that under certain conditions the successive transferring of S. lactis or S. cremoris resulted in the appearance of lac-cells. This is difficult to interpret, but one explanation is the loss of genetic material. Clark (2) observed that, after 61 successive transfers, Bacillus megaterium lost genetic material and became nonlysogenic. Lwoff (10) also demonstrated the loss of prophage by repeated transfers of the organism. Whether a similar phenomenon is responsible for the loss of lactose metabolism in lactic streptococci is as yet unknown. Lysogenic strains have been reported among the lactic streptococci (17), and it may be that this lysogeny is necessary to obtain lactose metabolism. The loss of the prophage would then result in a lac-variant. Hirsch (7) proposed the hypothesis that the lactic streptococci were of recent origin and, although milk is not considered to be a normal habitat of these organisms, he reasoned that they became adapted to this environment. His reasoning included the saprophytic nature of the organism, their lactose-fermenting ability, their habitat, and their antibiotic-producing ability. Could the lactic streptococci have acquired this ability to ferment lactose via transfer of genetic material from another genus? It is known that non-lactose-fermenting Salmonella (3) and Proteus (21) strains become lac+ upon acquisition of extrachromosomal deoxyribonucleic acidcarrying lac genes. Shigella dysenteriae, normally a lactose-negative organism, has also been shown to be converted to a lactose-positive strain by incorporation of prophage (1). This study also revealed that there can be selection of cells which fail to ferment lactose during continuous culture growth of lactic streptococci. This is the first report providing evidence that alterations in the characteristics of starter strains are produced by continuous culture techniques. Thus, if one is preparing concentrated cultures to be used for direct inoculation into the bulk starter tank or cheese vat, certain precautions must be taken to prevent a high incidence of lace cells. This is particularly true if continuous culture techniques are used for the preparation of the cells. Spontaneous lace mutants were found at 21, 32, and 37 C so temperature alone was not the primary factor in conversion. The presence of lactose as the sole carbohydrate source in the fermentation media would probably help reduce the frequency of the lacmutants. If the fermentation media contains glucose, the lacmutants are capable of growing as well as the lac+ parent cells. The presence of lactose alone, however, does not prevent the appearance of lacmutants, as was noted in Table 3.

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Aflatoxin Production in Peanut Varieties by aspergillus flavus Link and Aspergillus parasiticus Speare Levels of aflatoxin produced in peanuts differed with the genetic variety of plant and with the species and strain of invading fungus. Possibilities for identifying groundnut varieties partially resistant to aflatoxin production are discussed. Aflatoxin contamination in food grains is now well recognized as a public health hazard (6). Several approaches towards alleviation of the problem are being attempted (6). One promising method is to obtain varieties of peanuts (Arachis hypogaea L) (12, 13; A. Z. Joffe, Final Rep. PL. 480 Project, 1968, p. 142, the Hebrew Univ. of Jerusalem, Israel), sorghum (R. J. Anandam, M.Sc. Ag. thesis, A.P.A.U., Hyderabad), and corn (10) that do not serve as favorable substrates for the production of aflatoxins. In the first report of genetic resistance of peanuts to aflatoxin production, Rao and Tulpule (12) screened 60 varieties of peanuts; no toxin was produced on one variety, US-26 (PI. 246388), although growth of the fungus in the kernels was normal. On the other hand, Howell (8) and Doupnik (4) reported that aflatoxin was produced in variety US-26. Doupnik's (4, 5) data, however, indicate wide variations in toxin production among the 20 breeding lines of peanuts investigated. In view of the importance of the genetic approach for the prevention of aflatoxin accumulation in stored peanuts, an attempt was made to study in greater detail the interactions of plant variety and species and strain of fungus on aflatoxin production. Aflatoxin production was examined in two varieties of peanut, TMV-2 (the most common Indian variety) and US-26 (PI. 246388). Three isolates of Aspergillus flavus, (NIN 25, NIN 163, NIN 169) and two isolates of Aspergillus parasiticus (NRRL 2999 and RIB 4002; designated as Aspergillus toxicarius by Murakami [9]) were used. The toxin-producing potentials of these isolates were first graded on a synthetic medium described by Adye and Mateles (1). Twenty-gram lots of each variety of peanut were rehydrated with 10 ml of water, sterilized at 121 C for 15 min, and inoculated with 1 ml of a spore suspension (approximately 6 x 105 spores/ml) of a fungus. The flasks were incubated at 28 C for 7 days, sprayed with 95% alcohol, and dried overnight at 80 C. The dried samples were first defatted with n-hexane and then extracted with methanol. The aqueous methanol extracts were treated with basic lead acetate for removal of pigments. The toxins were then extracted into chloroform and the CHClI extracts were appropriately processed for screening by thin-layer chromatography using CHCl3-MeOH (95:5) as the developing solvent system. Aflatoxin B1 was quantitated as described by Pons et al. (11). This method' is capable of detecting as little as 0.3 part per billion (ppb) of aflatoxin B,. Chemical confirmation of aflatoxin was made by spraying the chromatograms with 10% HCl in ethanol, as described by Crisan (2). Toxin production by different fungal isolates varied considerably as is shown in Table 1. Isolates of A. flavus used in this study produced only B1 and B2, whereas A. parasiticus produced B1, B2, G., and G2. The two species can be readily determined by light microscopy. The conidia of A. flavus show only minute echinulation as compared to prominently echinulate conidia of A. parasiticus. The results indicate clearly that there are species and varietal differences in toxin production ( Table 2). The variation in toxin production appears to be intimately related to the inherent ability of the fungal isolate to produce the toxin. The fungal isolate used by Rao and Tulpule (12) was perhaps so low in toxin-pro- ducing capability that no detectable toxin could be detected when it was grown on US-26, although measurable amounts of the toxin were produced on other varieites of peanuts. The toxin production was also related to the species of Aspergillus used; A. parasiticus always produced greater amounts as compared to A. flavus. Such differences have also been observed on liquid media and other natural substrates (7). Despite these variables, the difference in toxin production attributable to the genotype was always demonstrable. This is also apparent from the data of Doupnik and associates (4,5). It is intriguing that certain varieties of peanuts support low toxin production, whereas other varieties support maximal production. This difference is possibly related to certain basic biochemical characters such as protein (10) or possibly vitamin E (3) besides cultivar practices. From the point of view of prevention, the challenge, therefore, appears to be to identify peanut genotypes that will support minimal toxin production by a number of fungal isolates of A. flavus and A. parasiticus. A. parasiticus is well recognized to be powerfully toxigenic. The prevalence of A. parasiticus in stored food grains appears to be not as sufficiently well investigated as A. flavus, although Hesseltine et al. (7) suggest that the occurrence of A. parasiticus is restricted primarily to the tropics. However, in our preliminary studies on more than a hundred isolates of Aspergilli from stored peanuts and other food grains, A. parasiticus was not encountered. An exhaustive study by Raghavendra Rao et al. (Final Report US-PL. 480 Project, Regional Research Labs, Hyderabad, India, 1970) on cotton seed mycoflora, involving about 2,500 isolates, also did not reveal the presence of A. parasiticus. The rare occurrence of A. parasiticus contami- We are thankful to C. Gopalan (Director, National Institute of Nutrition) and P. G. Tulpule (Head of the Toxicology Division, National Institute of Nutrition) for keen interest and valuable suggestions. We are also deeply indebted to L. A. Goldblatt (New Orleans) for generous supply of pure aflatoxin standards, to S. Raghavendra Rao

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Effects of Changes in Feed Level, Starvation, and Level of Feed After Starvation Upon the Concentration of Rumen Protozoa in the Ovine1 Four rumen fistulated sheep were used in five experiments to investigate the effect of feed level upon the concentration of rumen ciliate protozoa. The sheep were fed once daily 650 g of a pelleted diet composed of corn cobs, 45%; alfalfa meal, 35%; oats, 12.5%; cane molasses, 5%; urea, 0.4%; and vitamins and minerals, 2%. The concentration of protozoa reached minimum and maximum values at 5 and 22.5 h after feeding, respectively. Thus, to estimate apparent generation rates, concentrations of protozoa were determined at 5 and 20 h postfeeding. Apparent generation rate/h = natural log of ([concentration of protozoa at 20 h divided by concentration at 5 h] divided by the time interval, [T20 to T5]). Alteration of the feed to protozoa ratio by starvation and by changing the level of feed (200 to 900 g/day) showed that as the ratio of feed to protozoa increased, generation rate increased. Measurements of liquid turnover rates in the rumen showed that turnover rate decreased as feed level decreased. Turnover rate was near zero when the sheep were starved. Small quantities of soluble substrates, added directly to the rumen of starved sheep, maintained the protozoal population when rumen turnover was minimal. Furthermore, as rumen turnover rate increased with increased levels of feed, the effect of substrate on maintaining the protozoal population was negated. Thus, at high feed levels, turnover rate may be the dominant factor controlling the establishment and concentration of protozoa in the rumen. Feed level has been suggested as one of the factors which influences the ciliate protozoal population in the rumen. The proportion of dividing been [T20 to T5]). Alteration of the feed to protozoa ratio by starvation and by changing the level of feed (200 to 900 g/day) showed that as the ratio of feed to protozoa increased, generation rate increased. Measurements of liquid turnover rates in the rumen showed that turnover rate decreased as feed level decreased. Turnover rate was near zero when the sheep were starved. Small quantities of soluble substrates, added directly to the rumen of starved sheep, maintained the protozoal population when rumen turnover was minimal. Furthermore, as rumen turnover rate increased with increased levels of feed, the effect of substrate on maintaining the protozoal population was negated. Thus, at high feed levels, turnover rate may be the dominant factor controlling the establishment and concentration of protozoa in the rumen. Feed level has been suggested as one of the factors which influences the ciliate protozoal population in the rumen. The proportion of dividing entodinia has been observed to increase as the level of ration increased, whereas the concentration of entodinia showed no consistent relationship with feeding level (10). In comparison to full feeding, feeding at two-thirds of full feed resulted in a decreased rate of passage and increased protozoa numbers (2). Several other studies have also shown that feeding level affects the concentration of protozoa (1,3,4). The present investigation was an attempt to obtain additional information on how feed level affects the concentration of protozoa in the rumen. Particular emphasis has been placed on the short-term effect, in contrast to previous ' studies where the responses measured have usually been induced at least a week prior to the measurements. MATERIALS AND METHODS Four wether lambs weighing approximately 40 kg were prepared with rumen fistulas and housed singly in wooden pens which had a common water trough. They were fed 650 g per day of the following pelleted feed: corn cobs, 45%; alfalfa meal (17% crude protein), 35%; oats, 12.6%; cane molasses, 5%; urea, 0.4%; and minerals and vitamins, 2%. Samples of rumen contents for ciliate protozoa counts were obtained with a plastic tube through the cannula, immediately fixed in Formalin and then counted (8). Flagellate protozoa were not counted in these studies. Rumen volume and liquid turnover rate estimates were made by using polyethylene glycol (PEG), molecular weight 4,000. PEG in the rumen fluid was determined by the technique of Hyden (11). A preliminary investigation was conducted to de- IN THE OVINE collecting rumen samples from the four lambs at 0, 2, 5, 8, 11, 15, 21, 22.5, and 24 h after the regular feeding time for 2 consecutive days on two occasions 10 days apart. On both occasions the lambs were fed their regular level of feed (650 g) the first day and then starved the second day. In subsequent experiments samples of rumen contents for determining protozoan numbers were collected at 0, 5, 20, and 24 h after feeding. In experiment one, samples were collected on four consecutive days. On day 1 all animals were fed 650 g, and on the subsequent days they were fed either 200, 400, 650, or 900 g of feed. This experiment was replicated three times with each sheep receiving a different feed level in each replicate. Experiment two was similar to experiment one with the exception that the collection period was 5 days in length as a result of adding a day of starvation between the normal intake day and the three days of feeding at the different feed levels. Experiment two was also replicated three times. Experiment three differed from experiment two only in that all animals were fed 200 g of feed on the first day after the day of starvation and then fed at the different levels of feed for the next 3 days. Experiment four consisted of rumen volume and liquid passage rate measurements on all four sheep. Samples were collected over a 5-day period with a dose of PEG infused into the rumen, 1 h before feeding time on each day. Rumen volume estimates were made on the basis of marker concentration 1 h after dosing. Liquid rumen turnover rate was estimated from the change in marker concentration between the prefeeding and 23 h after feeding samples. On day 1 the sheep were fed 650 g of feed, whereas on day 2 they were left unfed. On days 3, 4, and 5, two sheep received 200 g of feed, whereas the other two sheep were fed 900 g. In experiment five, feed was replaced by a 250-ml intraruminal infusion of soluble substrates. Two levels of soluble substrates were used. The low level included 20 g of starch, 10 g of glucose, and 10 g of casein, whereas the high level was 40, 20, and 10, respectively. Rumen samples were collected at 0, 2, 5, 8, 13, 22, and 24 h after the infusion. Two animals were given each level of infusate. Figure 1 shows the change in the concentration of rumen protozoa in the 24-h period after either normal feeding or starvation. The mean concentration of protozoa at 24 h after feeding in this preliminary study was 7.4 x 10' per cm3. All lambs contained Entodinium and Diplodinium species of protozoa, whereas Dasytricha was found in only two lambs and Isotricha in one. A typical generic distribution as calculated from 1 day during the preliminary study was 91.7% Entodinium, 7.7% Diplodinium, 1.0% Dasytricha, and 0.6% Isotricha. A definite cyclic pattern was observed in the fed animals which had its minimum and maximum at 5 and 22.5 h postfeeding, respectively. Feeding caused a rapid decrease in the concentration of protozoa during the first 5 h, probably due to dilution by feed, saliva, drinking, and passage of ingesta from the rumen. During the 5-to 22-h postfeeding period concentration of protozoa increased in the fed lambs and represents the primary period during which protozoal cell division occurred. During the last 2-h period protozoan concentrations decreased slightly. This may have resulted from substrate shortage, increased rate of passage, or some other factor. In comparison, the concentration of rumen protozoa in unfed animals showed a steady decline which by the end of one day had decreased to 1.3 x 101 protozoa per cm3, which was equivalent to a five-fold decrease. RESULTS AND DISCUSSION From these data, the time period between 5 and 20 h postfeeding was selected for use in calculating an apparent generation rate. The term-apparent generation rate was used since it was impossible to correct for dilution associated with feeding and rate of passage from the rumen. This was calculated as follows: natural log of ([concentration of protozoa at 20 h divided by concentration at 5 h] divided by the time interval [20 to 5 h]). The apparent genera-tion rate for the cycle of fed animals in the preliminary trial shown in Fig. 1 was 0.0395 generations per h. The effect of changing feed level from 650 g per day to either 200, 400, 650, or 900 g per day upon the concentration of protozoa is depicted in Fig. 2. Decreasing the feed level to 200 g per day resulted in a decreased concentration of protozoa during the first 2 days and a partial recovery of the population on the third day. The apparent generation rates calculated from the 5-and 20-h samples reflect this decrease in concentration, being -0.0117, 0.0113, and 0.0282 generations per h for the 3 days, respectively. In comparison, the change from 650 to 400 g of feed showed only a minor decrease in protozoan concentrations and apparent generation rates of 0.0128, 0.0210, and 0.0193 were obtained for the 3 days, respectively. The generation rates for the animals fed both 200 and 400 g per day were both less than the rates observed in the animals fed 650 g per day. This suggests that generation rate is influenced by feed level, and agrees with previous observations of Warner (10) which indicated that the proportion of dividing entodinia increased with feed level. Increasing the feed level to 900 g did not increase the concentration of protozoa, which also agrees with the results reported by Warner (10). He concluded that there did not appear to by any consistent effect on protozoan concentrations with increased feed intake, especially at the higher levels of intake. Furthermore, the calculated generation rates were lower than those observed in the lambs fed 650 g. It is believed that generation rates are of more value than simple concentrations, since they reflect 90 E 7.0s the rate of increase in the protozoal population at the various intake levels. On the other hand, apparent generation rates could be misleading; for example, a negative generation rate or decrease occurred on the third day of 900-g feeding. Failure of the protozoal population to increase in concentration under the influence of more feed may have been masked by an increased rate of turnover from the rumen at this higher feed level (5). In other words, the actual generation rate may have increased but is not reflected by concentrations alone, since turnover rate increased with increased feed level ( Table 1). The relationship between apparent generation rate and rate of passage will be discussed later. Experiment two was an attempt to obtain additional information relating feed level 'to increases in the concentrations of protozoa. This was accomplished by using the same four feed levels in conjunction with a protozoal population which was reduced in concentration; the reduced population was obtained by starving the lambs for 1 day. The effect of these same feed levels upon a smaller initial protozoal population are shown in Fig. 3. All feed levels increased the concentration of protozoa. The increase observed in the lambs fed 200 g of feed was less on all 3 days than the increases observed with the other feed levels. In addition, the apparent generation rates were lowest on the 200-g feed level. Equal concentrations of protozoa were observed after 3 days of feeding either 400, 650, or 900 g of feed. However, the apparent generation rates were highest on the 650-g feed level, being 0.0550, 0.0742, and 0.0550 generations per h for the 3 days, respectively. The increase in generation rate obtained with on May 7, 2020 by guest http://aem.asm.org/ Downloaded from the 650-g feed level over that recorded on the 400-g level can be explained on the basis of more substrate. Failure of the 650-g feed level to result in a larger population at the end of 3 days is probably due to a faster rate of passage from the rumen. An additional increase in the rate of passage from the rumen would explain the apparent decrease in generation rate observed when feed level was increased to 900 g per day. In comparing the generation rates of experiments one and two, several points should be considered. First, the generation rates obtained in experiment two were considerably higher. This can probably be attributed to the larger ratio of feed to protozoa; however, rate of passage of ingesta from the rumen may have been reduced from the day of starvation. Second, in general the apparent generation rates recorded on day 2 of the differential feeding periods in experiment two were higher than those from days 1 and 3. This was not expected; the largest generation rates were expected on day 1 since the population was smallest at that time. However, this unexpected result may indicate that some protozoa were being counted which were not viable and thus unable to contribute to an increase in cell division on day 1. Experiment three incorporated 1 day of feeding 200 g of feed on the day after starvation and prior to the 3 days at different feed levels. This was an attempt to eliminate any countable nonviable protozoa. The results of experiment three are shown in Fig. 4. In general, there were no major differences between the results obtained in experiments two and three. Thus it appears that either the 1 day of 200 g feed did not eliminate countable nonviable protozoa or that factors other than this are involved. Two minor differences between experiments two and three were noted. First, the apparent generation rates were lower in this experiment and second the generation rates observed on the 900-g treatment were above that from the 650-g treatment on days 1 and 3. We have no explanation for these differences. The effect of feed level upon apparent generation rates in these three trials are summarized by Fig. 5. The top left portion of Fig. 5 refers to the average response on all 3 days, whereas the other quadrants depict responses on days 1, 2, and 3, respectively. In general, as feed level increased to 650 g, generation rate increased, whereas the increase to 900 g resulted in a slight decrease in generation rate. Furthermore, imposing the starvation treatment prior to feeding different levels (experiments two and three) resulted in higher generation rates. Thus, this figure depicts the change in generation rate as the ratio of feed to protozoa changed. Increasing the feed to protozoa ratio (experiments two and three versus experiment one) by starvation increased the generation rate. This, along with the increase in generation rate with the different feed levels up to 650 g, suggests that feed level is a primary factor controlling protozoa concentration up until the time that feed level caused rumen turnover to increase and override the effect of increased substrate. This conclusion supports in vitro data (9) which showed increased protozoa concentrations with increases in starch levels. Since apparent generation rates do not take into account the passage of protozoa out of the rumen with the ingesta, attempts were made to obtain rate of passage information for sheep on the different feed levels. Experimental procedures designed to measure rate of passage of particulate matter proved unsatisfactory; however, rumen volume and liquid turnover rate data were collected for conditions which replicated experiment two, except that only the 200and 900-g feed levels were used after the starvation period. These data, which indicate a general increase in turnover rate as feed level increased, are shown in Table 1. Rumen volume was decreased by starvation and further decreased after feeding 200 g of feed. In comparison, the 900-g feed level increased rumen volume above the poststarvation volume but not above the volume originally measured on day 1. The liquid turnover rate after the normal 650-g feeding was 2.00 turnovers per day. This compared very well with other reported values (5). In comparison, the turnover rate after starvation was a -0.17 per day. It is important to note that the value was calculated from samples collected 23 h apart. In starved sheep, the PEG concentration did decrease slightly for about 14 h and then during the last 9 h its concentration in the rumen fluid actually increased, resulting in a negative turnover rate. Complete interpretation of occurrences in the rumen during starvation are not obvious at this point; however, the data suggest that the liquid turnover rate is minimal or nonexistent. In reference to changes in the concentration of protozoa noted in the previous experiments, it thus appears that the decreases in the concentration of protozoa with starvation were in fact due to nutrient depletion and not rumen washout. Experiment five will deal with this further. The liquid turnover rates observed when sheep were fed 200 or 900 g per day show that a reduced feed intake results in a reduced rumen turnover. The liquid turnover rate during the feeding of 200 g per day was approximately 0.95 turnovers per day, whereas the turnover rate in sheep fed 900 g averaged 2.14 turnovers per day. The turnover rates obtained for the sheep fed 900 g increased on each of the 3 days after 900-g feeding, and may be indicative of the fact that feed residues in the rumen were lower during days 1 and 2. These data suggest that at reduced levels of feed, rumen turnover rate is reduced and thus may be of little importance in regulating the protozoal population. Consequently, establishment of increased concentrations of protozoa should occur more readily. This agrees with previous reports (2, 3) which suggested that protozoan concentrations increased when rumen turnover rate was reduced by reduced feed intake. The effect of nutrients upon the concentration of protozoa was further investigated by infusing small quantities of soluble substrates into the rumen of unfed sheep. It was assumed that the turnover rate in the rumen was essentially zero as suggested in experiment four. These results are shown in Table 2. Data from the preliminary trial for starved and fed animals are included for reference. During starvation without added nutrients, protozoan concentrations fell to 19% of the normal prefeeding level in 24 h. In comparison, the low and high infusions of starch, glucose, and casein maintained protozoa numbers at levels equivalent to 68 and 94%, respectively, of the normal prefeeding concentrations. Since 70 g of soluble substrate was able to maintain the total rumen protozoal population when turnover was minimal, it is suggested that substrate could be the major factor controlling the protozoal population at low feed levels. b The low and high soluble treatments consisted of 20 g of starch, 10 g of glucose, 10 g of casein, or 40 g of starch, 20 g of glucose and 10 g of casein, respectively. Each were mixed in 200 ml of water before adding to the rumen. The effect of feed level upon protozoan concentrations and liquid passage rates have both been determined, thus it should be possible to calculate a more reliable estimate of protozoal generation rate than the rates based solely on protozoan concentrations at two different time intervals. The sum of the turnover rate constants for the 200 and 900 g per day feed levels obtained in experiment four (Table 1) and apparent generation rates from experiment two (Fig. 3) should give an adjusted generation rate closer to the true value for the three-day period following a day of starvation. Hungate et al. (6) have shown that species of Entodinium pass out of the rumen at a rate similar to PEG, and since 90% or more of the protozoa in the animals studied were in this genus, the use of fluid turnover rate as an estimate of protozoan passage appears warranted. The turnover rate constant is negative in sign, but this has been ignored in the present calculations since we are estimating an increase in protozoal generation rate. These adjusted generation rates are presented in Table 3. The marked affect of turnover rate on apparent generation rate at the 900-g feed level clearly emphasizes the importance of this parameter in estimating overall protozoal growth at high feed levels. In contrast, turnover rate at the low feed level was fairly constant over the 3 days, suggesting that feed level was of more importance than passage rate in controlling protozoan concentrations. The results of experiment five on the infusion of soluble substrates would appear to support this conclusion. The liquid turnover rate when all four animals were fed 650 g per day was fairly close to that obtained at the 900 g per day level (Table 1). Although low, the relative relationship between apparent generation rates would be valid when turnover rates are similar. Using the adjusted generation rate on day 3 of the 900-g feed level, a division rate for the protozoa of once each 6.8 h is obtained. This compares quite well with a division time of 6.55 h calculated from the total turnover of rumen contents on that day (2.66 times), which assumes all of the protozoa passed out of the rumen at the same rate as PEG. These values are similar to previously reported division times (5,6).

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Direct Fluorescent-Antibody Technique for the Microbiological Examination of Food and Environmental Swab Samples for Salmonellae Comparative studies of a modified fluorescent-antibody procedure and the 5 to 7 day method used by the Association of Official Analytical Chemists for the detection of Salmonella were made on 151 samples of wheat products and 183 swab samples. The agreement between the two methods for the 334 samples tested was 92.5%. Food samples yielded 94.7% agreement, whereas the swab samples yielded 90.7% agreement. There were 7.5% false positives for the total number of samples tested. No false negatives were obtained by using the fluorescent-antibody method. The fluorescent-antibody (FA) technique introduced by Coons et al. in 1942 (2) as a rapid serological method for detecting microorganisms is now widely used in microbiology. Many attempts have been made to develop techniques for the detection of salmonellae with the FA procedure. Thomason and Wells (12) applied the FA technique for the detection of salmonellae by using pure cultures. The use of the FA technique for the detection of salmonellae in foods has been reported by Arkhangelskii and Kartoshova (1), Silliker et al. (11), Georgala and Boothroyd (4, 5), Haglund et al. (6), Fantasia (3), Insalata et al. (8), and others. In this study, a combination of the Association of Official Analytical Chemists (AOAC) standard cultural method (9) and an FA method (7) was used to screen wheat products and swab samples collected from in-process equipment in the wheat handling operation. MATERIALS AND METHODS Samples. The samples consisted of 151 wheat products and 183 swab samples. For each swab sample, a sterile swab from a tube containing 10 ml of lactose broth (Difco) was used to swab 10 square inches (64.5 cm 2) of equipment, food contact surfaces, or surrounding areas. AOAC cultural method. The conventional AOAC cultural method was followed through the selective enrichment phase so that a direct comparison of the FA results could be made with the standard AOAC method for the detection of salmonellae. Samples were pre-enriched in lactose broth for 24 h at 35 2 C. A 1:10 ratio of sample to broth was always main-tained. After incubation of the pre-enriched samples, a 2-ml amount was transferred to 18 ml of selenitecystine broth, which was incubated for 24 h at 35 ± 2 C. After incubation, the selenite-cystine broth cultures were streaked onto Brilliant Green, SS, and XL agar plates (Difco) for evaluation by the AOAC method. Pooling phase. Occasionally, to minimize the number of FA slides that had to be read in 1 day, a pooling phase was included (10). At the same time of the AOAC pre-enrichment transfer to selenite-cystine, a maximum of five pre-enrichment broths were also pooled by transferring an additional 2 ml from each of the five pre-enriched samples into a single selenite-cystine broth. A 1:10 ratio of sample to broth was still maintained. Elective enrichment phase. After incubation of the selenite-cystine broth cultures from which an FA slide was desired, 2 ml was transferred to 18 ml of preheated (35 ± 2 C) FAS broth (Difco) and incubated for 5 h in a 35 i 2 C still-water bath. The procedure used for FA slide preparation, FA staining, and microscope examination were identical to those described by Insalata et al. (7). After the slides were prepared from the elective enrichment broth, the FA cultural (FAC) confirmation procedure was performed by streaking a 3-mm loopful on selective media plates as outlined in the AOAC method. Of the 151 wheat products tested, 19 FA slide positives were found. Eleven of these were confirmed positive by the AOAC cultural method or the FAC method, or both. Therefore, eight (5.3%) of the FA slide-positive samples were not detected by either of the cultural methods; thus, the slides were considered false positives. RESULTS AND DISCUSSION In one instance in which five 2-ml amounts from five pre-enriched samples were pooled into one 90-ml selenite-cystine broth, four of the individual enriched samples were negative and one was positive when run by AOAC methodology. However, the pooled selenite-cystine broth culture failed to recover salmonellae. This failure of the pooled broth to recover salmonellae may indicate that the 2-ml amount of preenriched samples which is transferred should be increased in volume, or it may be necessary in instances of low numbers of salmonellae to pool fewer samples. Wheat samples (132) were negative for both the cultural and FA slide methods. The FA technique yielded 94.7% agreement with the AOAC method. No culturally positive wheat samples were recorded as negative by the FA method. The 183 swab samples analyzed yielded 42 FA slide positives, of which 25 were confirmed positive by using the AOAC or FAC methods, or both. Seventeen (9.3%) of the FA slide-positive samples were negative by using either of the cultural procedures. It is possible that the swab samples, because of the exposure to concomitant microflora sharing the same antigens as salmonellae, may produce more false-positive results than are obtained when certain food samples are analyzed. Swab samples (141) were negative for both the FAC and FA slide methods. The FA technique yielded 90.7% agreement with the AOAC method. Again, no culturally positive swab samples were recorded as negative by the FA method. The percent correlation of the FA method as compared to the AOAC method on the total 334 samples tested was 92.5%. The total false-positive results obtained in this study was 7.5%. In most instances, the samples recorded as FA slide positive showed large numbers of fluorescing organisms. In no instance did the FA method fail to detect a sample proved salmonella positive by cultural methods. This study has demonstrated the high degree of correlation of the FA technique in comparison to the 5 to 7 day AOAC method, when applied in the screening of samples contaminated with salmonellae. Also of significance is the degree of success which has been demonstrated by the FA method when applied against swab samples representing the sanitary indices of a process. INSALATA, MAHN The advantages of the application of the FA technique are reflected in shorter test times of 52-h elapsed time, shorter storage times for final products awaiting clearance, and the ability to rapidly determine the effectiveness of sanitation clean-up procedures in industrial processes to maintain good manufacturing practices. This study has also demonstrated that pooling suspect samples is possible to permit larger numbers to be tested simultaneously by this method. For this work, one trained laboratory technician was capable of preparing and examining 15 sets of FA slides daily, representing a maximum of 75 individual product or environmental samples. This figure may be increased if the work is segmented into a sample and slide preparation phase performed by one technician and the microscopy examination phase performed by a second technician. An undetermined number of false positives encountered while using the FA technique were caused by the nonspecific staining of a strain of Enterobacter agglomerans identified by B. M. Thomason of the Center for Disease Control. To reduce the number of false positives, there are several aspects of the procedure that require further development. These include: (i) increasing the specificity and sensitivity of the antiserum, (ii) cultural improvement, and (iii) definition of observations made when using the microscope in the diagnostic phase, to minimize subjective interpretations.

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2020-12-10T09:04:12.900Z

1973-02-01T00:00:00.000Z

237234603

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Duration of Viability and the Growth and Expiration Rates of Group E Streptococci in Soil In irradiated and nonirradiated feedlot and pasture soils inoculated with group E streptococci, the organism was not recovered 17 days postinoculation from either the irradiated or nonirradiated feedlot soils incubated at 37 C, but survived in the irradiated pasture soils for 24 and 31 days postinoculation. The streptococci survived in irradiated and nonirradiated soils incubated at 4 C for 116 days and in one irradiated feedlot soil for 165 days. The population of streptococci did not increase in either irradiated or nonirradiated soil, and the expiration rate was greater in the soils incubated at 37 and 25 C than at 4 C. With the relatively prolonged duration of viability of group E streptococci in soil at 4 C, it is suggested that soil contaminated with exudate from draining abscesses of infected swine could act as a source of infection during the colder season. In irradiated and nonirradiated feedlot and pasture soils inoculated with group E streptococci, the organism was not recovered 17 days postinoculation from either the irradiated or nonirradiated feedlot soils incubated at 37 C, but survived in the irradiated pasture soils for 24 and 31 days postinoculation. The streptococci survived in irradiated and nonirradiated soils incubated at 4 C for 116 days and in one irradiated feedlot soil for 165 days. The population of streptococci did not increase in either irradiated or nonirradiated soil, and the expiration rate was greater in the soils incubated at 37 and 25 C than at 4 C. With the relatively prolonged duration of' viability of group E streptococci in soil at 4 C, it is suggested that soil contaminated with exudate from draining abscesses of' infected swine could act as a source of infection during the colder season. Recently, it was reported that swine which carry group E streptococci (GES) do exist and possibly are a major factor in the propagation of streptococcic lymphadenitis in swine (SLS) (3, 4; J. A. Schmitz, Ph.D. thesis, Univ. of Missouri, 1971). However, it is also possible that the contamination of soil with exudate from draining abscesses containing GES could constitute a method of infecting a swine herd. This phenomenon could have occurred on a farm where the disease was not eradicated by depopulation of infected swine, disinfection of premises, or the introduction of specific pathogen-free swine (2). It had been observed previously that streptococci, such as Streptococcus agalactiae, S. dcysqualactiae, S. pyogenes, and S. uberis, did survive in damp soil for more than a year (1). This study was designed to determine the duration of viability and the growth and expiration rate of GES in sterilized and unsterilized soil at several temperatures. MATERIALS cylinder. The interval between irradiation treatments was 72 hr. (iii) Inoculation and incubation. Soil samples, in 500-ml plastic containers without lids, were inoculated with approximately 5 x 109 colony-forming units (CFU) of GES (strain 3X29a) and hydrated to 80% moisture holding capacity (MHC) with sterile isotonic saline solution (ISS). (Strain 3X29a from R. D. Shuman, National Animal Disease Laboratory, Ames, Iowa. Cells were washed twice in isotonic saline after growth for 24 hr at 37 C in Todd-Hewitt broth.) Three irradiated and three nonirradiated samples of each of the four soils were incubated at 4 C and 37 C. Humidity was maintained in the incubator at 37 C by bubbling air entering the chamber at approximately 2 liters/min through a reservoir of water in the chamber. Humidity was maintained in the incubator at 4 C by keeping a pan of water on a lower shelf. 180 (iv) Recovery of GES. Each sample was cultured for GES at various intervals postinoculation (PI) by inoculating each of three tubes containing 30 ml of blood azide-crystal violet (BACV) broth (8) with 2 g of soil ( Table 2). The broth cultures were incubated at 37 C for 24 hr and then centrifuged at 600 x g for 15 min. The sediment was streaked on BACV agar (8) and incubated for 24 to 48 hr at 37 C. One characteristic colony from each plate was used to confirm the serologic grouping of GES (7,9). Experiment 2: Growth and expiration rate of GES in soil. (i) Soils. Irradiated and nonirradiated soils of the Sarpy and Huntington types obtained in Missouri were used (Table 1). (ii) Sterilization. Three 80-g, oven-dried samples of each soil were irradiated twice with 450,000 R of gamma radiation per hr for 2.5 hr at an interval of 96, hr in a cobalt 60 cylinder. (iii) Inoculation and incubation of soils. Soil samples were inoculated with approximately 107 CFU of GES (strain 3X29a) and hydrated to 80% MHC with sterile ISS. Irradiated and nonirradiated specimens of each soil type were incubated at 37 C, 25 C, and 4 C in 100-ml glass containers with lids ajar. (iv) Quantitation of GES. The number of CFU of GES per gram of soil was determined by streak plate colony counts performed on BACV agar. Counts were made on days 0, 1, 2, 4, 8, 16, 23, 30, and 37. Incubation of the plates for 24 to 48 hr at 4 C after the initial incubation at 37 C for 24 to 36 hr enhanced beta-hemolysis and facilitated counting the GES colonies. One characteristic colony from each plate was used to confirm the serologic grouping of GES (7,9). RESULTS Experiment 1. With the feedlot soil incubated at 37 C, GES was detected in the nonirradiated specimens at 4 days PI and in the irradiated specimens at 10 days PI; however, GES could not be isolated from either irradiated or nonirradiated samples at 17 days PI (Table 2). With the pasture soil at 37 C, GES was isolated from nonirradiated samples at 4 days PI but not at 17 days PI. The GES was isolated from all irradiated pasture samples at 24 days PI and from the pasture A sample at 31 days PI. This sample was negative for GES at 52 days PI ( Table 2). The shortest survival time for GES at 4 C was in the soils of feedlot B where the final isolations were made at 31 days PI from the nonirradiated samples and at 59 days PI from the irradiated samples ( Table 2). The GESwas isolated from all of the remaining three soil types, both irradiated and nonirradiated, at 116 days PI. At 165 days PI, the irradiated soil of feedlot A was positive for GES while all other samples were negative. The feedlot A sample was negative for GES at 200 days PI. Experiment 2. The number of CFU of GES per gram of soil was between 106 and 101 in all samples on the day of inoculation. The number of GES in the soil samples incubated at 37 C decreased rapidly, and at 37 days PI only the irradiated Huntington soil contained viable organisms. At 25 C, the expiration rate of GES was slower, with viable organisms present in all but the nonirradiated Huntington soil at 37 days PI. The expiration rate of GES in soil incubated at 4 C was significantly less (P < 0.05, analysis of variance of means) than at 37 or 25 C, with high concentrations of GES in all specimens at 37 days PI (Fig. 1). At each temperature of incubation, there was no significant difference (P > 0.05) in the expiration rates of GES between Sarpy or Huntington soils or between irradiated or nonirradiated soil. DISCUSSION The significant finding in this study was that GES survived longer in soil, specifically at lower temperatures, than these investigators had anticipated. It appeared that there was little or no multiplication of GES in soil, but rather the population of organisms decreased at a rate which was related to temperature. Unquestionably, the technique used in quantitating the GES in experiment 2 resulted in considerable error; however, all specimens were quantitated in the same manner, thus justifying comparisons within the experiment. Irradiation of soil, which was designed to sterilize the soil without greatly altering its physical qualities (5), had less influence on the expiration rate on GES in experiment 2 than was anticipated, although there was an ap-preciable difference in the duration of viability in experiment 1. It is possible that oven-drying the soil samples in experiment 2 reduced the microflora significantly whereas air-drying in experiment 1 did not alter the microflora, thus accounting for the comparable rates of expiration of GES in irradiated and nonirradiated soils in experiment 2 and also for the marked difference in the duration of viability of GES at 37 C between the two experiments. However, even within experiment 1, it appeared that GES survived better in some soils than in others. This variability in the survival time of bacteria in different soils has been observed previously (6). The than pH or organic matter since the survival time of GES was considerably shorter than in the remaining soils of comparable composition. The greater survival time of GES in the pasture soils than in the feedlot soils at 37 C does not agree with a previous report in which survival of enteric bacteria was enhanced by increasing the organic content of soil (6). This disparity may be related to differences in the physiological properties of GES and the enteric bacteria or to the higher incubation temperature. The latter appeared likely considering the prolonged GES survival time of GES in the irradiated soil of feedlot A at 4 C. The isolation of GES from soils incubated at 4 C for 116 and 165 days, in this study, suggests that soil contaminated with exudate from draining abscesses of swine infected with GES could serve as a source of infection for swine during the late fall, winter, and early spring. There is the possibility that the sodium azide and crystal violet in the BACV, which was used as a selective medium, could have had an inhibitory effect on the growth of GES. In a previous study, there was a reduction of approximately 20% in the isolation of GES from BACV as compared to blood agar (unpublished data). However, in the same study, GES was isolated from BACV agar that had been streaked with an inoculum that contained less than fo CFU on blood agar. ACKNOWLEDGMENTS This work was supported by cooperative agreement no.

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2020-12-10T09:04:12.320Z

1973-06-01T00:00:00.000Z

237235277

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Improved Yield of Aflatoxin by Incremental Increases of Temperature Increasing the initial temperature of the rice fermentation of Aspergillus parasiticus NRRL 2999 from 15 to 21 C after 24 h of incubation and then to 28 C after 48 h resulted in about a fourfold increase in total aflatoxin over the usual fermentation which is held constant at 28 C for 6 days. The percentage of aflatoxin B1, the most toxic component, in the total aflatoxin was also increased. Increasing the initial temperature of the rice fermentation of Aspergillus parasiticus NRRL 2999 from 15 to 21 C after 24 h of incubation and then to 28 C after 48 h resulted in about a fourfold increase in total aflatoxin over the usual fermentation which is held constant at 28 C for 6 days. The percentage of aflatoxin B1, the most toxic component, in the total aflatoxin was also increased. The method of Shotwell et al. (7) for the production of aflatoxin offers several advantages, particularly for experiments requiring the continual feeding of aflatoxin to animals. The rice substrate is cheap, the fermentation has no interference in the common analytical methods, the toxic kernels are easily ground to a fine though not dusty powder which mixes easily and thoroughly into a variety of diets, and the yield is good enough so that usually the amount of toxic rice powder added to the diet is too small to alter the nutrient quality. Other advantages are the ease of extraction of the aflatoxin and its purity. Nevertheless, the quantities of aflatoxin consumed during feeding experiments are so large (880 mg during a typical dose response experiment with chickens [9]) that any improvement in yield represents appreciable savings in effort and expense. The present investigation was prompted by the observation of an unusually high yield of aflatoxin from a fermentation exposed accidentally during early incubation to subnormal temperatures as the result of an electric power shortage. A more systematic inquiry into the effect of temperature alterations during the fermentation resulted in a modification which gives about a fourfold increase in the yield of aflatoxin over that obtained with the method of Shotwell et al. (7). Except for the variation in temperature, the rice fermentations were done by the method of Shotwell et al. (7) in the flasks described by Smith and Hamilton (8). The contents of at least 40 flasks were combined for each determination. The results reported are the means of four replicate experiments. The replicate means were subjected to an analysis of variance in which an F-ratio was calculated, and because a significant ratio was obtained, the least significant difference between treatment means was determined (1). The total aflatoxin content of the fermented rice was determined by the method of Nabney and Nesbitt (4) with the modification of Wiseman et al. (10). The flasks were incubated on platform shakers in rooms where the temperature was controlled within 0.5 C of the stated temperature. The percentages of aflatoxin B1, B2, G,, and G2 were determined calorimetrically (4) after separation on thinlayer chromatograms (7). The effect on aflatoxin yield of varying the temperature of the fermentation during the initial 48 h of incubation is shown in Table 1. When the temperature was held at 15 C for 48 h before raising it to the normal 28 C for the remainder of the 6-day incubation period, the yield was increased highly significantly (P < 0.01) to 0.77 mg/g as compared to a yield of 0.46 mg/g when the temperature was maintained at 28 C throughout the fermentation. When the initial temperature of 15 C was increased to 21 C after 24 h and increased to the normal 28 C at the end of 48 h, the yield was increased still further (in a highly significant fashion; P < 0.001) to 1.85 mg/g. When the initial temperature of 15 C was raised to 28 C after 24 h and then to 32 C at 48 h, the yield was decreased highly significantly (P <0.01) from the control value to 0.07 mg/g. The percentages of aflatoxins B1, B2, G1, and G2 in the total aflatoxin are shown in Table 1 for the control fermentation in which the temperature was constant and for the fermentation conditions which gave the highest total yield. The percentages of 71, 9, 16, and 4 for B1, B2, G1, and G2, respectively, in the control fermentation agree closely with those obtained by Shotwell et al. (7). When the temperature was raised from an initial 15 C to 21 C after 24 h and then to 28 C after 48 h, the percentages were altered to 88, 2, 9, and 1, respectively. The enhancement of the B: G ratios of the toxins by changing the temperature agrees with earlier observations. Schroeder and Hein (6), Diener and Davis (3), and Schindler et al. (5) found increased production of aflatoxin B in relation to aflatoxin G when the fermentation was done at constant elevated temperatures. Schroeder and Hein (6) obtained evidence that the diminution of aflatoxin G relative to B at elevated temperatures was the result of accelerated catabolism of G at higher temperatures. Our data, in which the final temperature was the same in the two experiments, suggest instead that an enhanced production of aflatoxin B occurred in our experiments. This improved yield of aflatoxin by the use of incremental increases of temperature during fermentation has permitted a considerable savings in the time and effort needed for the production of aflatoxin for feeding trials. An additional benefit has been the enhanced percentage of B1 aflatoxin in the total aflatoxin since aflatoxin B1 is recognized as the most toxic of the components (2). LITERATURE CITED

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2020-12-10T09:04:12.368Z

1973-09-01T00:00:00.000Z

237229107

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Escherichia coli Field Contamination of Pecan Nuts More pecan samples collected from grazed orchards were contaminated with Escherichia coli than were samples from nongrazed orchards. No differences in frequency of contamination between mechanically and manually harvested nuts occurred. Nutmeats from whole uncracked pecans that were soaked for 24 h in a lactose broth solution containing E. coli did not become contaminated. Twentyfour percent of the whole pecans soaked in water for 48 h to simulate standing in a rain puddle developed openings along shell suture lines which did not completely close when the nuts were redried. Nutmeats as a potential source of microbial contamination of foodstuffs was not reported until 1927 when Weinzirl (12) found that candies containing nuts had more bacterial contamination than candies without nuts. In a later study (13) he indicated that the nutmeats were the source of the contamination, but attributed this primarily to unsanitary practices by the vendors handling the nuts. Further investigation revealed that, insofar as pecans were concerned, there was no one area in pecan processing that could be specifically cited as a contamination trouble spot, but that the entire process, involving tempering, cracking, shelling, storage, and handling, contributed to contamination (3,9,10,11). Standard methods for decontamination of nuts at shelling plants were proposed (11). It has been shown that some Escherichia coli on walnut hulls remain through the tempering process and subsequently contaminate the nutmeats (8). The walnut industry is becoming aware of the potential hazard caused by maintaining livestock in orchards (1). Chipley and Heaton (4) obtained shelled pecan samples from commerical operations and found that 20% of them tested positive for E. coli. This is of direct concern both to the producer, as evidenced by Food and Drug Administration seizures of such contaminated pecans (2), and to the consumer, as pecans are often eaten raw or entered raw into already cooked foods such as candy. Kokal and Thorpe (7) followed the E. coli contamination levels of almonds throughout the growing season and found that the level began to rise during the mechanical harvesting operation and peaked during the precleaning process. These processes are similar to those employed in pecan harvesting, which began to change to mechanical harvesting in the 1960s. This, coupled with the widespread practice of grazing cattle in pecan orchards, added a new dimension to problems of nut contamination by introduction of fecal material. We observed clumps of dried manure in some bins of mechanically harvested nuts from grazed orchards. In general, cattle are removed from pecan orchards several weeks prior to harvest. Sometimes the land is further prepared for mechanical harvest by disking the vegetation and manure under and smoothing the land. However, more often the grass cover is maintained as a permanent pasture and is therefore only mowed prior to harvest. Currently there is no testing or control of the amount or type of microbial contamination present on in-shell pecans before they enter the shelling plants. The objectives of this study were to compare the degree of E. coli contamination on nuts harvested from grazed and nongrazed orchards and to investigate some related problems in the orchard at harvest time. Samples were collected over a 2-year period. The 1970 harvest season was considered wet, and the 1971 season was considered dry. MATERIALS AND METHODS E. coli on pecans from grazed versus nongrazed orchards. Pecans were aseptically collected in sterile Whirl-pak bags (7.5 by 3 inches; 19.05 by 7.62 cm) during the harvest season from grazed and nongrazed pecan orchards in Georgia and Alabama. One sample per orchard in 1970 and five random samples from each orchard in 1971 were obtained. Each sample consisted of 100 to 150 g of in-shell pecans, out of which 50 g were rinsed in sterile buffer by the method of Hyndman (6). Serial dilutions ranging from 1 to 10-5 ml were made of the washings, inoculated into tubes of lauryl sulfate tryptose broth (LST), and incubated at 35 C for 48 h. After 24 and 48 h of incubation, all samples showing gas production were inoculated into tubes of EC medium and incubated at 45.5 C (5). Gas-positive samples after 48 h of incubation were inoculated into eosin methylene blue agar plates and incubated at 35 C. Different morphological type colonies on the eosin methylene blue plates were tested for indole-methyl red-Voges Proskauer-citrate (IMVIC) reactions. Penetration of unbroken shells by E. coli. Five replications of five nuts each of the pecan cultivar Cape Fear, having no visible cracks, were placed in lactose broth inoculated with E. coli. After 24 h at room temperature, the nuts were removed, surface sterilized in a 0.1% mercuric chloride solution, rinsed three times with distilled water, and aseptically cracked. Pieces of nutmeat and middle septum were removed and placed directly into tubes of LST broth. A sterile cotton swab was moistened in sterile buffer, wiped across the inside surface of the shell, and then placed in LST broth. These tubes were incubated at 35 C and observed for gas formation after 24 and 48 h. RESULTS AND DISCUSSION E. coli on pecans from grazed versus nongrazed orchards. There were about six times as many E. coli-contaminated samples collected from grazed orchards than from ungrazed ones ( Table 1). This proportion of contaminated samples was obtained during both the wet (1970) and the dry (1971) years, but the levels of contamination were greater during the wet year, with over one-third of the nuts being contaminated. A comparison of harvesting methods in these orchards revealed no differences in frequency of contamination between mechanically and manually harvested nuts ( Table 2). Penetration of unbroken shells by E. coli. After 48 h of incubation at 35 C, none of the LST broth tubes containing nutmeat, middle septum, or cotton swabs developed gas, indicating that the bacteria did not move through the intact shells to the pecan kernels. Formation of cracks and induced suture openings. Of the 25 nuts soaked in water for 1 week, 6 opened along the shell suture lines. None cracked anywhere else on the shell. These openings developed within the first 48 h of soaking, and none subsequently closed completely when the nuts were dried. Nuts sometimes stand in water for several days in rainy periods during the harvest season. In a grazed orchard, cracks induced by water absorption or mechanical harvesting could provide an easy entryway of E. coli penetration to pecan kernels. Cleaning processes employed by growers and shellers do not include removal of cracked but otherwise intact nuts. Such nuts also may not be decontaminated by currently used procedures prior to shelling and subsequently could contaminate shelling equipment. This study suggests that, even though E. coli would not move through unbroken shells of pecan nuts, it is possible for these shells to open along suture lines or be cracked in the field and thereby present an entrance for contamination into the nutmeats. This, in combination with the fact that there is a sixfold increase in contamination on nuts from grazed orchards over those from nongrazed orchards, would indicate that greater care will have to be exercised in pecan orchards than is currently being done to avoid E. coli contamination. The eventual permanent removal of cattle from pecan orchards may become a necessity.

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2020-12-10T09:04:16.707Z

1973-05-01T00:00:00.000Z

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Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation. Release of staphylococcal enterotoxin B (SEB) into the culture supernatant fluid during cultivation of Staphylococcus aureus is affected by the culture medium and relative concentration of its constituents (6,8,10,13,15). Factors such as pH (1,5,12), aeration, and agitation (3,10) also cause changes in the time of appearance and quantity of SEB released. This paper describes experimental production of SEB in a fermentor system in contrast to previous studies which utilized shaker-grown cultures. Superior advantages include precise, continuous measurement and control of pH, temperature, agitation, and aeration. Such precise control of the latter three factors allowed independent evaluation of other parameters such as pH and medium constituents, which also influence the excretion of SEB. MATERIALS AND METHODS Microorganisms, media, and cultivation. S. aureus strain 10-275, derived from strain S-6 for the production of maximal amounts of SEB, was employed. Stock inocula were prepared by lyophilizing 0. 1-ml samples of 18-h growth harvested from Trypticase-soy-agar plates in 10% skim milk. A separate sample was utilized for each experiment. Lyophilized organisms were inoculated into 4% N-Z Amine A (Sheffield Chemical, Norwich, N.Y.) and 1% yeast extract-broth (Difco), and they were incubated for 18 h at 37 C in a shaking water bath. After a second passage in this medium, 100 ml of an 18-h culture was inoculated into a 50-liter volume of medium in the fermentor (Fermentation Design, Bethlehem, Pa.). Media for the fermentor were designated as (i) AY medium consisting of 4% N-Z Amine A and 1% yeast extract-broth or (ii) AYD medium consisting of AY medium with 0.2% dextrose. All cultures were grown at a constant temperature (37 C), automatic foam control, and agitation rate (400 rpm). Sterilized air was sparged through the culture at the rate of 10 liters/min. Bacterial growth was estimated at hourly intervals by measuring the optical density of a 1:10 dilution of the culture at 540 nm. Plate counts of viable organisms were made concomitantly with turbidity measurements. Toxin assay. Cultures were sampled at hourly intervals, and the concentration of SEB in supernatant fluid was estimated from measurements of radial immunodiffusion reactions in serum-agar (8). Antiserum was produced in goats by hyperimmunization with highly purified SEB and was incorporated in 1% agarose gel (Marine Colloids, Inc., Springfield, N.J.) adjusted to pH 8.3 with 0.063 M borate buffer in 0.033 M NaCl; serum-agarose gels 2.5 mm in thickness were prepared on glass microscope slides. .0 resulted in optimal yields of SEB. In AYD medium, SEB concentration tripled within 3 h and increased to 580 Mig of SEB/ml by 10 h (Fig. 2); during this time viable counts increased from 1.0 x 108 to 1.1 x 1010 organisms/ml. Optical density measurements again paralleled viable counts. In AY medium, SEB concentrations remained at, or below, 0-h levels for 3 h; by 4 h a 10-fold increase in SEB occurred with maximal concentrations of 255 Mg of SEB/ml reached in 8 h (Fig. 3). Toxin release was unaffected by addition of 100 g of dextrose to AYD cultures after 5 h of fermentation. (ii) At pH 6.0 initiation of toxin production in AYD medium was somewhat delayed (Fig. 4). A fourfold increase occurred in 4 h; the concentration at 10 h was 268 Mg of SEB/ml. During this time, viable counts increased from 5.7 x 10' to 5.8 x 10' organisms/ml. (iii) At pH 8.0 toxin concentrations remained unchanged for 7 h after inoculation, and less than a twofold increase was noted at 8 h (Fig. 5). Toxin yield at 10 h was 32 Mg of SEB/ml. During toxin release, viable counts increased from 1.46 x 10' to 4.1 x 109 organisms/ml. A summary of hourly production of SEB under various conditions is presented in Ta VOL. 25, 1973 dard conditions, the effects of pH and dextrose addition could be studied independently of the effects of nutritional deprivation. ITOXIN Morse et al. (12) found that addition of 00 dextrose to a 1% protein hydrolysate medium caused a marked pH change during cultivation. Although the growth of the organism was not affected, there was a diminution in enterotoxin production. In our studies with a 4% protein hydrolysate medium, the inclusion of dextrose produced a lowering of the pH to 5.8 for a brief period, followed by a rapid return to neutral and later to alkaline. There was no disturbance in production of SEB, because pH rather than mal production of SEB requires a high concentration of casein hydrolysates (2,6). The medium, in addition to furnishing growth requirements necessary for production of SEB, provides adequate buffering capacity to prevent extreme acidity which may occur with the use of 1 or 2% protein hydrolysate media. By using a complete medium and rigidly controlled stan- dextrose appeared to be the limiting factor in the release of enterotoxin. Optimal production of staphylococcal alpha toxin was found by Duncan and Cho (4) to require 0.2% dextrose in the medium. Addition of dextrose 5 h after inoculation of the fermentor with pH control at 7.0 did not inhibit release of SEB. Optimal production of enterotoxin was achieved with AYD medium by controlling the pH at 7.0 (Table 1). Final viable cell counts revealed that at 10 h there was approximately 0.5-log less bacteria in either pH 6.0 or 8.0 controls. The pH 8.0 culture contained only minimal amounts of enterotoxin, and a significant amount was not produced until 8 h. At pH 6.0, fermentation produced only 45% of the toxin that was produced at pH 7.0. 771 In previous studies, the release of SEB into the culture supernatant fluid has been reported from mid-log to stationary phase (7,8,11,12). Two factors can explain these discrepancies: differences in media constituents and the requirement for an accurate assay of enterotoxin in the microgram range. The addition of 0.2% dextrose to medium rich in protein hydrolysate promotes good production of enterotoxin B. Significant amounts of SEB were produced when a medium consisting of 4% protein hydrolysate, yeast, and 0.2% dextrose was used. Cultures grown under these conditions produce enterotoxin in mid-log phase if pH is controlled at 7.0.

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2020-12-10T09:04:12.355Z

1973-03-01T00:00:00.000Z

237234630

s2

Optimum Temperature for Enterotoxin Production by Staphylococcus aureus S-6 and 137 in Liquid Medium The temperature for maximum toxin yield by Staphylococcus aureus S-6 and 137 in liquid medium cultured under aerobic conditions was determined to be 40 C. The optimum temperature for growth of Staphylococcus aureus is usually considered to be 37 C. Most studies involving enterotoxigenesis have been carried out at 37 C, although conclusive studies on optimum temperature for toxin production have not been made. Tatini et al. (Bacteriol. Proc., p. 17, 1971) reported that production of enterotoxins A, B, C, and D in Brain Heart Infusion broth was stimulated by incubation at 40 and 45 C. The present study describes the effect of temperature on toxin yields for two strains of S. aureus. S. aureus strains S-6 and 137 (obtained from the Food Research Institute, University of Wisconsin, Madison) were used as test organisms for enterotoxin B and C production, respectively.. For toxin yield studies, 1% inoculum was grown in 50 ml of liquid media (contained in 200-ml-size flasks) incubated in a water bath shaker (Precision Scientific Co., Chicago, Ill.) for 24 and 48 h at different temperatures. Temperatures were monitored by a Mettler digital thermometer: fluctuation was about +0.2 C throughout the experiments. For growth and toxin production kinetic studies, cells were grown in 75 ml of liquid media contained in nepheloflasks (300 ml; Bellco Glass, Vineland, N.J.) At regular intervals growth was measured by Klett-Summerson colorimetry (540 nm), and 1-ml samples were aseptically removed for toxin analysis. Culture samples were centrifuged at 20,000 x g for 15 min to obtain cell-free supernatant fluids. Toxin analysis, both qualitative and quantitative, was performed by using the capillary tube technique of Fung and Wagner (1). Two to four replicate samples were tested for each temperature studied. Among the 12 protein hydrolysates tested (individually and in combinations; 3% of each hydrolysate) with S-6 at 37 C, PHP-NZ-Amine NAK (2), PHP-Edamine S, PHP-NZ-Amine E, and PHP-soy peptone medium (Table 1) were found to support more than 400 gg of enterotoxin B production/ml. Effect of incubation temperature (10, 15, 20, 25, 30, 35, 37, 40, 42, 44, 45, and 50 C) on enterotoxin yields by S-6 in the four PHP media and a beef broth and by 137 in the PHP-NZ-Amine NAK medium ( Fig. 1 and 2) showed that the highest toxin yield after 24 h was at 40 C. Toxin B yields by S-6 in the four PHP media and beef broth were 450 to to.oos, 16 = 3.252 Conclusion: The difference between the toxin yield from the two temperatures is significant at an 0.5% level. a Analysis to test the difference between the means of two independent samples was performed. Effect of incubation at 37 and 40 C on growth and enterotoxin release by S. aureus S-6 and 650 jig/ml and 60 ,g/ml, respectively. After 48 h of incubation of each medium at each temperature (data not shown) a detectable amount of toxin B was obtained from 15 C and an increase of about 5% toxin yield from other incubation temperatures. About 20 to 40% decrease in toxin yield was obtained at 30, 35, 37, and 42 C compared to 40 C, and about 80 to 95% decrease at 15, 20, 25, 44, and 45 C. No toxin was detected at 10 and 50 C after 48 h of incubation. Similar findings were observed for 137 in the PHP-NZ-Amine NAK medium (Fig. 2). The maximum toxin C yield at 40 C was 200 ,gg/ml. Statistical analysis of enterotoxin B yields at 37 and 40 C by S-6 grown in PHP-NZ-Amine NAK medium showed that the difference between the enterotoxin yields at the two temperatures is significant at a 0.5% confidence level, with higher yields at 40 C ( Table 2; reference 3). The kinetics of growth and toxin production of S-6 and 137 at 37 and 40 C for the first 12 h are presented in Fig. 3. The total growth yields after 24 h (about 600 Klett units) and growth rate constants ( Fig. 3; about 1.5 per h) of the two strains were almost identical for the two incubation temperatures, with a shorter lag occurring at 40 C. Both S-6 and 137 released their respective enterotoxins earlier and in larger quantities when incubated at 40 C compared to 37 C. These data showed that 40 C is the optimum temperature for enterotoxin yields for the two strains tested. Another strain, S. aureus 217 (data not shown), also exhibited the same phenomenon. We thank M. S. Bergdoll for the supply of cultures, enterotoxins B and C, and their specific antisera. The assistance of Richard D. Miller and Lawrence Kingsley are appreciated.

v3-fos

2017-10-19T14:38:16.022Z

1973-12-01T00:00:00.000Z

10954670

s2

Virus Particles from Conidia of Penicillium Species Virus particles and their component double-stranded ribonucleic acid (dsRNA) have been isolated from conidia and mycelia of certain Penicillium species. The conidia and mycelia of P. stoloniferum NRRL 5267 contained 75 and 85 ,ug of dsRNA/g (dry weight), respectively. Of the total dsRNA released from NRRL 5267 conidia, 10% was nonencapsulated. Conidia of P. brevi-compactum NRRL 5260 and P. chrysogenum Q-176 contained 2 and 120 ,ug of dsRNA/g (dry weight), respectively, whereas mycelium from the two species contained 3 and 95 ug of dsRNA/g (dry weight), respectively. No viruses were isolated from conidia Polyhedral virus particles have been reported in a number of fungal species (5,12). Ellis and Kleinschmidt (11) were the first to present evidence that viruses occur in Penicillium stoloniferum. Banks et al. (3) isolated and characterized viruses and their component double-stranded ribonucleic acid (dsRNA) from P. stoloniferum. Bozarth et al. (6) described two serologically distinct viruses having different electrophoretic mobilities from P. stoloniferum NRRL 5267. Wood et al. (20) isolated a virus from P. brevi-compactum and demonstrated the double-stranded nature of its component RNA. The component dsRNA of a virus from a penicillin-producing strain of P. chrysogenum was characterized by Lemke and Ness (14). Viruses have been observed throughout the cytoplasm in thin sections of conidiospores of P. stoloniferum and P. brevi-compactum (13), of mushroom basidiospores (10), and of the zoospores of Plasmodiophora (1). However, the isolation and biochemical characterization of virus from fungal conidia have not been reported. We have isolated and characterized viruses and their component dsRNA from conidia of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, and P. chrysogenum Q-176. Although Banks et al. (4) have described an efficient pilot plant-scale isolation of viral dsRNA from several species of Penicillium and Aspergillus and Moffitt and Lister (17) have recently reported a rapid method for surveying fungal isolates for dsRNA-containing viruses, we developed a method for both the detection and isolation of viruses and dsRNA from subgram quantities of conidia and mycelia. The release of macromolecules, with time of disruption of conidia, provides the information necessary for quantitatively comparing concentrations of viral dsRNA in conidia and mycelia. The comparison provides an insight into the possible mode of transmission and rate of replication of viruses from the conidial to the hyphal stages of growth. MATERIALS AND METHODS Production and harvest of conidia. Cultures of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, P. chrysogenum Q-176, and P. stoloniferum NRRL 859 were furnished by the Agriculture Research Service Culture Collection, Northern Regional Research Laboratory. These cultures were maintained on potato-dextrose agar (PDA). Conidia of the organisms were produced by the method of Sansing and Ciegler (unpublished), as follows. White bread, containing no preservatives, was cut into 1.5-cm cubes; 200 g of the cubed bread was placed in a 2.8-liter Fernbach flask and autoclaved for 15 min. The bread was inoculated with 20 ml of a conidial suspension from PDA slant cultures and incubated for 10 days at 28 C. Conidia were harvested from the bread by adding 800 ml of a 10-'% solution of Triton X-100 and shaking the flask, after which the conidial suspension was filtered through cheesecloth to remove the bread cubes and the suspension was filtered through glass wool to remove bread fines. Conidia were pelleted by centrifugation at 2,000 x g for 15 min. The conidial pellet was suspended in 0.1 M potassium phosphate buffer, pH 7.2, and recentrifuged. This step was repeated twice to remove any starch particles remaining from the bread. Disruption of conidia. Known concentrations of conidia (as given under individual sections) were suspended in 0.1 M phosphate buffer (pH 7.2) and added to 75-ml glass Bronwill cell homogenizer flasks, each containing 45 g of 0.5-mm glass beads. Each sample was homogenized for a specific time (see assay sections) in a Bronwill mechanical cell homogenizer (Braun model MSK) at 4000 rpm under a cold CO2 stream. All homogenizing flask temperatures were maintained at 0 to 1 C. Samples of homogenates were examined microscopically to determine the percentage disruption of conidia. Disruption of mycelia. A 10% sucrose-2% yeast extract medium was inoculated with a conidial suspension and incubated at 28 C on a Brunswick shaker at 250 rpm in 2.8-liter Fernbach flasks (500 ml of medium) for 72 h. Mycelia were harvested by vacuum filtration and then suspended in 0.1 M phosphate buffer, pH 7.2 (5 ml of buffer/g [wet weight ] of mycelium). The suspension was added to 75-ml Bronwill flasks containing 45 g of 1.0-mm glass beads and homogenized for 3 min at 4000 rpm under a CO2 stream. Detection of virus. The homogenates resulting from the disruption of 1.5 g (dry weight) of conidia of each of the four Penicillium strains were centrifuged at 2,000 x g (10 min) to remove cell debris as described in Fig. 1. The supernatant fluid (SNF) was centrifuged at 105,000 x g for 2.5 h. The resulting pellet was suspended in 2 ml of 0.1 M phosphate buffer, pH 7.2, and centrifuged at 4,000 x g for 10 min. The supernatant was filtered through a 0.45-gm membrane filter (Millipore Corp.). The virus preparation was applied to carbon-coated Formvar grids, stained with 0.5% uranyl acetate, and rinsed twice with distilled water. The grids were examined by electron microscopy (RCA EMU-3 electron microscope) at an instrument magnification of x 32,000. The viruses were analyzed by polyacrylamide gel electrophoresis on 2.4% gels for 5 h (6 mA per tube) at 25 C as described by Loening and Ingle (15). Gels were scanned at 260 nm with a Gilford linear transport system. Isolation and quantitation of viral dsRNA. As shown in Fig. 1, two volumes of cold methanol were added to the SNF from each conidial homogenate, and the precipitates were sedimented by centrifugation at 2,000 x g for 10 min. Each precipitate was dissolved in 0.2 M sodium acetate and treated with an equal volume of aqueous 90% phenol containing 0.1% (wt/vol) 8-hydroxy quinoline (9). The mixture was shaken for 20 min at 25 C and centrifuged at 4,000 x g for 20 min. Nucleic acid was freed from phenol by repeated precipitation from 0.2 M sodium acetate with equal volumes of cold methanol. The nucleic acid precipitate was dissolved in a minimal volume of 0.15 M NaCl-0.15 M sodium citrate (SSC) solution, pH 7.4. The RNA samples were incubated with 1.0 gg of ribonuclease B (Sigma Chemical Co.) per ml of 0.3 M STE buffer (0.3 M NaCl, 0.01 M tris(hydroxymethyl)aminomethane, and 0.001 M ethylenediaminetetraacetic acid) at 37 C for 30 min. The remaining RNA was precipitated with cold methanol, the mixture was centrifuged at 8,000 x g for 10 min, and the pellet was redissolved in SSC buffer and subjected to electrophoresis. Mobilities of RNA components were compared to those of a purified dsRNA preparation (P. stoloniferum NRRL 5267) of known concentration and standard yeast transfer RNA (Sigma Chemical Co.). The dsRNA was measured by integration of the areas under the electrophoretic peaks compared to standards. Time release of macromolecules. Three 1.5-g (dry weight) samples of P. stoloniferum NRRL 5267 conidia were homogenized for 1, 2, and 4 min, respectively, by the previously described method. Three 3.0-g and three 6.0-g samples (dry weight) of conidia were homogenized for the same time intervals. Cell debris was removed from homogenates by centrifugation. The SNF was quantitatively assayed for total RNA, deoxyribonucleic acid (DNA), protein, and trehalase by the following methods. Quantitation of total RNA, DNA, and protein in conidial homogenates. Supernatants from centrifuged conidial homogenates were assayed for RNA by the orcinol method of Brown (7) and for DNA by the method of Burton (8). The pellet resulting from the hot trichloroacetic acid treatment of homogenates was dissolved in 1 N NaOH by heating at 90 C for 30 min. This solution was quantitatively assayed for protein by the method of Lowry et al. (16). Determination of trehalase activity. Trehalase activity was assayed by adding 0.1 ml of SNF to test tubes containing 2.0 ml of trehalose (2.0% wt/vol) in 0.1 M potassium phosphate buffer (pH 5.6) and incubating them at 25 C for 1 h. Glucose was assayed by the method of Nelson (19). Ratio of encapsulated to nonencapsulated dsRNA. Five 3.0-g (dry weight) samples of P. stoloniferum NRRL 5267 conidia were homogenized for 2 min. Cell debris was removed, and the resulting SNFs were pooled and centrifuged at 105,000 x g for 2 h to pellet virus particles. The virus-free SNF was quantitatively assayed for dsRNA. The virus pellet was suspended in 10 ml of 0.1 M phosphate buffer (pH 7.2), and 5 ml was assayed for viral dsRNA. The remaining suspension was diluted to 45 ml with buffer and homogenized in a Bronwill flask containing 45 g of 0.5-mm glass beads, as described for disruption of conidia. This homogenate was centrifuged at 105,000 x g for 2 h, after which SNF was assayed for dsRNA. Detection of virus and dsRNA in conidia. Analyses of conidial homogenates of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, and P. chrysogenum Q-176 by electron microscopy demonstrated the presence of isometric virus particles similar in size to those reported from mycelia (2,6,11,13,14,18,20). Their electrophoretic mobilities were identical to those determined from mycelial sources in this investigation. No particles were detected in conidial or mycelial homogenates of strain NRRL 859 of P. stoloniferum. Electrophoretograms of viral dsRNA isolated from conidia of the three Penicillium species, after treatment with ribonuclease, are depicted in Fig. 2. The five dsRNA bands observed for the fast-and slow-moving virus species of P. stoloniferum are identical to bands observed from mycelial extracts (6). No attempt was made to determine concentrations of the fastand slow-moving dsRNAs. The virus particles from P. brevi-compactum conidia contained the same three dsRNA species reported for mycelial extracts of the organism (20). Similarly, the three distinct bands observed for the virus from conidia of P. chrysogenum correspond to those reported for mycelial extracts (18). The three bands of P. brevi-compactum have electrophoretic mobilities similar to those for P. chrysogenum. The concentration of viral dsRNA in conidia and mycelia of the three species is compared in Table 1 Electrophoretic profiles (polyacrylamide gel) of dsRNA extracted from conidia of Penicillium species. The isolated RNA from the three fungal species were separated electrophoretically on polyacrylamide gels (2.4%) for 3 h at 6 mA/tube. almost twice that from P. stoloniferum (75 ig/g dry weight) and 60 times greater than in P. brevi-compactum (2 Mg/g dry weight). The concentration of viral dsRNA in the conidia of these species is quite comparable to that found in their mycelial forms. Time release of macromolecules. The relationship between concentrations of macromolecules (RNA, DNA, and protein), concentrations of conidia, and disruption time is shown in Table 2. Optimal breakage of conidia (88 to 94%) occurred at 4 min. At all conidial concentrations the percent of breakage increased with disruption time. The release of macromolecules also increased with increased disruption time at all three conidial concentrations; maximal release came at the 4-min time intervals. Trehalase activity increased as a 95 a These values were obtained by analyses of RNA extracted from 1.5 g (dry wt) of conidia of NRRL 5267 and Q-176, and 15 g (dry wt) of conidia of NRRL 5260. b These values were obtained by analyses of RNA extracted from mycelia harvested after 72 h of growth at 28 C as a shake culture. function of disruption time, with maximal activity occurring at 4 min. The amount of dsRNA released from disrupted conidia represents 0.8 to 1.0% of the total RNA released and is 0.003 to 0.004% of the total spore mass on a dry weight basis. Nonencapsulated dsRNA in conidia. Quantitative analyses of 15 g of conidia (dry weight) from P. stoloniferum NRRL 5267 indicated that 10% of the dsRNA exists free in the conidia. Homogenization of virus with subsequent repelleting resulted in no detectable dsRNA in the SNF. This failure to detect nonencapsulated dsRNA indicated that viruses are not being disrupted as a result of mechanical homogenization. DISCUSSION This report describes the first isolation and characterization of virus particles from fungal conidia. Electron microscope comparisons of particles isolated from conidia or mycelia of a particular isolate show that particles from either source are structurally identical. Biochemical and physical comparisons of mycoviral dsRNA isolated from conidia or from mycelia of a particular isolate show that the dsRNA is qualitatively the same. Quantitatively, the ratio of viral dsRNA per gram (dry weight) of conidia is equal to the ratio of viral dsRNA per gram (dry weight) of mycelia. The presence of virus in conidia explains a mechanism for viral sustainment during nonvegetative stages of the life cycle of fungal isolates.

v3-fos

2020-12-10T09:04:17.322Z

1973-09-01T00:00:00.000Z

237230590

s2

Viability of Actinomycetales Stored in Soil About 1,800 Actinomycetales stored in soil for up to 20 years were checked for viability. About one-half were viable. Since the early 1950s, we have used soil culture, agar slant culture, and lyophilization concomitantly for preservation of all strains of aerobic Actinomycetales, mostly streptomycetes and streptoverticillia. The large number of strains accumulated over the years has forced abandonment of routine transfer on agar slants and, more recently, the use of soil cultures. The decision to abandon preservation by soil culture prompted examination of these materials to determine how many had survived storage at room temperature over periods up to about 20 years. The information obtained is presented herein, along with some miscellaneous observations relating to this collection. Three different kinds of soil cultures had been prepared. An initial lot had been made by adding suspensions of spores to sterilized loamy soil. These preparations, stored in a refrigerator under humid conditions, became contaminated with molds and, for the most part, had to be destroyed. A second lot of soil cultures was prepared by adding broth cultures to sterilized prairie black loam soil and allowing them to dry at room temperature (2) with subsequent storage at room temperature. In 1959, a third lot of cultures was prepared in a silica sand-CaCO, amended soil (screened, air-dried prairie black loam soil, 10,000 g; white silica sand, 7,500 g; and CaCO,, 25 g; mixed by hand and screened). Because of the possibility of mite infestation, the cotton stoppers of one lot of tubes were treated with a miticide (0.1% HgCl, in 95% ethanol containing identification dye). Unfortunately, this lot was additionally sealed with rubber stoppers over the cotton plugs to facilitate numbering of tubes for rapid retrieval from large storage racks. Many of these cultures died, possibly because of the exclusion of air or permeation of the soil with ethanol, or both. IThis study was presented, in part, at the 73rd annual meeting of the American Society for Microbiology, Miami Beach, Fla., 6-11 May 1973. New soils had to be prepared for each of these after only short periods of storage (up to 1 year). With the passage of time, much dust accumulated on the cotton stoppers. Despite this, few cultures were found to be contaminated (Table 1). About one-half of the tested soil cultures contained viable Actinomycetales ( Table 1). Age of storage seemed to have little effect on the number of strains viable. The results suggest some other reason for death of the cultures, e.g., the method of preparation, the drying out process, or exposure to fluctuating room temperature. Our experiences with these soil cultures and with maintenance on agar slants is an argument in favor of lyophilization as a means of preservation. Lyophilized preparations have shown much higher percentage of survival and less risk of contamination of the cultures based on the experiences of the several curators of the Agricultural Research Service Culture Collection over a 25to 30-year period. B. P. was an Agency for International Development scholarship trainee, Department of Health, Bangkok, Thailand.

v3-fos

2019-03-19T13:06:09.757Z

1973-01-01T00:00:00.000Z

82808100

s2

The Change of Amount of Inhibitors Inducing Dormancy in the Dutch Iris Bulb Although many studies have been done on flower forcing in Dutch iris (Iris hollandica cv. Wedgwood) by the cooling treatment, references to the problem of endogenous growth regulators in the same plant material have been rather few. It is well known to floriculturists that flower forcing in this plant necessitates a previous break of the dormancy of the bulb before cooling. Kimura and Stuart (1972) carried out experiments to find an appropriate method for breaking dormancy, applying various time-temperature combinations. The present study was aimed at proving the existence of endogenous growth inhibitors which may regulate the dormancy of Dutch iris bulbs, to identify them chemically, and to clarify the role of high temperature in breaking the dormancy of iris bulbs. Experiment 1. Bulbs of Dutch iris were obtained from Fukuoka Prefecture on May 30, 1970. They were divided into two lots, each of which was stored at 100 and 20°C respectively until late August. The bulbs from each lot were bored through the central part containing the main bud, using a cork borer. The bulb columns thus produced were 2 cm in length and 1 cm in diameter. The boring of the bulbs was repeated at weekly intervals from June 7 to the end of August. The total weight of ten columns was about 17 g. These columns were ground into powder while they were cooled with dry ice-methanol. Two grams of the powder were mixed with 90% methanol to extract the inhibitor. The extraction was repeated twice. The ethyl acetate-soluble acidic and neutral fractions were obtained by the procedure shown in Fig. 1. These fractions, which were dissolved in 3 ml of ethyl acetate, were spotted on Toyo No. 51 filter paper and developed with iso-propanol : ammonia : water (10 :1 :1 v/v). The activities of the endogenous growth inhibitor in 11 segments of the chromatograms were measured using Nitch's straight growth test of Avena coleoptile (Nitch and Nitch 1956) with the modification by an addition of 0.1 r IAA*) in each test sample. It was shown that the addition of IAA in this test is effective in enlarging the difference among the inhibitions. genous growth inhibitors which may regulate the dormancy of Dutch iris bulbs, to identify them chemically, and to clarify the role of high temperature in breaking the dormancy of iris bulbs. Experiment 1. Bulbs of Dutch iris were obtained from Fukuoka Prefecture on May 30, 1970. They were divided into two lots, each of which was stored at 100 and 20°C respectively until late August. The bulbs from each lot were bored through the central part containing the main bud, using a cork borer. The bulb columns thus produced were 2 cm in length and 1 cm in diameter. The boring of the bulbs was repeated at weekly intervals from June 7 to the end of August. The total weight of ten columns was about 17 g. These columns were ground into powder while they were cooled with dry ice-methanol. Two grams of the powder were mixed with 90% methanol to extract the inhibitor. The extraction was repeated twice. The ethyl acetate-soluble acidic and neutral fractions were obtained by the procedure shown in Fig. 1. These fractions, which were dissolved in 3 ml of ethyl acetate, were spotted on Toyo No. 51 filter paper and developed with iso-propanol : ammonia : water (10 :1 :1 v/v). The activities of the endogenous growth inhibitor in 11 segments of the chromatograms were measured using Nitch's straight growth test of Avena coleoptile (Nitch and Nitch 1956) with the modification by an addition of 0.1 r IAA*) in each test sample. It was shown that the addition of IAA in this test is effective in enlarging the difference among the inhibitions. A comparison of quantitative changes of endogenous acidic inhibitors is shown in Fig. 2. Two inhibitory zones, inhibitors a (Rf. 0.1-0.4) and j3 (Rf. 0.5-0.7), were clear in both lots until mid-July; then the amount of inhibitor 3 was slightly greater than that of inhibitor a. A gradual decrease of inhibitor j9 occurred in the material of the 20°C lot after mid-July, while no changes in inhibitor j3 were noted in the 10°C lot. In the data on August 9, inhibitor j3 in the 20°C lot disappeared completely and a small amount of promoter appeared. On the day of assay sampling, bulbs were dissected and the length of the first leaf in the bulb was recorded. The results are shown in Fig. 3. A clear difference between the two lots was recognized. While the first leaf of bulbs stored at 20°C showed remarkable elongation, that of bulbs stored at 10°C elongated only slightly, but with thickening, indicating that new bulbing was occurring. Bulbing continued until the completion of a new bulb formation inside the the old scales, which became thin white membranes at 10°C. Newly formed bulbs remained dormant over one year. first leaf in bulbs ance coincided with the elongation of the first leaf formed in Experiment 1, it was tried to obtain inhibitor j3 from bulb (16 kg) by fractionation with ethyl acetate and the mass paper chromatography in late June, 1971. Crude inhibitor p obtained was purified successively by charcoal and silicic acid-celite adsorption chromatography, and the optical rotatory dispersion of its methanol solution was measured. The result showed that there was a typical cotton effect of ABA.*) Then the gas chromatographic analysis was performed, using a Hitachi K53 gas chromatograph. The result of the analysis was that the main inhibitory substance is identified with ABA ( Fig. 5). On the other hand, the inhibitors in the neutral fraction, whichh was extracted from bulbs (1972) of about 100 kg, were purified by column chromatography. N.M.R., I.R., and Mass spectra were recorded for these substances. The inhibitors in the neutral fractionn were identified with capric acid and related compounds. Discussion and conclusion. Beij er (1952) reported his experimental results obtained from the long term storage of the iris bulb. Bulbs stored at 25.5° and -0.5°C exhibited no growth for a long period. Bulbs stored at a high temperature (25.5°C) were able to be used for forcing without any difficulty, but no flowering resulted *) abscisic acid in the bulbs stored at a low temperature (-0.5°C). The data of the present study and of another series of experiments done in the same laboratory (Tsukamoto and Ando, 1973) are in line with that of Beij er. It is interesting to note that the new bulb formation in a mother bulb stored at 10°C for a long period is similar to the pupation of the freesia corm, on which Mansour (1968) conducted an intensive experiment. Aoba (1972) also reported the similar facts foundd in freesia corm. However, in these papers there was no elucidationn of this phenomenon from the standpoint of an endogenous regulator. Judging from the data on the quantitative changes in inhibitors found'd in the present study, it may be concluded that the breaking of the dormancy of iris bulbs is due to a decrease of inhibitors, which releases the sprouting activity from the dormant state. The authors clarified that the first inhibitor inducing dormancy is ABA and the second ones are certain fatty acids. Recently, some other inhibitors have been found in the neutral fraction. For example, Hashimoto et al. (1972) reported that neutral inhibitors, batatasins, induce the dormancy of bulbils in yam. This is the first information that a fatty acid plays an inhibitory role in dormancy Tso (1964) studied the inhibitory erect of the methyl ester of a fatty acid on the lateral shoot elongation in the tobacco plant, using a f oliar spray. He concluded that the methyl ester of capric acid is the strongest in its inhibitory effect. Poidevin (1965) tested the inhibitory activities of saturated fatty acids on the germination of mustard seeds, and came to the conclusion that only certain fatty acids, which contain 8 to 11 carbon atoms, show inhibitory activity. These results coincide with the data of the present study. Lenton et al. (1972)

v3-fos

2020-12-10T09:04:12.816Z

1973-07-01T00:00:00.000Z

237230035

s2

Stimulation of Lactic Acid Bacteria by a Micrococcus Isolate: Evidence for Multiple Effects Growth of, and rate of acid production by, six cultures of lactic acid bacteria were increased in the presence of Micrococcus isolate F4 or a preparation of its capsular material. Concentrations of hydrogen peroxide found in pure cultures of the lactic acid bacteria were not detectable, or were greatly reduced, in mixed culture with Micrococcus isolate F4. The capsular material was not as effective as whole cells in preventing accumulation of H2O2. Catalase stimulated growth of, and the rate of acid production by, the lactic acid bacteria, but not to the same extent as Micrococcus isolate F4 in some cultures. The existence of two mechanisms for micrococcal stimulation of the lactic acid bacteria is postulated. One mechanism involves removal of H2O2; the other has not been characterized. Acid production by lactic acid organisms in milk can be stimulated by the addition of various plant and tissue extracts (3, 4, 9, 12-14, 20, 23). Addition of ferrous ions and catalase to milk also stimulated acid production by certain lactic streptococci, apparently due to the destruction of metabolically produced peroxide which is inhibitory (7). However, in mixed populations there may be many factors which govern the growth of an organism. Some strains of Streptococcus lactis and Streptococcus cremoris produced a heat-labile inhibitor of Lactobacillus casei (1), whereas a peptide from cell extracts of S. lactis stimulated the growth of L. casei (2). Growth of propionibacteria was stimulated by lactobacilli in cheese (17) and by micrococci in lactate agar and Emmental cheese (18,19). Stimulation of acid production and growth of lactobacilli by capsular material from micrococci has been demonstrated (16). Lactic acid bacteria, important in the production of certain fermented foods, have also been shown to have an antagonistic effect on staphylococci and salmonellae, food-borne pathogens (8), and on a variety of food spoilage organisms (5), including sporeformers (10). In the present study we have investigated the interaction between a Micrococcus isolate and several lactic acid bacteria. The involvement of hydrogen peroxide is described. MATERIALS AND METHODS Cultures. The isolation of Micrococcus F4 used in these experiments was described previously (16 Measurement of growth, peroxide, and acid production. Experiments were carried out in nonfat milk solids reconstituted to 10% and steamed for 30 min. Where indicated, freeze-dried capsular material was added to milk (1 mg/ml) before steaming. The capsular material was prepared from Micrococcus isolate F4 as described previously (16). Catalase (2,000 U per mg; Sigma Chemical Co., St. Louis, Mo.) was filter sterilized at a concentration of 1 mg/ml and added to the milk, where indicated, to a final concentration of 0.01 mg/ml. An equal volume of sterile distilled water was added to control cultures. Experiments were started by inoculating 75 or 100 ml of tempered milk in a 250-ml flask to 107 to 7 x 107 colony-forming units (CFU) per ml with a 16-h litmus milk culture of streptococci or lactobacilli. Micrococcus isolate F4 was collected by centrifugation and suspended in phosphate buffer before addition, where indicated, to a final concentration of 107 to 2 x 107 CFU per ml. Cultures were incubated without shaking at the appropriate temperature for growth of the lactic acid bacteria. At intervals, samples of the culture were removed for analyses. CFU were determined by plating on Standard Methods Agar with and without 5% NaCl. The addition of 5% NaCl was found to prevent growth of streptococci but not micrococci during a 24-h incubation period. To measure acid production, 5-ml samples of culture were added to 5 ml of cold (5 C) distilled water and titrated to pH 8.4 with 0.1 N NaOH. The rate of acid production is expressed as the maximum slope of the curve obtained by plotting volume of 0.1 N NaOH required for titration against time (A ml 0.1 N NaOH per h). The enzymatic method of Gilliland (6) was used to determine residual peroxide in 10-ml samples removed from the culture at intervals during incubation. RESULTS The rate of acid production by several lactic acid bacteria was increased on the addition of Micrococcus isolate F4 to milk cultures (Table 1). There does not appear to be any correlation between the wide range of rates of acid production (0.06 to 0.74 ml 0.1 N NaOH per h) in pure culture and the extent of the increase in the presence of F4 (15-180%). The effect of F4 on growth also varied widely ( Fig. 1 and 2, Table 2) and can account for the increase in acid production. The increase in acid production was directly proportional to the percentage of increase in cell number (Fig. 3) (assuming the cell number is proportional to CFU). F4 added to cultures of S. lactis C10 increased the rate of growth, but not the final cell yield (Fig. 2). Both the final cell yield (Table 2) and the rate of growth was increased when F4 was added to L. casei ( Fig. 4) and to S. cremoris Cl. Addition of F4 to S. lactis 5 ( Table 2) and S. thermophilus ( Fig. 1) increased the final cell yield but not the rate at which the culture grew. Growth of F4 alone in milk did not result in a change in pH. However, growth of F4 under the conditions of these experiments was usually poor. The growth rate ranged from no growth to In three separate experiments, the rate of growth of S. thermophilus was 4.0 doublings per h. The rate and extent of growth of F4 appeared to depend at least on the rate of acid production by the lactic acid bacteria and the temperature of incubation. At temperatures above 30 C or during rapid acid production, growth of F4 was inhibited. The amounts of hydrogen peroxide formed by the cultures also varied (Fig. 5). L. casei did not produce detectable quantities of hydrogen perroxide. S. thermophilus, in one of three experiments, produced 0.115 ttg of H202 per ml at 6 h, but no peroxide was detectable over the remainder of the time interval measured (0-6 h and 25 h). The concentration of H202 (0.8-1.8 gg/ml) that occurred in pure cultures of S. lactis 5, S. cremoris Cl, and S. lactis C10 were reduced to less than 0.05 gg of H202 per ml (essentially background) in mixed culture with F4. The relatively high concentraions of H202 produced by L. bulgaricus were markedly reduced in the presence of F4, but remained detectable during the periods of maximum production. To see if F4 was simply removing H202 from the medium, the effects of catalase and F4 on growth, acid production, and H202 production were compared. Except in one case, no H202 was detectable in medium inoculated with lactic acid bacteria in the presence of 0.01 mg of catalase per ml. In cultures of L. bulgaricus to which catalase was added, low levels of H202 were detectable during the period of maximum production equivalent to the levels found in the presence of F4. Acid production by L. bulgaricus was stimulated by catalase to almost the same extent as by addition of F4 (data not shown). Catalase and F4 were equally effective in stimulating acid production by S. thermophilus (Fig. 6); however, the final cell yields were higher in flasks containing Micrococcus F4 ( Fig. 1 and Table 2). Addition of catalase to cultures of S. lactis C10 also stimulated acid production by, and growth rate of, the culture to almost the same degree as F4 (data not shown). The final cell yield was not increased by either catalase or F4 ( Table 2). Although no H202 was detected in cultures of L. casei, the addition of catalase caused some stimulation both of acid production (Fig. 7) and growth rate (Fig. 4). However, F4 was much more stimulatory. This is seen by comparing increase in acid production, rate of cell growth, and final cell yield ( Table 2). These results imply a second means of stimulation by F4 in addition to stimulation by removal of H202. The capsular material prepared from Micrococcus isolate F4 is a heat-stable substance containing riboflavine, N-acetylhexosamine, sialic acid, glutamic acid, and an allo-isoleucine (16). The abilities of capsular material and whole cells to stimulate growth and acid production of S. lactis C10 were compared. The stimulation of acid production (Fig. 8) and growth (Fig. 2) by capsular material was as great as that by whole cells. However, the capsular material was not as effective as whole cells in preventing an early accumulation of H202, although some activity was evident (Fig. 9). Capsular material did not reduce the concentration of H202 added to uninoculated milk or to 0.01 M acetate, pH 4.5. This suggests again that capsular material, and presumably the whole cell, stimulates S. lactis C10 by some means in addition to removing H202. 3. Relation between increase in rate of acid production and increase in cell number. The rate of acid production in the presence and absence of F4 was measured, and the percentage increase was calculated ( Table 1) and is plotted against the percentage of maximum increase in cell number (Table 2) DISCUSSION The data presented indicate that whole cells of Micrococcus isolate F4 and its capsular preparation stimulate growth and acid production in several lactic-acid-producing bacteria. There appear to be two ways in which the addition of whole micrococci stimulate cultures of lactic acid bacteria. The presence of F4 cells prevents accumulation of detectable levels of H202 and the addition of catalase results in stimulation of acid production and growth of the bacteria tested. We conclude, therefore, that the ability of the micrococci to remove H202 is at least in part responsible for stimula- tion. However, we postulate the existence of at least one other mechanism of stimulation to account for the greater stimulatory activity of F4 compared with catalase in cultures of S. thermophilus (Fig. 1) and L. casei ( Fig. 4 and 7). A second means of stimulation is also implicated in experiments with capsular material added to the culture of S. lactis C10. In these experiments, increased growth occurs even though the H202 accumulation is either not reduced (Fig. 9) or only slightly reduced (data not included). The data also suggest that, in S. lactis C10, either removing H202 or affecting the unknown target is sufficient to stimulate the culture. It is evident that under certain conditions H202 may not be inhibitory. Organisms without catalase that are not particularly sensitive to H202 have been reported (15). Gilliland and Speck (7) described a L. bulgaricus strain that was not stimulated by catalase even though in the absence of catalase it produced larger quantities of H202 than did a S. lactis culture which was stimulated by catalase. This is another situation where H202 may not be inhibitory. Our culture of L. bulgaricus, however, was stimulated by addition of cata- lase. It may be pointed out here that stimulation of acid production by addition of catalase does not necessarily mean that the lower rates of acid production were due to an inhibitory concentration of H202. The peroxidatic activity of catalase has been demonstrated with substrates such as ethanol acting as hydrogen donor (11). It is possible that increased growth is due not to removal of inhibitory concentrations of H202, but to secondary effects involving utilization of H202 to oxidize another compound and render it stimulatory or to effects involving utilization of H202 to remove a different inhibitory compound by oxidation. However, demonstration of the inhibition of lactic acid bacteria by added H202 in several laboratories (21,22) including our own supports our conclusion that stimulation results at least in part from removal of an inhibitory concentration of H202. Also, a culture of L. bulgaricus to which both F4 and H202 were added was not stimulated to a greater extent than one to which F4 alone was added (data not shown). Greater stimulation might be expected if both factors were involved in the formation of a stimulatory compound. Ranges of sensitivity to H202 have been reported from 5 ,ug/ml for single-strain lactic streptococci and commercial lactic starter cultures (21) to 100 to 200 Mg/ml for strains of L. Iactis (22). In our experiments, cultures of L. casei and S. thermophilus did not produce concentrations of H202 detectable by our assay (<0.1 Ag/ml), but were stimulated by catalase. Again, stimulation in this case may have been rdue to peroxidatic activity of catalase. In the experiments of Keilin and Hartree (11) high concentrations of H202 were decomposed to water and oxygen, whereas low concentrations could be used to oxidize ethanol, catalase functioning as a peroxidase under these conditions. Stimulation by catalase could also be accounted for if a small amount of H202, capable of inhibiting the L. casei and S. thermophilus cultures, is masked to the assay by a component of the milk. It has been demonstrated with the assay used for this work that H202 added to milk cannot be totally recovered as measured by the assay (6). This may be due to masking of the peroxide as well as to decomposition by organic matter. In addition, the data could be explained if the small amounts of H202 responsible for inhibition, and accessible to catalase in the growing culture, are bound in some way to the cells and removed with them during centrifugation before assay, or are decomposed during this procedure. The concentration of catalase (0.01 mg/ml or 20 Sigma U/ml) added to cultures of L. bul-garicus did not remove all the H202 formed. This concentration is expected to decompose 680 ,g of H202 per ml per min at pH 7 and 25 C. The milk system was at pH 6 to 5 at 37 C and contained about 2 ,ug of H202 per ml, compared to approximately 340 ,ug of H202 per ml present during estimation of activity of the enzyme preparation. Perhaps these differences in conditions are responsible for the residual H202 in spite of the very high concentration of catalase. To insure that catalase concentration was not limiting stimulation of L. casei, the effect of a threefold increase in concentration of catalase was determined. The higher concentration did not result in greater stimulation of either growth or acid production, indicating that sufficient or excess catalase was present for maximum stimulation by this means. Our data agree with the results of Gilliland and Speck (7) that indicate that the lactic streptococci can produce significant quantities of H202. In their experiments, maximum accumulation of H202 occurred during the time of most rapid decrease in pH and dropped before the rate of acid production (measured as pH change) decreased. In the experiments described here, where detectable, the H202 levels increased or remained constant as cell growth began to slow down. The second means by which micrococci stimulate lactic acid bacteria is not known. The capsular material has limited ability to remove H202 and therefore might be acting on this second target. More specific characterization of the composition of this material is underway. This may give some indication of how it functions.

v3-fos

2014-10-01T00:00:00.000Z

1973-01-15T00:00:00.000Z

10002873

s2

Realized genetic parameters following selection in tribolium castaneum in two environments SUMMARY Heritabilities and genetic correlations were estimated in a population of T y ibolium casta-neum, where larva weight and offspring number were measured in one base and eight selected generations. Control lines (each represented by 50 single-pair matings) and lines selected for larva weight or offspring number (each represented by 25 single-pair matings) were replicated twice in both a wet and a dry environment. The results indicated a decline over generations in the realized heritability of larva weight in both the wet and dry environments. The realized genetic correlations between larva weight in the wet as opposed to the dry environment indicated that genotypes affecting this trait were not specific to either environment. The realized genetic correlations between larva weight and offspring number were positive and decreasing under selection in the wet environment. By contrast, in the dry environment, the estimates of this realized correlation were near zero and increasing slightly under selection. The simultaneous improvement of more than one trait in animal or plant populations has prompted wide use of different versions of the selection index as formulated by HAZEL (i 943 ). Estimates of parameters represented by either genetic correlations and heritabilities or by genetic and phenotypic variances and covariances are needed in constructing such indexes. The model of L ERNER ( 195 8) indicated that selection would tend to decrease positive genetic correlations, even allowing for a shift from positive to negative. Trends in genetic correlations, observed by FRIARS,B OHR >a N and McKEAN ( 19 6 2 ) in a selection experiment with chickens, support the hypothesized changes of I,!RN!R. However, B OHR E N , HILL and R OB E R T SON ( 19 66) developed models and conducted computer simulated selection experiments which suggested that the direction of change in genetic correlations was, in fact, gene frequency dependent. A pertinent conclusion of these latter workers was that, with existing theory, the prediction of correlated responses is likely to be much poorer than the prediction of direct responses, unless genetic parameters are estimated in each generation. In this connection, the degree to which inaccuracies of estimates affect genetic progress has been dealt with by M A o ( 1971 ). Ultimately, genetic progress is reflected in realized heritabilities and genetic correlations. The study reported here represents an investigation of the behavior of realized heritabilities and genetic correlations in a controlled selection experiment with Tribolium castaneum. Experimental Details of the experiment have been presented by FRIARS, N AYAK , Jm and R AK TOE ( 1971 ). In general, control lines comprised of 50 single pairs, each contributing a daughter and a son to the next generation, were maintained by random mating in one base and eight selected generaions in each of a wet ( 75 ± 3 p. 100 relative humidity) and a dry environment ( 50 ± 5 p. 100 RH). Simultaneously, four lines (selected for larva weight in wet, selected for larva weight in dry, selected for offspring number in wet, and selected for offspring number in dry) were maintained with 25 single pair matings in each generation. Each of the four lines were tested in both the wet and dry environments in each generation. Two replicates of the experiment were separated by a time interval of approximately six weeks. Paramete y Estimation Realized heritabilities were estimated by using the difference in the means of control and selected lines in each generation divided by the selection differential cumulated across generations. Realized genetic correlations were estimated by using the deviations of the selected from the control lines as measures of direct and correlated responses to selection in modifications of the formula of FALCONER, ig6o) where : v A = estimated realized genetic correlation, CR ---correlated response, R = direct response, 7 = cumulated standardized selection differential, h = square root of realized heritability. Subscripts C and R are for correlated and direct traits respectively. 9 k = realized genetic correlation estimated by reciprocating the traits in which direct and correlated responses to selection were observed. The product ofl%1 ' rA t R x RY ' 1/2 provided a joint estimate of the genetic correlation Rx Ry between the two traits X and Y. ' Heritabilities The heritability estimates of larva weight derived by means of mid-parentoffspring regression in the control populations (table i) fluctuate considerably bet ween generations. However, the realized estimates of heritability (fig. i) are slightly higher in the dry environment and in agreement with the control population estimates although the differences between the wet and dry environments were not significant in either case when a paired t-test was applied. Trends in the estimates of heritability were not significant (P > . 05 ) in the control populations (table i). However, a definite decline was noted in the wet and dry environments ( fig. i), where linear and not quadratic regression accounted for a significant proportion of the sums of squares (P < . 05 ) in the dry environment as opposed to both linear and quadratic, after linear effects, being significant (P < . 05 ) in the wet environment. Genetic Correlations No trends, either between or within the two sets of estimates of the genetic correlation between larva weight in the two environments, were significant (P < . 05) when tested by regressions and t-tests. Opposite linear regressions from the two estimates of realized genetic correlations in each of the two environments (table 3 ) were significantly different from each other (P < . 05 ). However, the geometric means of the two type of estimates deviate from one in only two cases and a paired t-test of the estimates revealed a non-significant difference (P < . 05). Estimates of the realized genetic correlation between larva weight and offspring number differ consistently between the wet and dry environments (fig. 2 ). The estimates in the dry environment were close to zero and showed a slight increase while a definite decline from an initial estimate of about .6 was noted in the wet environment. The opposite slopes were both significant (P < . 05 ). He y itabitities The standard errors of heritabilities estimated in the control populations would be expected to be higher than the realized heritability estimates according to the derivations of HILL ( 1971 ). Such would appear to be the case through comparisons of table I and figure I . The cumulation of selection intensities over generations has the effect of generating a lack of independence between the generation estimates of realized heritabilities. Similarly, the differences between the regressions in the selected and control lines are not independent from one generation to the next. The values of estimated realized heritabilities used here suggest a declining rate of realized progress. These results coud be reflecting the error of the difference between the regressions of the control and selected lines (FRIARS, N AYAK , J UT and RAKTO!, I g 7I ) where for instance errors in the estimates of genetic gain would be enlarged in the later generations as differences between the predicted values in the control and selected lines increased. However, a similar result was noted in both the wet and dry environments. Genetics Correlations The lower values of the genetic correlations, as estimated by the product moment correlations between full sib means in the wet and dry environments (table 2 ) as opposed to the realized estimates (table 3 ), are reasonable in light of the fact that mean family size ranged from about five to nine. In fact, R OB E R T SON ( 1959 ) suggested much larger numbers per family to reduce the error of such correlation estimates to acceptable magnitudes. The small year to year variation in realized genetic correlations, similar to realized heritabilities, reflects the cumulative effects between years ( fig. 2 ). The reverse trends in the two types of correlation estimates (table 3 ) could, similarly to the suggested bias in the realized heritability estimates, be attributed to differences in regressions of control and selected lines in either the direct or correlated traits. Hence, one can only conclude that the genotypes selected for high pupa weight in the wet environment produced high pupa weight equally well in the dry environment, or vice versa. Contrary to any consistent trend in estimates of the genetic correlation between larva weight in the wet and dry environment, the realized genetic correlation between larva weight and offspring numbers in the wet environment ( fig. 2 ) showed a decline. This trend is similar to that where the genetic correlation between juvenile body weight and egg production switched from positive to negative in the course of a nine generation selection experiment with chickens (FRIARS, BO HR E N and McKEArr,zg62). However, McNARY ( 1959 ) found no such trends in similarly estimated genetic correlations between pupa weights measured in wet and dry environments. In contrast to the wet environment, the most striking feature of the realized genetic correlations between larva weight and offspring number in the dry environment is the close proximity to zero, with a slight tendency toward an increase ( fig. 2 ). The dry environment yielded much lower means than the wet for both traits (F RIARS et al., 1971 ). The possibility that a high level of performance is needed to allow full expression of the genetic correlation between two traits is one tenable hypothesis. Such a hypothesis is supported by the findings of HICKS ( 195 8) in chickens where the genetic correlation between egg number and body weight was negative in years favouring good performance but positive in poor years. Re!u your publication en décembre 1972. ACKNOWLEDGEMENT The financial support of the Ontario Ministry of Agriculture and Food and the National Research Council of Canada is gratefully acknowledged. Les résultats indiquent un déclin, au cours des générations, de l'héritabilité du poids des larves à la fois dans l'environnement humide et dans l'environnement sec. Les corrélations génétiques entre les poids larvaires dans les milieux humides et secs indiquent que les génotypes affectant ce caractère n'étaient pas spécifiques pour l'un ou l'autre des deux milieux.

v3-fos

2018-04-03T00:32:11.896Z

1973-09-01T00:00:00.000Z

12829731

s2

Trends in Wort Carbohydrate Utilization A gas chromatographic method suitable for any type of low-molecular-weight carbohydrate analysis has been utilized to determine the individual wort sugars in corn adjunct wort from a Western Canadian brewery. The fluctuations in each sugar during primary lager fermentation have been graphed. "End fermented" wort has been shown to contain some maltotriose, a small amount of maltose, and the nonfermentable carbohydrates, and dextrins. including maltotetraose, maltopentose, Qualitative and Qualitative and quantitative carbohydrate analyses of wort have become increasingly more important to the brewer to detect differences in the composition of wort from brew to brew, mainly to predict or ensure the degree of attenuation (10). For a number of years, however, reliable analytical work on wort or beer carbohydrate composition depended on the timeconsuming separation of individual carbohydrates by paper chromatography followed by elution, and the application of a colorimetric and quantitative chemical assay (15,16,25). Most often, therefore, only fermentable carbohydrate (3, 10), or total carbohydrate by the anthrone method (9,17,21) or by specific gravity determination (5), is followed. The desire to eliminate tedious paper chromatographic methods has led to interesting developments in gas-liquid chromatographic (GLC) analysis (22). The application of this newer method to carbohydrates, however, awaited the contribution of Sweeley et al. (24), who prepared and chromatographed a number of carbohydrates in the form of volatile trimethylsilyl derivatives. Since that time, a number of other papers have appeared (for review, see references 20 and 22). For the brewing industry, the reports by Marinelli and Whitney (12,13), Otter and co-workers (18,19), and Clapperton and Holliday (4) have been most significant. The application of this method for individual sugar analysis, except in quality control of initial wort, or final beer, has been more limited. For example, the work of Harris and co-workers (7, 8) with paper chromatography has not been extended to show the disappearance of individual sugars in wort during commercial fermentation by using the more sensitive and more rapid GLC method, even though Griffin (5, 6) has used the method with the top fermenting ale yeast, Saccharomyces cerevisiae, growing in a stirred laboratory fermenter. A gas-liquid chromatography system in the present study has been used to qualitatively and quantitatively determine the fermentable carbohydrate levels in beer wort throughout the course of a Western Canadian commercial lager fermentation. We have made use of this method to apply the results toward a study on the effect of yeast environment on flocculation of S. carlsbergensis (manuscript in preparation). MATERIALS AND ME'rHODS Fermentation. A typical closed 8,600-gallon (39,000-liter) brewery fermentor was used, maintained at 57 F (14 C) throughout primary fermentation until the cooling stage was reached. Samples were taken from a lower port and a higher port (upper) near the center of the tank. Only the lower port samples have been discussed and plotted. Gas chromatography (12,13). All GLC analyses were performed with a Hewlett Packard model 5750 B chromatograph equipped with a thermal conductivity detector at 355 C with a 120-mA bridge current. The column used was copper (2 ft by 1/4 inch [60.96 by 0.635 cm] outside diameter) packed with 3% SE-52 Silicone gum rubber on 60/80 mesh Chromosorb W AW-DMCS (Chromatographic Specialties, Brockville, Ontario). The carrier gas was 80 ml of helium per min. The oven program from 150 to 350 C was carried out at a rate of 6 to 10 degrees per min with the injection port at 375 C. An attenuation of 1 was usually used. The strip chart recorder was a Hewlett Packard model 7127A run at 0.5 inch (1.27 cm)/min. The method described by Marinelli and Whitney (12,13) was used to prepare trimethylsilyl derivatives but with the incorporation of phenyl-ft-D-glucopyranoside as an internal standard for quantitation. To each vial containing 0.5 ml (approximately 60 mg solids) of wort was added 1.0 ml of pyridine (silylation grade, PATEL AND INGLEDEW Pierce Chemical Co., Rockford, Ill.) containing 5 mg of phenyl--D-glucopyranoside per ml. Then 0.9 ml of 1,1,1,3,3,3-hexamethyldisilazane and 0.1 ml of trifluoracetic acid (Aldrich Chemical Co., Milwaukee, Wis.) were added. The vials were shaken for 30 s and then allowed to stand at 20 to 22 C for 15 min with occasional shaking. Reactions are quantitative using this method (20). When insoluble components were seen, the reaction mixtures were warmed prior to injection. Injections of 10 to 50 liters were done in duplicate by using a Hamilton syringe (Hamilton Co., Whittier, Calif.). Peak areas were calculated by cutting and weighing xeroxed peaks. The retention times were obtained by injection of standard solutions of sugars found in wort. Relative response values were also calculated for all sugars of lower molecular weight than maltotriose. Yeast dry weight determinations. Wort samples were centrifuged by using a Sorvall SS-1 super-speed angle centrifuge (Ivan Sorvall Inc., Norwalk, Conn.) at a relative centrifugal force greater than 5,400 x g. Supernatant fluid was discarded, and cells were washed twice and resuspended in 0.05 M phosphate buffer (pH 6.4) in h6s the volume of the original sample. Triplicate 2-ml samples of the resuspensions and of the resuspending buffer were transferred to preweighed aluminum foil pans. Dishes were dried to constant weight at 105 C, and cell mass/ml of wort was calculated. Chemical determinations. Total N in wort was determined by the Kjeldahl method as adapted by Bremner (2). Prior heating of samples in 0.5 ml of concentrated H2SO4 was used to prevent excessive foaming during digestion (1). The distillate was titrated with standard H2S04 to an end point of pH 4.9 by using a Radiometer automatic titrator type TTT1 (Radiometer, Copenhagen, Denmark). Protein nitrogen in wort was estimated by using the method of Lowry (11) with crystalline (X3) egg albumin (Nutritional Biochemical Corp., Cleveland, Ohio) as standard. Results were read at 750 nm (9) on a Spectronic 20 colorimeter (red phototube and filter). Triplicate anthrone tests for total carbohydrate in wort and cell samples were carried out by the method outlined by Herbert et al. (9), adapted for more reproducibility from Morris (17). Standard curves were prepared for each essay. A Beckman B spectrophotometer at 625 nm rather than a colorimeter was used as advised by Herbert et al. (9). (14). Primary fermented wort in Table 1 is actually beer after the initial 5 to 6 day fermentation and cooling cycle. At this time, all fructose, sucrose, and glucose, most of the maltose, and 80% of maltotriose have disappeared from the wort (94% of all fermentable sugar). Maltotetraose, maltopentose (detected but not quantified by this GLC method), and the larger-molecularweight, nonfermentable dextrins remaining after primary fermentation are not attacked by S. carlsbergensis, but were measured by anthrone. RESULTS AND DISCUSSION In Fig. 1, the trends of total fermentable carbohydrate, wort nitrogen levels, and yeast mass in suspension have been recorded. The decrease in total nitrogen is almost entirely a decrease in low-molecular-weight nitrogen compounds (amino acids, small peptides, and inorganic nitrogen). Protein nitrogen, for example, as measured by the Lowry method (11), shows little decrease during the fermentation, with some of this decrease due to utilization of Lowry-positive amino acids. Yeast mass throughout this time increases dramatically (450%) in the wort. In the later stages the decrease in mass is due to the phenomenon of flocculation, as yeast sink by the lower sampling port to sediment at the bottom. Figure 2 is a representation of the fermentable carbohydrates in a typical chromatogram of corn adjunct brewers wort. By using peak weights (areas) and the calculated relative response values in Table 2 thoughout primary fermentation. Figure 3 reflects the ease of following environmental changes in a medium by this method compared to previous studies (7, 8). Sucrose decreased rapidly to an undetectable level during the first 5 h of the fermentation. The sucrose probably is hydrolyzed by invertase (sucrase), a yeast enzyme situated between the cell wall and cell membrane (23). However, Harris et al. (7) state that glucose is used first from wort, followed by fructose and then sucrose. In this experiment, the sucrose was hydrolyzed first. Fructose levels increase to the 5-h sampling period, reflecting hydrolysis of su- Phenyl-ft-D-gluco-4.38 Internal standard pyranoside a RRV/mg of sugar = (area sugar peak x attenuation/area internal standard x attenuation) mg of sugar in 0.5 ml of freeze-dried simulated wort. Amount of sugar (mg/100 ml) in unknown wort = (200 x area sugar peak (wort) x attenuation/area internal standard x attenuation) RRV/mg of sugar. b The maltose response factor may be used for maltotriose and maltotetraose, since it is difficult to purchase these compounds in pure state (Marinelli, personal communication, 1972). 24 Glucose, 16%, is unchanged for the first 4 to 5 h, probably because its rate of utilization is comparable to its rate of formation from sucrose. However, by 24 to 48 h, measurable levels of glucose disappear. Maltose and maltotriose hydrolysis to glucose must therefore be slower than glucose utilization. Maltose (66% of fermentable sugar) is not significantly attacked during the first few hours, presumably because maltase permease or the hydrolysis reaction are glucose repressed. A very rapid utilization is seen between 10 and 50 h, eventually resulting in 93 to 96% utilization. Maltotriose utilization is similar to maltose, eventually leading to 75 to 80% metabolism by the yeast. Residual maltose and maltotriose are attacked in subsequent fermentation and aging steps in the brewing process, since bottled beer from this process contains only nonfermentable carbohydrate and small amounts of maltotriose. The ability to monitor wort carbohydrates or any other carbohydrates by gas chromatography is certainly an advantage to a quality control or research laboratory. The method is rapid, quantitative, and can be applied to a large variety of studies, including lactose in milk, maltose and glucose ratios in syrups, and enzymatic attack on carbohydrates. It has already been used to detect glucose, fructose, and sucrose levels in tomato, cabbage, apple, carrot, and potato tissues (22

v3-fos

2020-12-10T09:04:16.932Z

1973-09-01T00:00:00.000Z

237229691

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Sand Beach Bacteria: Enumeration and Characterization Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans. Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans. Bacteria are considered to be an important component of the sand beach community (5-7, 12, 14). The productivity of the sand beach is ultimately limited by nutrient input. From laboratory studies, it appears that nutrients (carbon) pass first through the bacterial community and then into the protozoan and metazoan community (12). To initiate studies on sand beach bacteria in Lebanon, a tideless, fully exposed marine beach was chosen: Sindbad beach, 30 km south of Beirut. The beach has no obvious signs of pollution. Small recreational swimming areas are located on the north and south quarters of its 1.5-km length. In the center part, samples were taken repeatedly, to enumerate bacteria and also to isolate several hundred individual strains to determine their biochemical, morphological, and cultural characteristics. MATERIALS AND METHODS Enumeration of bacteria in sand. The total number of bacteria per gram of sand was obtained by: (i) collecting a small sand sample in the field (kept chilled until return to the laboratory); (ii) aseptically adding 6 to 7 g of the wet sand to a pretared 18-by 250-mm sterile screw-cap tube for obtaining the wet weight of sample; (iii) adding sterile 0.1% peptone (Difco) in sea water or distilled water to the 5.0-ml mark of the tube; (iv) vortex shaking for 60 s (longer shaking did not increase the number of bacteria detected); and (v) taking samples from this tube for further dilution and plating on peptone-yeast extract medium (PYE). PYE contained: peptone (Difco), 0.5%; yeast extract, 0.05%; and agar (Difco), 1.5% made with either sea or distilled water. Other media (increasing or decreasing the peptone concentration 1Present address: Biology Department, University of Jordan, Amman, Jordan. with the exclusion of yeast extract, or replacing the yeast extract with glucose or phosphate) supported the growth of fewer bacteria. Interstitial bacteria (not attached to sand grains) were collected with an interstitial water sample (13) which had been thoroughly rinsed with the sample. For enumeration, 1, 5, or 25 ml of the interstitial water was then suctioned through HA-Millipore membranes (pore size, 0.45 Am, in Millipore field monitors). PYE broth was then aseptically added to the absorbant pad below the membrane. All incubations were done at room temperature (25 C). The number of colonies on PYE agar plates were counted after 4 days of incubation, and those on Millipore membranes were counted after 36 h of incubation, the latter with the aid of a binocular dissecting microscope. Total (direct microscope) counts were made from untreated interstitial water and sand samples that had been shaken in 0.004 N NaOH. Samples of either were suctioned through Millipore membranes, and 2% erythrosin in 5% aqueous phenol was then added to the absorbant pad to fix and stain the bacteria present on the membrane. After removal of excess stain, the bacteria retained on the filters were counted with the aid of an oil immersion lens. Sites. During 1971 the wave wash zone (WWZ) was sampled at the highest point that the waves kept the sand saturated with water. Also, the water table (WT) was sampled at the point inward from the WWZ where there was 40 cm of sand covering the water table. The WT was usually 3 to 6 m from the WWZ. In 1972, samples were taken from the WWZ, 10 and 20 m inwards on a transect perpendicular to it. Bacterial isolates. All pure cultures were obtained by repetitive streaking on PYE-sea water agar plates and were stored on PYE-sea water agar slants at 4 C. Biochemical and cultural determinations. Sugar fermentation was determined by the method of Hugh and Leifson (9); presence of deoxyribonuclease was determined by the method of Lachica et al. (11); hydrolysis of gelatin and starch was determined as suggested by Skerman (16); the Voges-Proskauer reaction was determined by the method of Barrit (1); indole production, motility test, catalase, and oxidase tests were determined by the methods of Skerman (16); and the flagella stain was done by the Leifson procedure (4). Sea water was used to prepare all media except for the Hugh-Leifson medium, which contained 3.0% NaCl, 0.3% K2HPO4, and 0.1% MgSO4 in place of sea water. The ability to grow aerobically on single carbon sources was tested by a modification of the method of Stanier et al. (17), in which sea water was used in place of distilled water. Control plates included no added carbon source besides agar and whatever soluble carbon was present in the sea water. No growth was observed on these plates. The ability to grow at various temperatures (50, 37, 25, 15, 5, and 0 C) and at various pH values (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) was checked on PYE-sea water. All test plates were inoculated by the replica plate method from normal PYE-sea water agar master plates. RESULTS AND DISCUSSION The number of bacteria per milliliter of interstitial water accounted for 22 to 46% of the total number of bacteria per gram of wet sand (Fig. 1). The determination of the total number of bacteria in the interstitial water did not involve shaking, and therefore aggregates may have been counted as one cell. Therefore, this number may be underestimated and could be twice as high (8). The numbers of viable bacteria were 10-4 that of total bacteria (Fig. 1). These results suggest that a large proportion of the bacteria active in the ecosystem are not grown in the culture medium and indicate the need for further studies of the efficacy of various nonselective media for isolation and enumeration of these bacteria. The numbers of viable bacteria per milliliter of interstitial water were lower than the numbers of viable bacteria per gram of wet sand. The latter was determined by a method involving shaking; the former did not. Thus, both the total (direct microscope count) and viable numbers of bacteria per milliliter of interstitial water were probably representative of the number of bacterial aggregates, whereas the numbers of bacteria per gram of wet sand (direct and viable counts) were probably more indicative of the number of individual cells. Microscope study by simple (18) and fluorescent staining (2) revealed no bacteria attached to the sand grains in repeated attempts. Table 1 shows that most of the viable bacteria were evenly distributed in the water-saturated zones of the WWZ and WT. The number of viable bacteria varied by one order of magnitude. It increased 10-fold during a local storm. Marine bacteria seemed to be predominant in the samples, with an exception of the sample taken at on August 19, i.e. higher numbers of bacteria were grown on the medium containing sea water. In the spring and summer of 1971, 362 randomly chosen isolates were cultured and maintained as stock cultures for characterization. Table 2 shows the distribution into six arbitrary, physiological groups based on the ability to ferment various sugars. Group 1 isolates were versatile fermenters and comprised only 17% of the isolates; in group 2, the number of isolates capable of limited fermentational versatility was also small, comprising only 23% of the isolates. Rods were the major morphological type (88% of all isolates) and comprised 96% of the major group, group 3, the oxidative group (lacking the ability to ferment). The fact that oxidative organisms are the dominant group (59%) is consistant with the selective effect on aerobic plates and the presence of oxygen in interstitial water. Makemson (unpublished data) has shown that the percent saturation of oxygen was only once as low as 27% (1.41 ml of 0/liter), at 20 m inwards from the top of the WWZ. The normal values of oxygen saturation were between 50 to 75% in the WWZ as well as in the fresh water area in the water table (10 and 20 m inwards). Although anaerobic pockets can exist in aerated soils (8), it seems that in the Sindbad beach the chance to develop such an anaerobic environment is rare, since the interstitial water is in constant flux. Rapid washing of the WWZ with sea water (15) combines with a substantial fresh water movement to the sea through the WT. The later causes a rather steep salinity gradient: 10 m inwards from the WWZ it was common to find a value of 18 to 30% salinity. Thus, it is doubtful that anaerobic pockets could develop in the sand to make free-living anaerobic (fermentative) bacteria a dominant component of the total bacteria present. However, meiofauna and other metazoans in the beach may provide anaerobic environments in their gut cavities, thereby selecting and enriching for anaerobic or fermentative bacteria. This may account for the significiant number of fermentative bacteria (41%) in our collection. Out of the eight carbon sources tested to support the aerobic growth of the isolates as sole carbon sources, lactate appeared to be the more universal carbon source, which 43% of the isolates could utilize (Table 3). Although this study did not determine what carbon sources these isolates are utilizing in situ, these data show that a large percentage of the versatile fermenters have the ability to utilize lactate. Although the number of sole carbon sources tested was not as extensive as the 146 single carbon sources studied in aerobic and fermenta- Klug and DeMoss (10), in which case the total number of isolates does not apply.°N umber of isolates in each category. Aerobic are groups 3a and 3b, and fermentative are groups la, lb, 2a, and 2b in Table 2. c ND, Not determined. These open marine isolates appear to be more versatile than those from Sindbad beach. For example, 100% of the fermentative and 65.8% of the oxidative marine bacteria used glucose as a sole carbon source, compared to only 30% of the aerobic and 22% of the fermentative beach bacteria. Except for the utilization of acetamide, the open ocean marine bacteria were nutritionally more versatile than beach bacteria. Starch was not tested in the papers by Bauman et al. (3,4). The presence of amylase was more prominent in the aerobic beach bacteria than those from the open sea. The reverse was the case when comparing the fermentative isolates. The apparently higher nutritional versatility of the offshore isolates compared to beach isolates may have been caused by the different modes of isolation. Indole production has been demonstrated from 55 to 78% of bacteria isolated from marine invertebrate guts (10). In comparison, indolepositive organisms seem to be less prevalent in estuarine sediments (30-40%), in estuarine water (14-18%), in open ocean inhabitants (4-5%), and in open ocean water (0.07-1.5%). Over half of the bacteria isolated from Sindbad beach were indole positive ( Table 4). The trait was widespread among the physiological groups (Table 5). If it is correlated with invertebrate gut inhabitation, the data would suggest a symbiotic association of our isolates and the resident invertebrates. The beach isolates were deoxyribonuclease positive, gram negative, motile, and oxidase positive. All had a growth preference for 15 to 37 C and pH 7 to 9. The motile organisms were all polarly flagellated. Only one aerobic isolate was peritrichously flagellated, and none of groups la, lb, 2b, and 3b was motile. Only a few of the isolates liquefied gelatin, reduced nitrate to nitrite, or could grow below pH 5. Roughly 25 to 34% of the isolates could grow on PYE made with distilled water. This test was performed over a year after initial isolation on PYE-sea water media. From fresh sand, only 9.4% of the bacteria which grew on PYE-sea water agar also grew on PYE-distilled water media. The variation in these counts (excluding the August 19th sample) was remarkably consistant at both the WT and WWZ (3.1 to 15.6%). In the August 19th sample, the bacteria which grew on PYEdistilled water agar were 43.8% of those growing on sea water agar in the WWZ and 225% in the WT. Although the number of samples was limited, bacteria capable of growth on fresh water medium seem to be a minor component located on the fresh water side of the beach. The percent of bacteria growing on sea water agtir averaged 70% of the total viable bacterial count in the WT and WWZ. ACKNOWLEDGMENT The logistic and technical assistance of Neil Hulings was greatly appreciated by both authors.

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2020-12-10T09:04:17.327Z

1973-07-01T00:00:00.000Z

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Production of Ochratoxins A and B on Country Cured Ham Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm. Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm. During a recent survey on the mycoflora on country cured hams in the Southeastern United States, Sutic et al. (17) isolated two aflatoxinproducing strains of Aspergillus flavus Link ex Fries as well as other potential mycotoxin-producing aspergilli. Among these strains, one was identified as A. ochraceus Wilhelm. In a similar study, Strzelecki et al. (16) recovered two strains ofA. ochraceus from country cured hams but were not able to show any production of ochratoxins. Ochratoxin A and its dechlorinated analogue, ochratoxin B, are metabolites of several members of the A. ochraceus group (6) and of Penicillium viridicatum Westling (13), a mold which was also frequently found on country cured hams by Leistner and Ayres (8). The toxicity of ochratoxin A is well established, whereas ochratoxin B was first reported to be nontoxic (15). Later Peckham et al. (11) indicated that the toxicity of ochratoxin B was one-tenth that of ochratoxin A to day-old chicks. No carcinogenic effect has been observed for either toxin. Scott et al. (13) and Shotwell et al. (14) described cases of natural occurrence of ochratoxin A in moldy corn, wheat, and other agricultural products. In the present investigation, country cured hams from curing plants in Georgia were surveyed for occurrence of A. ochraceus. Isolates of A. ochraceus and P. viridicatum from cured hams were then screened for production of ochratoxin A and B by using media known to be suitable for obtaining high-toxin yields. I Present Address: Department of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland. 27 Since it was not known if cured ham itself could sustain ochratoxin formation, or how deeply the toxin or mold mycelium would penetrate if the surface of a whole ham were contaminated with A. ochraceus or P. viridicatum, these factors were also checked. MATERLALS AND METHODS Organisms. From five Georgia processing plants, 166 swabs were taken from 153 hams aged for 1 to 12 months. Sampling was restricted to colonies that appeared to have morphological and cultural characteristics of aspergilli. Each swab was inoculated initially on the Czapek-Dox agar plus 10% NaCl, thus favoring the growth of osmophilic species of Aspergillus which includes A. ochraceus (7), and then onto malt agar for final identification. From the 166 swabs, one strain of A. ochraceus (H 33) was found. This strain, as well as A. ochraceus D-1, isolated by Sutic et al. (17) Culturing. (i) On rice, defatted peanut meal, and corn. Spores (3 x 106) of each strain of A. ochraceus and P. viridicatum and 75 ml of sterile water were added to 150 g of rice or defatted peanut meal in 500-ml Erlenmeyer flasks and incubated at 25 to 27 C for 14 days. In addition, P. viridicatum was cultured on popcorn under the same conditions. These conditions were reported by Schindler and Nesheim (12) to yield maximal amounts of toxin. After steaming the cultures to facilitate extraction of ochratoxins, they were transferred to Mason jars and extracted. (ii) On country cured ham to screen for ochratoxin production. Boneless slices of fully aged country cured ham (0.5-1.0 cm thick) were procured from various commercial curing plants. Excessive fatty parts were removed and the slices were cut to a weight of 100 to 150 g. They were surface sterilized by dipping them into 1% NaOCl solution for 1 min, rinsing with sterile water, and blotting dry with sterile cheese cloth. For inoculation, the slices were swabbed with 0.5 to 1.0 ml of a spore suspension containing 106 spores per ml, thus obtaining a spore load of 0.5 x 106 to 106 spores per slice. The ham slices were then suspended by a string in sterile 1-qt (0.946-liter) Mason jars which were covered with three layers of no. 1 Whatman filter paper instead of Mason lids. The jars were incubated for various lengths of time at 5, 15, 25, 30, and 37 C and at a relative humidity of 70 to 75%. If necessary, saturated aqueous NaCl solution (relative humidity 75% at 20 C [10]) was added to the jars. After incubation, the ham slices were cut into small pieces for extraction. (iii) On country cured ham to determine toxin and mold penetration. From the center section of fully cured and aged hams, slices (5 cm thick) each weighing about 1,000-g were cut, surface sterilized as described above, and placed into sterile culturing chambers. The top surface (crossing the bone, 150 to 180 cm2) was inoculated with approximately 103 spores per cm2, whereas the edges were kept sterile by repeatedly cleaning them with NaOCl solution. Cultivation was at 25 C and 70 to 75% relative humidity for 21 days. The mycelial mat was then scraped from the surface and the slices were cut into layers (0.5 cm thick) which were assayed individually for ochratoxins. Also, slabs were cut from the slices in different depth, again surface sterilized, and incubated on rose bengal-streptomycin agar RBM-2 (18) at 25 C. Assays. Official methods of the Association of Official Analytical Chemists (1) were used to determine moisture and salt content of cured hams. To quantitate ochratoxins, all cultures were extracted with chloroform by using a Sorvall high-speed blender. The crude extracts were filtered through diatomaceous earth and concentrated to 50 ml. A clean-up step with column chromatography by the procedures proposed by Eppley (4) followed. The ochratoxins in the purified extracts were separated by thin-layer chromatography on Adsorbosil-1 (Applied Science Laboratories, State College, Pa.) with toluene-ethyl acetate-formic acid 5: 4: 1 (vol/vol/vol) as developing solvent (4). To exclude possible interference of 4-hydroxymellein with ochratoxin A, chloroform-acetone 93:7 (vol/vol) was used as a second solvent system (9). A Photovolt fluorodensitometer was used to compare intensity of fluorescence of the samples with that of standards obtained from the Bureau of Food Sanitation, Food and Drug Administration, Washington, D.C. It was possible to improve the sensitivity of this method by exposing the thinlayer chromatography plates to ammonia fumes for 2 to 4 min, a treatment which changes the fluorescence of both ochratoxin A and B from blue-green to intense blue. The reaction with ammonia is specific for both ochratoxin A and B. Additional confirmation was obtained by extracting the purified extract with 0.1 M aqueous sodium bicarbonate solution, acidifying with 2 M hydrochloric acid, and re-extracting with chloroform (4). This extract was concentrated and chromatographed as before. RESULTS AND DISCUSSION As the results of the screening tests in Table 1 indicate, both isolates of A. ochraceus recovered from country cured ham were able to produce ochratoxins A and B. Toxin yields were lower than those observed on shredded wheat by Schindler and Nesheim (12). Whereas P. viridicatum (ATCC 18411) produced ochratoxin A as expected, none of the isolates of P. viridicatum from cured hams formed detectable amounts of either ochratoxin A or B. This contrasts somewhat with the results of Scott et al. (13), who found only 5 nonproducers among 27 strains of P. viridicatum isolated from various grains, mixed feed, beans, and peanuts. Since the isolates of P. viridicatum from cured ham did not yield any toxin on rice, peanut meal, or corn, only A. ochraceus was grown on aged hams. Table 2 shows that again all three strains of A. ochraceus produced ochratoxins A and B. The temperature optimum for toxin production by strain H 33 was 25 to 30 C; very little toxin was recovered at 15 C and none was recovered at 5 or 37 C, at which temperatures only poor mycelial growth and almost no sporulation occurred. At 25 C, strain H 33 produced more toxin on hams containing 45% moisture than on those having 55%, the latter being above the equilibrium moisture at 75% relative humidity. These hams therefore lost 5% moisture during incubation. Aspergilli tend to show optimal growth at comparatively low water activities (8). Culturing at 90 and 100% relative humidity in ambient air resulted in poor growth and successful competition from other molds and bacteria present in hams. In aging rooms of commercial plants, 75% relative humidity prevails. In the study on mold and toxin penetration into ham (55% moisture, 5.1% NaCl), mycelial growth was observed as deep as 1 cm along the binding tissue where the meat often split open during the incubation period (21 days at 25 C and 75% relative humidity). In the lean muscle, growth occurred to a depth of approximately 0.5 cm. This figure was not very consistent, since contamination from other locations on the hams was difficult to prevent when the ham slabs were prepared for incubation. One-third (7 Mg) 28 APPL. MICROBIOL. of the total ochratoxin A produced was found in the mycelial mat on the surface of the ham, two-thirds (14 Mg) was found in the upper 0.5-cm meat layer (260 Mg of ochratoxin A per kg of meat), only traces were found in the second 0.5 cm, and none was found in the deeper layers. Ochratoxin was detected only in the layer in which mycelial growth was also observed. Ochratoxin apparently penetrates into the meat along with the mycelial growth, whereas physical diffusion of the toxin is limited, at least during the 3-week period of this experiment. This is different from cultures of A. ochraceus in liquid media, where a large proportion of toxin is always found in the culture filtrate. In studies of aflatoxin in bread, Frank (5) recovered the toxin only in zones where mycelial growth occurred. There is evidence that A. ochraceus is able to produce more than negligible amnunts of ochratoxins A and B on country cured hams under conditions which are often encountered in commercial curing plants. Mycelial growth is not restricted to the ham surface, and toxin can penetrate as far as 0.5 cm into the muscle of the meat. The three strains of A. ochraceus used in this study also produced penicillic acid on cereals. Penicillic acid does not seem to be a problem on meat because it reacts easily with amino acids to form much less toxic or nontoxic compounds (2). None of the strains of P. viridicatum isolated from hams produced measurable amounts of ochratoxin A or B. However, besides also producing penicillic acid (2), P. viridicatum is a source of the mycotoxin citrinin (13). Its occurrence on aged hams and its importance are still to be determined.

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2018-04-03T02:15:44.713Z

1973-02-01T00:00:00.000Z

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Growth of Hansenula holstii on cadavers. Growth of a yeast was observed on prosected cadavers used for demonstration purposes in a medical school. An asporogenous yeast was isolated and identified as an atypical form of Hansenula holstii by analysis of the extracellular polysaccharide. The isolate showed resistance to embalming fluid but was eventually eradicated by addition of picloxidine digluconate to the fluid. purposes at 4 C were never affected. This report concerns the identification of an unusual strain of Hansenula holstii Wickerham as the yeast involved and the methods used for preventing its development on cadavers. MATERIALS AND METHODS Isolation and identification of the yeast. The yeast was isolated on a number of occasions from two of the affected cadavers on 4% malt extract (ME) agar (9), and a representative isolate, NRRL Y-7178, was selected for study. The culture techniques used for identification were described by Wickerham (9,12). Later the composition of the extracellular polysaccharide was determined by the procedures for purification and analysis as described by Slodki et al. (7). The sensitivity of the cadaver yeast to the preservative picloxidine digluconate (Resiguard, Nicholas Laboratories, Ltd., Bucks, England), was determined by inoculating glucose-peptone broth containing this compound at concentrations from 1: 100 to 1: 5,000. All cultures were incubated at 24 C, checked for visual growth, and subcultured after 3 days. RESULTS Colonies of NRRL Y-7178 were white to greyish-white and quite mucoid. Budding was multilateral and the cells measured 2.0 by 2.5 ,gm to 3.5 by 5.0 tim. Neither hyphae nor pseudohyphae were produced on Dalmau plates (9) of yeast morphology agar or corn meal agar. There was no sporulation at 15 or 25 C on ME, yeast-malt (YM), V-8 (9), corn meal, or Gorodkowa agars (8), or on carrot, cucumber, and gypsum blocks. Addition of 2, 5, 10, and 20% glucose or sodium chloride to YM and V-8 agars to increase osmotic pressure did not induce sporulation. Analysis of the extracellular phosphomannan, however, was quite revealing. As shown in Table 1, the polymer formed from glucose (5 g/100 ml) was indistinguishable from phosphomannans elaborated by Hansenula holstii strains, especially the haploid strain NRRL Y-2154 (7). D-Mannose and D-mannose 6-phosphate were the sole products of vigorous acid hydrolysis (2 N HCl, 100 C, 1 hr). Autohydrolysis, i.e., conversion to the phosphomonoester form by heating decationized phosphomannan (pH 2.5, 100 C, 30 min) gave rise to a mixture of phosphorylated oligosaccharides and a monoester phosphorylated mannan fragment. The highand low-molecular-weight components were separated by gel chromatography (M. E. Slodki et al., Proc. 4th Int. Ferment. Symp., in press). The phosphorylated mannan fragment gave a strong precipitin reaction with concanavalin A, whereas the intact phosphomannan gave a weak reaction. As judged by paper electrophoresis in 0.05 M barbital, the oligosaccharide phosphates were a mixture of monosubstituted esters apparently containing 4 to 6 mannose units. The pentasaccharide phosphate ester was the predominant component. All these results are consistent with previous findings on the phosphomannans of H. holstii (4). As with most other phosphomannan-producing yeasts, an extracellular neutral mannan was alternatively formed when orthophosphate was omitted from the fermentation medium (5). The neutral mannan gave the same pattern of enzymatic degradation (M. E. Slodki et al., in press) observed when other H. holstii mannans were incubated with Arthrobacter a-mannosidase. Attempts to mate NRRL Y-7178 with the mating types of H. holstii NRRL Y-2154 and Y-2155 were carried out on ME agar according to Wickerham (10), and on the restricted growth (RG) medium of Herman (2). There was no evidence of conjugation or sporulation on either of these media. The cadaver yeast was significantly more tolerant to the embalming fluid than the other yeasts tested. It grew well in broth containing 2% preservative, a fungistatic effect was seen at 3%, and above 3% concentration the embalming fluid was fungicidal. Four out of the five test yeasts were killed by a concentration of 0.5% preservative and the fifth (C. albicans) by 1%; none showed growth above 0.2% concentration. Picloxidine digluconate was fungicidal to the cadaver yeast at concentrations as low as 1: 5,000. The compound was successfully employed as a preservative of cadavers at a 1: 100 concentration in the presence of 0.5% embalming fluid. DISCUSSION Peterson (3) was apparently the first to report the isolation of a yeast from cadavers. His isolate, extremely tolerant to embalming fluid, was described eventually as the new species Hansenula petersonii Wickerham (11). Both the fermentation and assimilation patterns of this species differ from those of H. holstii (12). A common habitat of H. holstii is the frass of coniferous trees and gums of fruit trees (10,12). Strain Y-7178 differs from the usual isolates not only in habitat, but by its failure to produce hyphae or pseudohyphae and by its inability to assimilate D-galactose, L-sorbose, and soluble starch. It also lacks mating competence but so also do many of the haploid isolates of H. holstii collected from frass of coniferous trees (12). Many isolates of this species also vary in their ability to assimilate carbon compounds and in the degree to which they produce hyphae and pseudohyphae. Nevertheless, Y-7178 could not, with any confidence, be assigned to H. holstii were it not that its extracellular phosphomannan corresponds to that of this species and is, in fact, indistinguishable from the phosphomannan of Y-2154, a sexually reactive haploid strain (4). The specificity of extracellular polysaccharides has clearly been demonstrated (6, 7), but the procedure has seldom been used for purposes of identification. Picloxidine digluconate has previously been shown to be useful in retarding various types of postmortem breakdown when incorporated in preserving solutions (1). The strain of H. holstii isolated from the cadavers was sensitive to concentrations as low as 1: 5,000 but because of other beneficial effects reported (1), the preserving solution was supplemented with picloxidine digluconate at a concentration of 1: 100. Since this recommendation was followed, no trouble with microorganisms attacking prosected specimens has been encountered for 3 years.

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2020-12-10T09:04:12.743Z

1973-07-01T00:00:00.000Z

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Microbial Penetration Through Three Types of Double Wrappers for Sterile Packs Microbial penetration of sterile packs was studied by using double-wrap (two layers each) muslin, single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC wrappers to wrap 20 gauze sponges (2 by 2 in.). These packs were stored on open shelves of a central sterile supply department of a hospital and processed for sterility at weekly intervals. Microorganisms penetrated the double-wrap muslin as early as 28 days, the single-wrap muslin and single-wrap two-way crepe paper combination in 77 days, and the single-wrap muslin and single-layer BAR-BAC combination in 63 days. A previous study on microbial penetration of sterile packs was conducted to determine, under actual hospital conditions of storage, safe times for sterile storage of four widely used single or double wrappers for packs (1). Viable microorganisms penetrated packs with single wrappers faster than packs with double wrappers, and the time for microbial penetration was less than half as long with open-shelf versus closed-cabinet storage. On the basis of this study, single wrappers were not recommended for sterile packs (1). An extension of that study has been conducted to evaluate the safe time for storage of sterile packs with two additional types of double wrappers to compare them with the most effective wrapper previously studied, double-wrap (four layers) muslin. Because storage on open shelves is common in hospitals, and because such storage resulted in most rapid microbial penetration in the previous study, the comparison of the three types of wrappers was conducted only with open-shelf storage. MATERIALS AND METHODS Standard packs approximately 8 by 10 in. (20.4 by 25.5 cm) consisting of 20 2 by 2 in. (5.1 by 5.1 cm) 12-ply gauze sponges were prepared and sterilized as described by Standard, Mackel, and Mallison (1). These packs were covered with double muslin (each two layers), single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC outer covering. Muslin wrappers used for the standard packs were 140-thread-count material, unbleached, dyed green, laundered, and ironed 1 to 10 times before use. BAR-BAC (Angelica Uniform Co.) wrappers were tightly woven cotton material, dyed green, laundered, and ironed at least one to three times before use. The paper wrappers used were commercially available two-way crepe paper (Dennison Wrap). All wrappers were approximately 24 by 24 in. (61 by 61 cm). The sterile packs were transported in sealed, sterile plastic bags to a hospital central sterile supply department (CSSD). They were placed on open shelves in the same area used for hospital sterile supplies. Test packs were picked up at random in groups of two or four at weekly intervals, placed in sterile plastic bags, and returned to the laboratory for microbiological assay by procedures previously described (1). A total of 252 test packs were assayed. Three series of 14-week evaluations were conducted. On the initial day of each series of evaluations, three packs wrapped in each type of wrapper used in that series were chosen at random, after placement on shelves of the hospital CSSD, and transported back to the laboratory in sterile plastic bags for an initial control assay to confirm that the packs were not contaminated during transportation. In addition, at the time of each weekly pick-up of study packs, two sterile double-muslin-wrapped (4 layers) packs were transported to the hospital and back to the laboratory in sterile plastic bags for assay as weekly transportation controls. A total of 108 initial control packs and weekly transportation control packs were assayed. Temperature and relative humidity were monitored throughout the study by use of 7-day recording hygrothermographs. These instruments were calibrated at weekly intervals with a sling psychrometer. Viable surface contamination settling on the outside of the packs was estimated by using stainlesssteel strips placed open on the storage shelves used for the packs, as described previously (1). RESULTS Microbial contamination was determined for packs with three types of double wrapping: double-wrap (each two layers) muslin, singlewrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC outer covering. Table 1 gives the time in days until the first contamination was found inside packs covered with each type of wrapping material. Contamination occurred as early as 28 days with double-wrap muslin, 63 days with the BAR-BAC and muslin combination, and 77 days with the two-way crepe paper and muslin combination. Tables 2 to 4 show the number of pack weeks of exposure and the number of Temperatures in the CSSD remained between 70 and 80 F (21.1 to 26.7 C) with few exceptions throughout the entire study, and weekly average relative humidities ranged from about 30 to 55%. Table 5 shows the total microbial counts from stainless-steel strips exposed on open shelves to estimate the amount of viable fall-out on sterile packs on the shelves. The microbial counts were calculated on the basis of an area measuring 80 square inches, equal to the exposed surface area of the packs used in the study. About 65% of the settled microorganisms were aerobes grown without heat shocking, about 20% were anaerobes grown without heat shocking, over 5% each were molds and aerobes grown after heat shocking, and less than 5% were anaerobes grown after heat shocking. Only one of the 108 control packs utilized during the study was found to be contaminated. Forty test packs were contaminated during the study. Table 6 shows the type and frequency of organisms isolated from the 41 contaminated packs. The most frequently isolated organisms were Aspergillus spp, Streptomyces spp, and gram-positive sporeforming rods. also was similar to the amount found in the previous study. Thus, a direct comparison of the results of the two studies was possible, and these results add to current knowledge on safe time of storage for sterile packs in hospital CSSD. It is possible that manipulation of hospital linen packs larger than those used in this study might hasten microbial penetration, but this possibility was not evaluated. DISCUSSION In this study, packs wrapped with the BAR-BAC and muslin combination or the two-way crepe paper and muslin combination gave at least eight weeks of sterile storage on open shelves. Thus, more than twice the safe storage time is possible with these two combination wrappers than with double-wrap (four layers) muslin. Clearly, an evaluation of the economics of the extra wrapping cost with these combination wrappers versus the value of extra possible length of sterile storage would be necessary for an individual hospital to determine the preferable wrapping system for its use. Longer sterile storage is possible if packs are sealed into sterile impervious plastic bags (1). Closed-cabinet storage will also offer improved protection for sterile packs. Single wrappers are not recommended.

v3-fos

2018-04-03T05:29:00.608Z

1973-03-01T00:00:00.000Z

42975553

s2

NOTES Method for Detecting Mycoplasma and Bacterial L-Form Colonies in Relief with an Ordinary Light Microscope by Means of Oblique Light A simple method is described for obtaining images in relief of fresh preparations with an ordinary light microscope by lowering the condenser and displacing the objective at an "off-clicked" position. The detection of the typical "fried-egg" colo- nies of mycoplasmas and L-forms grown in agar has become an obligatory first step for their recognition (2). For this purpose, their direct observation under low magnification through a stereoscopic microscope is widely used. If the source of light is obliquely transmitted, as recommended by Edward (1), the results are improved, since a relief view of anything grow- ing on the agar surface can be more characteristic and identifiable. Due a chance we found a very simple for similar images in relief means of a by oblique observing colonies of mycoplasma grown in agar, condenser kept in low position to contrast, as positioning nosepiece slightly "off-clicked" The detection of the typical "fried-egg" colonies of mycoplasmas and L-forms grown in agar has become an obligatory first step for their recognition (2). For this purpose, their direct observation under low magnification through a stereoscopic microscope is widely used. If the source of light is obliquely transmitted, as recommended by Edward (1), the results are improved, since a relief view of anything growing on the agar surface can be more characteristic and identifiable. Due to a chance observation, we found a very simple method for obtaining similar images in relief with an ordinary light microscope, by means of a special optic effect caused by oblique light entering the objective. While observing colonies of mycoplasma grown in agar, with the condenser kept in a low position to accentuate the contrast, colonies appeared as if seen in relief, due to a sort of lateral illumination. Upon further careful observation, this effect was found to be due to inadvertently positioning the revolving nosepiece to a slightly "off-clicked" position. This observation is explained as follows. When the condenser is kept low, it illuminates a wider area than in the normal, high position, and the laterally located objects in the area receive oblique light which causes the effect of relief. The laterally displaced nosepiece can then reach this obliquely illuminated area. To obtain this special illumination, two adjustments in the microscope should be made. (i) The condenser must be placed in a low position, sometimes in the 484 lowest one possible, depending on the microscope. (ii) The body of the objective to be used should then be intentionally deviated from the correct "clicked" position. The relief increases as the deviation from the correct position increases, and the contrast of the illumination is accentuated. There is always, however, an optimal median point at which a satisfactory relief is obtained before definition becomes poorer and the contrast too strong. In addition, the position of the condenser, being always low, should be adjusted to the best point as determined by the image quality. Once this is found for each microscope, the results are always excellent and reproducible. Only magnifications of an approximate total of x 100 can be obtained with this method with a concomitant loss in definition, since the image is viewed through the periphery of the lens. However, with the larger diameter (x4 and x 10 objectives) commonly used when looking for mycoplasma or L-form colonies the quality of the image is not substantially impaired ( Fig. 1-6). The primary advantage of this method, apart from the general one of getting a stereoscopic view through an ordinary light microscope, is the possibility of taking consecutive pictures of a specific specimen under different kinds of light. Thus, the details of its surface can be observed first with the "off-clicked" position of the objective; then, when the nosepiece is clicked to the normal position, the inner features of the specimen can be easily studied with the transmitted light, either fresh or after staining. Compensation for the shift in field of view, induced by moving the lens from the "off-clicked" to the "on-clicked" position, must be made visually through the ocular during the process to insure the correctness of the field being viewed. We think that this simple method, or rather a trick, as one might call it, will be an aid to persons working with mycoplasma and L-forms as a means of distinguishing between colonies and artifacts (2,3). It may also be useful to virologists using tissue cultures, since the characteristic cytopathogenic effect will take a tridimensional appearance (Fig. 6). In general, any observation of a surface permeable to light will benefit from the application of this method; although in our experience the examination of specimens on the agar surfaces of petri dishes is the most rewarding one. To our knowledge, a simple method for obtaining special contrast in fresh preparations with accompanying sensation of relief with an ordinary light microscope is described here for the first time. Because of its simplicity, convenience, and the quality of the images that can be seen or photographed in a wide range of circumstances without extra cost or special attachments, this technique should gain wide use.

v3-fos

2020-12-10T09:04:12.627Z

1973-07-01T00:00:00.000Z

237229988

s2

Patulin Production in Apples Decayed by Penicillium expansum Sixty isolates of Penicillium expansum were tested for patulin production in decaying apples. All the isolates were found to produce the mycotoxin patulin as determined by thin-layer chromatography. Since patulin is known to be stable in many apple products, the results indicate that apple products made partially from apples decayed by P. expansum will contain patulin which may present a health hazard. The results also suggest that patulin may be important in the decay of apples by P. expansum. furo[3,2-C]-pyran-2(6H)-one} (1). Patulin has been shown to be carcinogenic to rats when administered by subcutaneous injection (3) and is acutely toxic to animals (2). The properties of patulin were most recently reviewed by Ciegler et al. (2). The purpose of this study was to determine how much patulin may occur in apples rotted by P. expansum. P. expansum was isolated from apples decayed in refrigerated storage at 0 C. The isolates were collected in 1971 and 1972 from one orchard in Burlington, Vt. Sound McIntosh apples were wounded with a sterilized glass rod and then mycelial plugs from cultures growing on potato dextrose agar were placed in the wounds. The inoculated apples were incubated in perforated plastic bags at 25 C. When the apples were mostly decayed, 100 g of decayed tissue from each apple was blended for 3 min and then filtered through Whatman no. 1 filter paper. Fifty milliliters of this filtrate or 50 ml of cider was extracted with three 50-ml portions of ethyl acetate. After the ethyl acetate extract was evaporated to dryness, the residue was dissolved in 5.0 ml of ethyl acetate (6 ulin was added to apple juice and extracted in the same manner. There was a 55% recovery of patulin with this extraction procedure. Twenty iliters of this extract or 1 to 25 1Liters of the patulin standard (1 mg/ml) was spotted onto Mallinckrodt 7 GF silica gel plates. The patulin was a gift from Lederle Laboratories, lot no. 1129 C-127-1. The plates were developed with benzene-methanol-acetic acid (90:5:5) in unequilibrated, unlined tanks and then inspected under shortwave ultraviolet. The patulin appeared as a dark spot against a fluorescent background (4). The R. value (approximately 0.47), size, and difference in intensity of the standard spots and the background were recorded. Each sample spot was placed in a range between two standard spots by comparing size and difference in intensity. All isolates of P. expansum tested produced patulin. The amounts varied from a low range of 9 to 18 mg/liter (26.7% of the isolates) to a high range of 120 to 150 mg/liter (1.7% of the isolates). Table 1 shows that, in general, the number of isolates per range decreased as patulin production increased. In selected cases, two or three apples were inoculated with the same isolate and were assayed for patulin. In these tests patulin production was similar in all but two cases. One hundred samples of fresh apple cider purchased from several cider mills were also assayed for patulin. Up to 45 mg of patulin per liter of apple cider was detected in five samples of so-called "organic" apple cider. Inspection of the mill revealed that the organic apple cider was made from wild, unsprayed apples that were insect damaged and decayed. In other ciders tested, the estimated amount of patulin detected ranged from undetectable in 91 samples up to 25 mg/liter in four samples in 1971. The ciders containing the appreciable amounts of patulin were produced by cider mills where decayed apples were not sorted out or where the apples were stored in large bins for extended periods. These practices were changed, and no patulin was detected in these sample mills in 1972. The results show that all the isolates of P. expansum tested produced patulin, often in appreciable quantities. Since patulin is stable in apple products (5,7), the results indicate that products made from apples rotted by P. expansum will contain patulin which may present a health hazard. Up to 45 mg of patulin per liter in purchased apple cider was found only in cider produced by cider mills where decayed apples had not been sorted or where apples were stored in bins for extended periods. This allowed P. expansum ample time to decay the apples in the bins. Since patulin is highly toxic to plants as well as animals (2), it is possible that the patulin produced by P. expansum may play a role in plant pathogenesis. Brian et al. (1) stated that patulin is not always present in P. expansumrotted apples. From this they postulated that patulin is not involved in apple decay. Our results do not support this hypothesis, since all of our isolates produced at least 10 ,ug of patulin/ml of expressed juice from decayed apples. This amount may be sufficient to be cytotoxic to apple cells.

v3-fos

2020-12-10T09:04:12.641Z

1973-11-01T00:00:00.000Z

237229942

s2

Diacetyl, Acetoin, and Acetaldehyde Production by Mixed-Species Lactic Starter Cultures Citrate utilization and acetoin, diacetyl, acetaldehyde, and lactic acid production in milk at 21 C by five different mixed-strain starters, containing Streptococcus diacetilactis (D type), Leuconostoc (B type), and S. diacetilactis and Leuconostoc (BD type), were measured. BD and D cultures utilized citrate more rapidly and produced more diacetyl, acetoin, and acetaldehyde than B types. All cultures produced much more acetoin than diacetyl, with the BD and D cultures producing four to five times larger amounts of acetoin than the B cultures. Reduction of diacetyl and acetoin toward the end of the normal incubation period was characteristic of BD and D cultures, whereas a similar reduction of acetaldehyde was characteristic of BD and especially of B cultures. Continued incubation of B cultures beyond 17 h also resulted in reduction of diacetyl and acetoin. Addition of citrate to the milk retarded diacetyl and acetoin reduction. Mn2+ had no effect on diacetyl production by a BD culture but increased citrate utilization and, as a consequence, caused greater diacetyl destruction in one of the B cultures. The mixed-strain starter cultures used in the manufacture of many fermented dairy products contain lactic acid-producing bacteria, usually Streptococcus lactis or S. cremoris, or both, and diacetyl-producing bacteria, usually S. diacetilactis or different species of Leuconostoc. Depending on the nature of the aroma-producing bacteria, Galesloot and Hassing (4) divided starter cultures into three types: B type, containing only Leuconostoc species as an aroma producer; D type, containing only S. diacetilactis as an aroma producer; and BD type, containing both Leuconostoc and S. diacetilactis as aroma producers. Some differences in the behavior of these starters have been reported (4,5), but in many studies no distinction was made regarding the nature of the aroma producers. Speckman and Collins (15) have shown that diacetyl and acetoin are synthesized by two distinct pathways and state that this finding places in question the results of a large number of published papers in which measurements of acetoin plus diacetyl have been considered to indicate the total production of diacetyl. According to de Man and Galesloot (10), the addition of trace amounts of Mn2+ to milk stimulated production and reduction of acetoin plus diacetyl and increased the utilization of citrate by B type but not D type cultures. High numbers of S. lactis and S. diacetilactis in comparison to leuconostoc have been implicated as the cause of a green or yogurt-type flavor in buttermilk because of the accumulation of acetaldehyde (9). Since Leuconostoc species can convert acetaldehyde to ethanol in milk (6), it seems probable that B and D type cultures may behave differently in their ability to produce acetaldehyde. The purpose of the present study was to compare flavor production by cultures containing different aroma producers. MATERIALS AND METHODS Cultures. The B type mixed cultures (FR8 and 21) were from J. Stadhouders, NIZO, Ede, The Netherlands, the D culture (H) was obtained from Chr. Hansen's Laboratory Ltd., London, and the BD cultures (V and FD) were from Visby Maelkeri-Laboratory, Tonder, Denmark, and Flora Danica Laboratory, Odense, Denmark. All cultures were routinely cultivated in autoclaved (5 min, 121 C) 10% low-heat nonfat milk solids (NFMS) at 21 C by using a 1% inoculum. Identification of mixed strain cultures. Whether the cultures were of the B, BD, or D type was determined by a modification (Leesment, personal communication) of the method of Nickels and Leesment (12,13). In the modification, colonies surrounded by a clear halo were inoculated into litmus milk fortified with 0.3% yeast extract and incubated 820 at 25 C for 6 days, after which citrate and acetoin were determined by the methods given below except that 1 ml of the culture was used directly in the estimation of acetoin. Cultures which produced acetoin and utilized citrate were considered to be strains of S. diacetilactis, whereas those utilizing citrate and not producing acetoin were considered to be Leuconostoc. Experimental procedure. Previously chilled, autoclaved 10% NFMS was titrated to allow calculation of the percent developed lactic acid and inoculated with 2% of the culture being studied. After thorough mixing, the inoculated milk was divided into approximately 70-ml volumes in sterile screw-capped bottles (150-ml capacity) which were incubated at 21 C. One bottle was removed periodically for the various analyses. For the experiments containing added citrate, the control milk before inoculation was brought aseptically to the pH of the milk containing added citrate (pH 6.9). Citrate was added as sterile trisodium citrate to give a final concentration of added citrate in the milk of 0.5% (wt/vol) as citric acid. Analyses. Lactic acid production was measured by titrating 10 g of culture to pH 8.3 with 0.11 N NaOH by using a Radiometer pH-stat and expressing the results as percent lactic acid. Diacetyl and acetoin were measured in steam distillates by modifications of the Prill and Hammer and Westerfeld procedures, respectively (B. Walsh and T. M. Cogan, J. Dairy Res., in press). The method of Lindsay and Day (8) was used to measure acetaldehyde except that 20 g of culture was steam distilled in the apparatus described by Cogan (2) and a sample of the first 10 ml of distillate was analyzed. Citric acid was measured in 5-g amounts of the NFMS by the method of Marier and Boulet (11). All results are expressed on a per gram basis. RESULTS Accumulation patterns. A comparison of diacetyl, acetoin, acetaldehyde, and lactic acid production, and citric acid utilization by cultures H (D type) and 21 (B type) is shown in Fig. 1. Similar rates of lactic acid production were obtained in both cultures. However, the D culture produced more acetoin, diacetyl, and acetaldehyde than the B culture, but none of these compounds was produced at the same rate as lactic acid in either culture. Slight reduction of diacetyl and acetoin were noted in the D culture at the end of the growth period, whereas the B culture did not appear to have reached maximum production of either compound. Little acetaldehyde accumulation occurred in the B culture. More rapid utilization of citric acid occurred in the D culture, with complete utilization obtained after about 14 h of incubation. Citrate was more or less completely utilized within the pH range of 4.8 to 5.0 in the case of the D and BD cultures. The latter cultures gave results similar to the D culture, whereas the other B culture behaved like the one shown in Fig. 1. Acetoin accumulation patterns for all the cultures are shown in Fig. 2. Cultures FR8 and 21 (B type) produced about the same amount of acetoin which reached a maximum of about 85 Mg/g, whereas cultures FD, V, and H (BD and D types) produced between 400 and 520 gg/g. Reduction of diacetyl by D and BD but not B cultures was evident toward the end of the incubation period. In all cultures, much more acetoin than diacetyl was produced (e.g., culture V produced a maximum of 5 g). TIME, hours Acetaldehyde production (Fig. 4) VOL. 26,1973 DIACETYL, ACETOIN, AND ACETALDEHYDE PRODUCTION containing added citrate remained above 5.10 even after 16 h of incubation when the pH of the control culture was 4.70. In the absence of added citrate, reduction of accumulated diacetyl and acetoin occurred when the citrate level had fallen to approximately 100 jsg/g (i.e., 94% utilization of citrate). The reduction of acetoiu was retarded and diacetyl was delayed by increased levels of citric acid. A similar but not as dramatic effect was obtained in the case of the two B cultures. Effect of manganese. Mn2+ addition at a level of 5 ;sg/ml increased the rate of utilization of citrate by culture FR8 (B type) but resulted in lower absolute amounts of diacetyl and acetoin being produced with subsequent greater rates of destruction (Fig. 6). Acetoin reduction was affected much more than diacetyl reduc- tion. Similar rates of diacetyl and acetoin production were observed up to 15 h of incubation. Once the citrate level had fallen to 100 ,. g/g, reduction of both acetoin and diacetyl ensued. Mn2+ had no effect on diacetyl or acetoin production or citrate utilization by the FD culture (BD type). In the case of the other B type culture, addition of Mn2+ retarded citrate utilization and led to slightly lower absolute amounts of diacetyl and acetoin being produced. DISCUSSION The more rapid utilization of citrate by D and BD cultures compared to B cultures probably reflects faster growth of S. diacetilactis compared to leuconostoc strains. S. diacetilactis has been shown by Harvey and Collins (3) to possess an inducible citrate permease, which may be an additional factor in the utilization of citrate by D and BD cultures. In agreement with the present results, Galesloot and Hassing (4) also noted poor citrate utilization by B type cultures. In their studies, season had a pronounced -L---.L. seffect on citrate utilization, which was lowest in 16 April-and highest in October-produced milk. The D and BD starters also produced more I C by cul-diacetyl (up to 11 yg/ml) and acetoin (up to 500 1 V (-). Ag/ml) than the B type, again reflecting rapid uptake of citrate by S. diacetilactis, which has been shown by many workers to act as a precursor for diacetyl and acetoin production in milk. The acetaldehyde may also originate in citrate since Speckman and Collins (15) have shown that "active" aldehyde is involved in diacetyl biosynthesis. Seitz et al. (14) have shown that S. diacetilactis contains diacetyl reductase, which probably explains the reduction of diacetyl by cultures containing S. diacetilactis (D and BD types) observed in the present study. If this species also contains an active acetoin reductase, a similar explanation of the present results can be invoked. In this connection, Bryn et al. (1) have shown that the same reductase enzyme carried out both diacetyl and acetoin reduction in Aerobacter aerogenes. Much greater amounts of acetoin than diacetyl were found in all cultures, but especially in those containing S. diacetilactis. Both diacetyl and acetoin are synthesized by different metabolic routes (15), and whether or not the present results reflect a more active pathway for acetoin than for diacetyl biosynthesis or the presence of a highly active diacetyl reductase enzyme is not known. A diacetyl reductaseless mutant of S. diacetiflactis could be used with good effect to study this particular aspect. Despite the fact that diacetyl and acetoin are synthesized by different pathways (15), any factor which had an influence on diacetyl production also influenced acetoin formation in a similar manner. Acetaldehyde production by B cultures compared to BD and D cultures was low, probably due to the presence of an active alcohol dehydrogenase in the leuconostoc strains present, as suggested by the results of Keenan et al. (6). Unfortunately, no effort was made to quantify ethyl alcohol in the present study. Acetaldehyde production by BD cultures lies between B and D cultures, lending some weight to this conclusion. The acetaldehyde accumulation patterns for the BD and D cultures were similar to those obtained by Keenan et al. (7) for single-strain lactic streptococci. Diacetyl and acetoin reduction occurred only in the BD and D cultures and was not usually noted until late in the incubation period when virtually complete utilization of citrate had occurred. These experiments were limited to the usual incubation period given to cultures in industry, namely 14 to 16 h. Continued incubation of B cultures beyond 17 h also led to slight diacetyl and acetoin reduction (Fig. 6) which coincided with the presence of low levels of citrate. These findings suggest that the rate of biosynthesis of diacetyl and acetoin is greater than the rate of destruction so that destruction is not evident until synthesis ceases when the precursor compound (citrate) is completely utilized. Supporting evidence for this conclusion is found in the effect of Mn2+. When Mn2+ increased the rate of utilization of citrate, destruction of diacetyl occurred at an earlier time in the incubation period. That addition of citrate increased diacetyl production has been noted by numerous workers. No reduction of diacetyl or acetoin occurred in the presence of increased citrate (Fig. 5), and it is interesting to speculate that, if incubation had continued until all the added citrate were utilized, reduction of both diacetyl and acetoin may have occurred. Another explanation may be the effect of pH on diacetyl reductase, which may be more active at low pH than at high pH since the addition of citrate resulted in a final higher pH value of the milk. The compound(s) to which diacetyl and acetoin are reduced has not been identified, although Galesloot and Hassing (5) state that it is 2, 3-butylene glycol. In agreement with the results of Galesloot and Hassing (4), Mn2+ increased the rate of destruction of diacetyl and acetoin but in only one of the two B cultures examined. However, the effect is not a direct one on the rate of reduction of diacetyl and acetoin as suggested by these workers, but rather on the rate of utilization of citrate, since destruction of diacetyl, whether in the presence or absence of Mn2+, does not occur until all the citrate is utilized. Thus, as found in the present study, if Mn2+ retards citrate utilization, less destruction of diacetyl will occur, whereas if it promotes utilization a lower absolute amount will be produced and greater destruction ensues. This interpretation of the present results suggests that the role of Mn2+ and citrate in diacetyl and acetoin production by starter cultures needs further study.

v3-fos

2016-05-12T22:15:10.714Z

1973-07-15T00:00:00.000Z

17091072

s2

Field testing of young breeding pigs. I. – Description of the construction of a performance index SUMMARY In order to construct a performance index, that could be used in field testing of young breeding pigs, samples of gilts and of boars from the Dutch Landvace breed and the Dutch York-shire breed were measured 3 or 2 times. The sample sizes varied from 150 to 2 86. The index chosen was a linear combination of 2 scores : a score for weight and a score for backfat thickness. INTRODUCTION Farm testing of young breeding gilts of 5 i/2-8-months old started in the Netherlands at the end of 19 68 in the province of Limburg. During the last years this system became increasingly popular and at the moment about 10 ooo young animals have been tested. The method performed has been the usual one, weighing the animals and measuring their backfat thickness by means of ultrasonics. In order to rank the animals the weight was corrected for age ; backfat thickness was corrected for weight. The corrected values were transformed to scores and a linear combination of both scores gave the final index. (') Publication no. A 27 z of the Research Institute for Animal Husbandry « Schoon.oord ». In practice it turned out that the older animalsand consequently the heavier animalsusually got a higher index than the younger ones. A preliminary investigation showed this was caused by an inaccurate correction of weight for age, this correction having been obtained from the regression coefficient of weight on age, based on individual observations, with one observation per animal. It was assumed that the regression of weight on age within animals could be a better basis for correction. To estimate this regression coefficient a special measuring programme was performed, in which a sample of animals was measured more than once. MATERIAL A sample of young gilts and boars from two breeds (Dutch Landvace = DL and Dutch Yorkshire = DY), spread over many farms, was taken. The gilts were measured three times with a 3 o-day interval between each measurement, the boars were measured twice, also with a 30 -day interval. The average backfat thickness of four measuring point was used. The four measuring points were obtained by the following procedure. The posterior edge of the cartilage of the scapulum and the posterior edge of the last rib were palpated on the right side of the animal. Through each of these two points a line was drawn perpendicular to the midline of the back. The distance between the two intersection points with the mildine of the back was divided into 3 equal parts and was extended in posterior direction by the lenght of such a « third » part. Then the most anterior point (on the shoulder) was omitted. The backfat thickness was measured five cm lateral to the four remaining points. The numbers of animals and the means of the traits for each of the 3 or 2 measurements are given in table r. The table shows differences in means between sexes and also between breeds. At about the same weight the DY-animals have less backfat than DL-animals. This difference is not reflected in a difference of fat percentage, when carcasses of animals of both breeds are dissected. The reason for this discrepancy is a difference in the distribution of the fat layer between the two breeds. DY has more fat at the shoulder and less at the loin than DL. The fat thickness at the shoulder, measured by means of ultrasonics, however, is not very accurate and is therefore not included in the average ultrasonic backfat thickness. REGRESSION BETWEEN AND WITHIN ANIMALS The linear regression of weight on age, and of backfat thickness on weight was estimated between as well as within animals. Also the total regression was estimated. The between animals regression is the regression based on animal means. The within animals regression is based on the sum of products and sum of squares, estimated within each animal and pooled over animals. The results are shown in table 2 . The table clearly shows the difference between the two kinds of regression coefficient : between animals versus within animals, especially for the regression of weight on age. In the latter case the regression between animals is lower than the regression within animals, except for DY-boars. There is no doubt that the regression coefficient of weight on age within animals is reflecting the real growth rate in that particular age range much better than the regression between animals. The regression between animals will be affected by any preselection among the animals, and also by a less representative choice of the sample. The differences between the various « between animals regressions !· of table 2 are not in accordance with the growth data of these breeds and sexes, shown in progeny testing stations. Restricting ourselves to the regression within animals, then we see a clear sex difference : the regression of weight on age in boars is higher than in gilts. This is in accordance with the higher growth rate of boars. Within sexes there are no breed differences. For the regression of backfat thickness on weight the differences between the two types of regression coefficients (between versus within animals) are not so striking as for the regression of weight on age. The regression between animals is higher than the regression within animals, except for DI,-gilts. With regard to the regression within animals there are sex as well as breed differences. Boars have a lower regression then gilts and DY-animals have a lower regression than DI,-animals. CONSTRUCTION OF SCORES FOR WEIGHT AND BACKFAT THICKNESS The goal of the investigation was to construct a performance index, which should be a combination of two scores : a score for weight and a score for backfat thickness. The results of the analysis, presented in tables I and 2 , led to the conclusion that it was necessary to base the scores on the regression within animals. Furthermore different scores for each sex and breed should be used. Besides that it was found that animals that were heavier at a given age, had a higher regression of weight on age. Similarly, animals that had thicker backfat at a given weight showed a higher regression of backfat thickness on weight. In order to take this into account the following procedure was taken for the construction of the scores. This will be described for the construction of the score for weight. In an analogous way the score for backfat thickness, which is called score 2 , can be derived. In table 3 the necessary quantities for the construction of the scores are summarized. In the last column of table 3 the correlation ra b between the intercept a and the regression coefficient b of the individual regression lines are given. These correlations are not needed for the construction of the scores. CONSTRUCTION OF AN INDEX AND INDEX-TABLES In order to rank the animals on their performance a simple combination of both scores was taken. A high score for weight reflects a relative high growth rate and a low score for backfat thichness reflects a relative low backfat thickness. So a positive score for weight and a negative score for backfat thickness were desirable. Therefore the index was defined as : In this index both scores have the same weight, which is debatable. It is of course possible to construct a more sophisticated index, in which the scores are given weights that are in accordance with their respective economic values and heritabilities. A model calculation, attaching reasonable economic weights and heritabilities to both traits showed us that the decision to give both scores the same weight is not far from the truth. In this calculation it was taken into account that in practice most breeders are feeding their animals restricted, so there is a rather strong correlation between growth rate and food conversion. From the great number of animals on which they are based, both scores may be taken to have a distribution in the population with mean o and standard deviation z. If the scores were uncorrelated, the index would have a distribution with mean o and standard deviation !/i&dquo; + i = !/ 2 ! z . q.. An analysis showed that the scores were slightly unfavourably correlated. This is shown in table 4 . In gilts the correlation is lower than in boars. It seems that the correlation is decreasing when the animals are getting older (compare first and later measurements). These correlations are much lower than those found by S TANDAL (r 9 62), although it must be taken into account that S TANDAL used slightly different scores. His score for weight was based on the partial regressions of weight on age and backfat thickness and his score for backfat thickness was based on the partial regressions of backfat thickness on weight and age. The observed standard deviation of the index of gilts was almost i.q.. The index of the boars had a lower standard deviation (about i.2 5 ), because of the small unfavourable correlation between both scores in boars. For practical purposes the use of negative values for the index was not desirable. For this reason the original index distribution was rescaled to a distribution with mean io and standard deviation = 2 . 5 . This implied that in gilts an original value of -4 standard deviations = -5 .6 was rescaled to o and a value of -! 4 standard deviations = !-5 .6 was rescaled to 20 . The corresponding original values in boars were -4 standard deviations = -5 and -f-4 standard deviations = -!-5. With this rescaling almost the whole distribution of observed index values will fall between o and 20 . A value of o is indicating an animal with a very low performance and a value of 20 is indicating an animal with a very good performance. To facilitate the use of the index, tables were constructed. For each age class (comprising 5 days) a separate table was made. In the table the corresponding index value is given for each weight (in classes of 2 kg) and each backfat thickness (in mm). The ranges covered by these tables are : REPEATABILITY OF THE INDEX To get an idea of the reliability of the index constructed the repeatability of this index was estimated. This was done by computing the correlation between the different indices of the same animal for the successive 2 or 3 measurements. The results are shown in table 5.

v3-fos

2018-04-03T01:12:58.840Z

1973-01-01T00:00:00.000Z

28630358

s2

Effects of Some Disinfectants on African Swine Fever Virus Ten commercially available disinfectants were tested at high pH in 2% sodium hydroxide and low pH in 2% acetic acid as inactivants for African swine fever (ASF) in a protein-rich blood-spleen homogenate. As assayed in leukocyte cultures, sodium hydroxide and acetic acid, sodium meta silicate and Roccal did not inactivate ASF virus in 1 hr at 22 to 25 C. Some viricidal activity as assayed in leukocyte cultures was found with Weladol, Triton X-100 Amphyl, pHisoHex, sodium dodecyl sulfate, LpH, Environ, Environ D, and One-Stroke Environ. Of these, the last four appeared to be most promising. When assayed in pigs, only One-Stroke Environ (1/E) was viricidal. Concentrations of 1.0, 0.75, and 0.5 were effective, but, at 0.25%, virus was not inactivated. The minimal time to inactivate ASF virus by 1% 1/E is 60 min. A room contaminated with ASF virus was made safe for pigs after 1 hr by spraying with 1% 1/E. The most active component of 1/E is o-phenylphenol. Although another component of 1/E, i.e., o-benzyl-p-chlorophenol, also has some activity, the mixture of the active components of 1/E is most effective against ASF virus. One of the soluble antigens associated with ASF virus is destroyed by 1/E. antigens associated with ASF virus is destroyed by 1/E. African swine fever (ASF) is no longer an obscure disease of Africa. Since 1957 it has occurred in Portugal, Spain, France, Italy and-in June of 1971-Cuba (13). The causative agent is a deoxyribonucleic acid virus (1) that is sensitive to ether and chloroform (5). Since a safe and effective vaccine has not been produced, control of the disease depends entirely upon rapid diagnosis, slaughter of infected and exposed animals, and strict imposition of effective sanitary measures. This so-called "stamping out" procedure was successfully applied in France and Italy, and the disease was eliminated before extensive spread occurred. However, there is no published evidence that the measures applied would be adequate or feasible in areas having an intensive swine industry. It is known that ASF virus may remain viable for long periods of time in feces, blood, and soil and on wooden surfaces (9). It is also known that the virus suspended in a menstruum rich in protein may withstand rather extensive variations in pH (5). Furthermore, proteolytic enzymes have little or no effect on the virus (5). It is therefore likely that solutions commonly used to disinfect premises where animal diseases have occurred are not completely effective in destroying ASF virus; nor will a waiting period of several months assure that the premises are safe for restocking. Laboratory methods for inactivation of ASF virus have been reported (16). The methods employ fast-acting agents such as beta-propiolactone, acetylethylenemine, and glycidaldehyde. Even at low concentrations, these compounds are effective ASF virus inactivants, but, because of their instability in aqueous solutions, they are not suitable for use in the field where spraying affords the only practical means of covering the large areas that must be disinfected following an outbreak. The presence of extraneous organic matter may greatly reduce the effectiveness of some classes of chemical disinfectants. This is especially true of chlorine compounds and cationic detergents (17). A high concentration of organic matter is a feature common to the environments most likely to be contaminated with ASF virus during an outbreak of the disease. Since the work reported here was undertaken in an effort to find a disinfectant capable of rendering an infected premise safe for early restocking after an outbreak, the disinfectants were tested against ASF virus in the presence of a high concentration of proteins, and one agent that was effective under 115 these conditions was further used to decontaminate a room containing several days' accumulation of litter, feces, and urine of pigs that died of ASF. MATERIALS AND METHODS Virus. The Lisbon strain of ASF virus (11) was used. The stock virus suspension was prepared by I. C. Pan of this laboratory. Pigs infected by the intravenous route died of acute ASF 5 to 6 days later. Spleens were removed, homogenized in an equal volume of whole defibrinated blood, filtered through several layers of cotton gauze mesh, divided into several portions, and stored in a dry-ice chest. The stock virus suspension had a protein content of 21.3% as determined by micro-Kjeldahl analysis. A fresh sample was thawed for each experiment. Hemadsorption titers determined at the time of each experiment ranged from 101 to 107/ml. Virus assay. In all inactivation experiments, the stock virus and virus-inactivant mixtures were titrated by the hemadsorption reaction (10) in pig leukocyte cultures (LC) prepared as described elsewhere (7). Titers were reported as log of 50% hemadsorption per milliliter. The toxicities of the inactivants alone were simultaneously determined in similar cultures. Soluble antigens. Monolayer cultures of VERO cells were infected with ASF virus (5). When the cytopathic changes were well advanced, the cells were suspended by shaking and were collected by centrifugation. One volume of packed cells was suspended in two volumes of phosphate-buffered saline and sonically disrupted at 0 to 5 C, using ten 10-sec bursts of energy. The resulting suspension was clarified by centrifugation at 10,000 x g for 30 min, and the supernatant fraction was used as antigen. Agar gel diffusion precipitation tests. Plates were prepared with 0.85% Ionagar in 0.05 M borate buffer, pH 8.6. The distance between the reagent wells was 3 mm. ASF survivor pig serum was used as an antibody source. Normal pig serum and an extract of sonically disrupted, noninfected cultured cells were used as controls. Immunoelectrophoresis. The microtechnique (14) was employed with 0.025 M barbital acetate buffer, pH 8.6, and a constant current of 2 ma/cm for 90 min. A 0.1% alcoholic solution of bromophenol blue was used as an indicator of electrophoretic migration. The precipitin patterns were developed with ASF survivor pig serum. Inactivants. The sources of inactivants used were as follows: LpH, Environ, Environ D, and One- Stroke In addition to the commercially available germicidal agents listed above, 2% acetic acid and 2% sodium hydroxide were tested in the initial screening experiments, for these solutions have been widely used as viricidal agents (3). Each of the inactivants was made up as a 1% solution in both 2% acetic acid and sodium hydroxide. In later experiments, distilled or tap water were used as diluents to prepare 1% solutions and the concentration of the inactivant was altered as shown in the tables. Tests in animals. Adult pigs weighing from 100 to 200 kg were used in the ultimate determinations of virus inactivation. They were housed in rooms designed for maintenance of strict isolation (2), but, because of space limitations, only the pigs serving as virus controls were housed separately. The pigs receiving the virus-inactivant mixtures were housed together. All inoculations were by the intramuscular route. Temperatures were taken daily, and a rise to 103 F (39.4 C) or higher was regarded as the first sign of ASF infection. The first animal displaying the febrile reaction was regarded as having been infected by the inoculum given. Any other animal in the room that became febrile within the next 2 days was likewise considered to have been infected by the inoculum administered. If an animal's first febrile response occurred more than 2 days after that of the first reactor in the room, it was regarded as a possible contact infection. In any case, the delayed response would be indicative of either complete or substantial inactiviation of the virus in the inoculum given. Further, comparison of the day of fever or death among these pigs and the control pigs served as an additional basis for analysis of the test results. Spleen and kidney samples were taken from one or more pigs in each experiment and assayed for ASF virus by the hemadsorption test in LC. Tests were also made for the presence of soluble antigen by using agar gel diffusion precipitin reaction (6). Inactivant screening test. The purpose of this test was to eliminate any inactivant that would not reduce the titer of the test virus at least from 106 to 103 in 60 min at room temperature (22-25 C). One volume of the stock virus suspension was mixed with nine volumes of the test compound. After 60 min, 1 ml of the mixture was diluted 1:10 in culture fluid and the pH was adjusted to 7 to 8, by using 1 N NaOH or 1 N HCl. A 1-ml amount of the adjusted mixture was further diluted 1:10 in the culture medium, and four LC in short Leighton tubes were each inoculated with 1 ml of this dilution. In all cases, the pH of this second dilution of the virus-inactivant mixture was 7.3 to 7.4 as measured by a glass electrode. Each of the test inactivants was treated in the same way and assayed for toxicity. The LC were examined daily for signs of virus hemadsorption or toxicity. Assay of inactivated ASF virus in pigs. In the first of these experiments, four of the most promising inactivants, i.e., SDS, Environ, Environ-D, and One-Stroke Environ (1/E) were used at the 1% level with stock virus and a 60-min inactivation time. Nine volumes of inactivant were mixed with 1 ml of virus. After 60 min, pigs were inoculated with the virusinactivant mixtures. Minimum effective concentration. The second experiment with pigs was to determine the minimal concentration of disinfectant required to render the virus noninfective for pigs. A 1-ml amount of stock virus was mixed with nine volumes of 1.0, 0.75, 0.5, and 0.25% 1/E, and 60 min later two pigs were inoculated with each of the virus-inactivant mixtures and assayed. Two pigs housed in a separate room served as virus controls. Time of inactivation. The third experiment with pigs was designed to determine the minimal time required to inactivate ASF virus by 1/E. Nine volumes of 1/E solution were mixed with one volume of stock virus suspension, and a 5-ml sample was removed at 15, 30, 60, and 120 min. Two pigs were immediately inoculated at each time interval with the virus-inactivant mixture, and the virus was titrated in LC at the same time. Two virus control pigs were housed separately. Transmission and disinfection experiment. To establish that an ASF virus-contaminated area could be effectively disinfected, it was first necessary to show that the contaminant in the room could be lethal to pigs. Accordingly, a room housing ASF virus-infected pigs was not cleaned for 2 days. During this time, two pigs previously infected with ASF virus died of the disease. These pigs were removed and 4 hr later two susceptible pigs were placed in the room. One pig developed a fever in 3 days and died on the 9th day. The other pig appeared normal during this interval and was destroyed on the 10th day. Both pigs were removed, and the room was thoroughly sprayed with a 1% solution of 1/E prepared in tap water. All residual fecal material and feed were dispersed with 1/E. One hour later, four susceptible pigs were placed in the room. Viricidal activity of the active components of l/E. Each of the three active ingredients in 1/E, i.e., o-phenylphenol, o-benzyl-p-chlorophenol, and p-tertiary-amylphenol, was prepared at the same concentration and pH as found in a 1% aqueous solution of 1/E. Each was tested singly and in combination, by using the stock virus. Assay for virus in these mixtures was in LC and in pigs. RESULTS A summary of the results of the first screening test with 1% solutions of 10 disinfectants in both 2% NaOH and acetic acid as well as the latter reagents alone is shown in Table 1. Virus was detected by the hemadsorption reaction in LC cultures in both NaOH and acetic acid, sodium meta silicate at both high and low pH, Roccal in the alkaline, and Solvental in the acid solutions. With the other disinfectants, i.e., LpH, Environ D, Environ, Weladol, pHi-soHex, Triton X-100, SDS, and Amphyl, there was either no hemadsorption or the test was not readable because of various degrees of cellular degeneration caused by the disinfectant. The results of a second screening test employing disinfectants that appeared to be viricidal or toxic (Table 1) are shown in Table 2. These disinfectants were used as 0.1% aqueous solutions, and the virus-inactivant mixture was assayed in LC at the 102, 10-3, and 10-4 dilutions. Four of the nine disinfectants tested, Environ D, Environ, 1/E, and SDS, appeared to inactivate ASF virus at the 10-2 dilution. In contrast, hemadsorption occurred in the 10-2 dilutions of the LPH, Weladol, pHisoHex, Amphyl, and Triton X-100 virus mixtures. The three Environs and SDS were tested as ASF virus inactivants as 1.0 and 0.25% aqueous solutions. Pigs were used as the virus indicator to circumvent the cytotoxicity of the disinfectants in LC at this concentration. Table 3 shows the results of this test. Only the pig inoculated with the 1% 1/E-virus mixture failed to develop ASF from the inoculum. The pig inoculated with the 0.25% 1/E-virus mixture developed ASF as did the pigs inoculated with SDS, Environ, and Environ D-treated virus suspension. In determining the minimum effective concentration of 1/E, each virus-inactivant mixture was tested in two pigs. As indicated in Table 4, the two pigs inoculated with the 0.25% 1/E-virus mixture developed ASF and died in times comparable to the virus control animals. The pigs inoculated with the 0.50, 0.75, and 1% 1/E-virus mixtures had reactions indicative of infection by contact. It was therefore indicated that 1/E in concentrations of 0.5% and higher was able to inactivate the stock virus suspension. The results of the experiment to determine the minimal time to inactivate the stock ASF virus suspension are shown in Table 5. One of the two pigs (pig no. 21) inoculated after 15 min of inactivation developed ASF and died. Another animal (pig no. 23) that had received stock virus exposed to the disinfectant for 30 min reacted within 2 days after the initial reaction of pig no. 21. Complete inactivation of the stock virus at 30 min was therefore regarded as uncertain. However, inactivation at 60 and 120 min was unequivocal, and, compared to the virus controls, it was evident that substantial inactivation also occurred in the lesser time intervals. One of the two pigs housed in the contaminated room developed a febrile response 6 days later and died on the 9th day. ASF virus Test procedure: 1% solutions of each disinfectant were made in 2% acetic acid and 2% sodium hydroxide. Stock virus (a blood-spleen mixture containing 106.5 hemadsorbing units of ASF virus per ml) was diluted 10x in the disinfectant solution and held for 1 hr at room temperature (22-25 C). The mixture was then diluted 10 x in culture medium and adjusted to pH 7 to 8 with sodium hydroxide or hydrochloric acid. After another 10x dilution in culture medium, 1.0 ml was put on each of four leukocyte cultures. Duplicate tests without virus served as toxicity controls. h +, Positive hemadsorption reaction indicating presence of active virus; -, negative for hemadsorption indicating inactivation of more than 3 logs of virus; D, degeneration of leukocytes due to toxicity of disinfectant solution. " DPI, day postinoculation. Note: An initial febrile response occurring 3 or more days after that of the first responding animal in the room is indicative of a contact infection and cannot be attributed to the inoculum given. c Pig 16 did not have a fever and was still apparently normal at 30 DPI. 6 10 22 12 14 30 23 8 12 24 11 14 60 25 11 14 26 11 14 120 27 13 18 28 11 14 Virus control 29 4 8 30 4 7 a Stock virus was diluted 10x in 1% One-Stroke Environ and held at 22 to 25 C. Samples taken at the indicated times were injected immediately (1 ml intramuscularly). " Pigs 21 through 28 were housed together. Pigs 29 and 30 were held in a separate room. c DPI, day postinoculation. Note: An initial febrile response occurring 3 or more days after that of the first responding animal in the room is indicative of a contact infection and cannot be attributed to the inoculum given. The solutions were prepared with distilled water, and in each instance the compound was in the concentration it would be in a 1% solution of 1/E. The stock virus (106 5 hemadsorbing units per ml) was diluted 10x in the test solution and held for 1 hr at 22 to 25 C. The mixture was further diluted 100x in culture medium, and 1 ml was put on each of four leukocyte cultures. b +, Positive hemadsorption reaction, indicating presence of active virus; -, no reaction, indicating inactivation of more than 3 logs of virus. was recovered from its spleen. The other pig housed in the room did not develop a fever or signs of ASF and was killed on the 10th day as ASF developing at this time could be considered to originate from the first pig. The four pigs housed in the previously ASFcontaminated room that had been sprayed with 1% 1/E did not develop ASF, nor were ASF precipitating antibodies detectable in their sera when the experiment was terminated after 21 days. Table 6 shows the results obtained when the stock virus was exposed to each of the active ingredients of l/E and assayed in LC. There was no sign of hemadsorption in o-phenylphenol, o-benzyl-p-chlorophenol, or in mixtures containing these compounds, but hemadsorption did occur in the p-tertiary-amylphenol-virus mixture. Toxicity of the compounds was tested at the same time, and, although some cytotoxicity was evident, there was a sufficient number of intact leukocytes and red blood cells available to indicate the presence of virus by hemadsorption. The results of inoculating these compound-virus mixtures into pigs are shown in Table 7. Compared to the virus control pigs, there was no difference in the first day of temperature (3 days postinoculation [DPI ]) in the pigs inocu- After 30 min of reaction with 1% 1/E, one of the soluble antigens was greatly diminished, and after 60 min it could no longer be detected (Fig. 1). Electrophoretically, this antigen at pH 8.6 had very little migration in contrast to most soluble antigens that migrate toward the positive electrode. DISCUSSION Ideally, each virus-inactivant mixture should have been tested in pigs housed in separate rooms. Because of space limitations, this was impossible. Therefore, it was necessary to depend upon previous experience con-cerning contact transmission of ASF virus. The Lisbon 60 isolate was chosen for these experiments because it was involved in an extensive outbreak in domestic swine; it was known to persist and spread readily under field conditions; and its clinical manifestations were quite uniform and well defined. The onset of fever was a fairly reliable indicator of when infection had occurred. Since it was known that ASF virus may appear in pharyngeal and nasal excretions as early as 2 days before fever (4), it was reasonable to assume that an animal having its first febrile response more than 2 days after that of the first reactor in the room was either infected by contact with the first infected animal or had received an inoculum containing an extremely low level of active virus. The compounds tested were representative of the common classes of disinfectants and detergents, namely, halogen derivatives, substituted phenols, polyphosphates, quaternary amines, nonionic and anionic surface-active compounds. All were evaluated by the same criterion, i.e., the effectiveness of a 1% solution as an inactivant of ASF virus suspended in a proteinrich menstruum. From the data in Tables 1 and 2, it is evident that many commercially available disinfectants have a marked degree of viricidal activity against ASF virus. Among these are Amphyl, pHisoHex, Weladol, and Triton X-100, besides those tested in pigs FIG. 1. Effect of One-Stroke Environ (lIE) on the soluble antigens of African swine fever virus. Untreated soluble antigens are on the left, and the lIE-soluble antigen mixtures are on the right on each slide. A current of 2 ma/cm was applied for 90 min, and the precipitin patterns were developed with ASF survivor pig serum. One fast moving antigen was apparently destroyed immediately. The progressive disappearance of the antigen closest to the negative electrode during the 60-min treatment is evident. (Table 3). Under conditions of higher concentrations, longer time of inactivation, or a menstruum having a lower protein content, they may well be effective inactivants for ASF virus. It appears from Tables 6 and 7 that the most effective viricidal ingredient against ASF virus in One-Stroke Environ is o-phenylphenol. This compound is a common ingredient in many commercially available disinfectants and is effective in destroying hog cholera virus (18; H. S. Wright, Bacteriol. Proc., p. 10,1971). Its use for that purpose has been approved by the Animal and Plant Health Division of the U.S. Department of Agriculture (12). The 1% solution of o-phenylphenol recommended as a disinfectant for hog cholera is 10 times the concentration used in our experiments (Tables 6 and 7) and should likewise serve to inactivate ASF virus under field conditions. Since ASF virus is sensitive to ether and chloroform and its nucleic acid is readily extractable by both phenol and SDS (1), it is probable that the mechanism of its inactivation by phenolic derivatives is solubilization of the lipid segments of the virus envelope. When the antigenic components of the virus have been characterized, the observation made with immunoelectrophoresis will be more meaningful, and may perhaps be indicative of the mechanism of inactivation. Since certain Argasid ticks are capable of harboring and transmitting ASF virus for many months (8), disinfection of a previously infected premise may also require elimination of these arthropods before restocking is considered.

v3-fos

2017-07-29T04:25:02.436Z

1973-04-15T00:00:00.000Z

9307710

s2

Cost-effectiveness and population structure in cattle breeding programmes. Cost-effectiveness requires that a breeding programme be both genetically and economically well-planned. Planning consists of deciding between alternative courses of action. These decisions arise at both the strategic and tactical level. The point is made that the tactical choices have been well served by science, but that the more important strategic ones have not been. A formal framework is described in which the sequence of decision-making required for an animal breeding programme is treated systematically. Current work in the genetic/economic planning of cattle breeding schemes is reviewed. The interaction of breeding programmes and population structure is dealt with in some detail. INTRODUCTION The contribution which the science of animal genetics makes to the practice of animal breeding is to provide a basis for rational decision-making. That is to say that from a reasonably well-established knowledge of how inheritance works (both in individuals and in populations), we can select between individuals, strains and mating systems and predict with reasonable accuracy the genetic consequences of our decisions. If the economic cost of the alternative courses of action can be measured, and if their results can also be expressed in monetary terms, it becomes possible to calculate the cost-effectiveness of a breeding programme. This is to state ( 1 ) Invited report presented in the Study Meeting of the European Association for Animal Production, joint Session of Commission on Cattle Production and Commissionon Animal Genetics, Verona, Italy october 6th, 1972 . in very broad and simple terms, what can be an extremely complex situation. The following factors may all interact to produce this complexity : -multiplicity of breeding goals, -presence of both additive and non-additive genetic variation, -variation in production systems, -structure of the animal population concerned, -structure of the human organization concerned. However, the basis of any breeding programme remains the physical fact of Mendelian inheritance, and we can perhaps order this commplexity best by letting the biology lead the way. THE GENERAL STRATEGY O>! LIVESTOCK IMPROVEMENT There is a natural sequence of decisions which must be made in the logical development of any livestock improvement programme. I have attempted to describe them schematically in figure i. In the first place, we need an agreed choice of breeding goals. The practice has too frequently been to aim for an ill-defined mixture of aesthetic and economic merit. In fact it is probably fair to say that most of the dialogue between breeders in the traditional sense and scientists involved in breeding has never progressed beyond this initial question. Given that agreed objectives are possible, the next problem is that of measuring them. This poses no difficulty for some traits like backfat thickness or litter size, but can be a real obstacle for others, such as conformation, meat quality or disease resistance. The obstacle may not be solely the technical difficulty of measurement, but, as in the case of carcass composition in cattle, the cost. Associated with this complex of defining and measuring the selection goals is the question of placing economic values on them. With agreed and measurable goals it is reasonable to ask next wheter genetic improvement is possible. We look first for additive genetic variation so that we can estimate the probable response to selection. More scientist man years have been devoted to heritability estimation than to any other aspect of animal genetics. This was necessary in the same sense as mapmaking is necessary. For the future, periodic revision of the genetic parameters studied should be an almost automatic part of any well-designed breeding scheme. If the goals are sufficiently heritable, selection will be effect. If heritability is low, selection will not be effective. However, in this situation, evolution has frequently provided enough non-additive variation to make the exploitation of heterosis worthwhile. With complex goals, it may even be necessary to pursue both selection and heterosis responses. In general, the traits of major economic importance in cattle are sufficiently heritable to call for the selection alternatives. This is fortunate, because selection responses are cumulative, in contrast to those from heterosis, and in addition do not incur the extra costs which are sometimes required to support a population structure designed for crossing. If the genetic architecture of the population indicates that neither additive nor heterotic responses can be expected, then either a change of breeding goals or a change of population is needed before genetic improvement is possible. If usable heterosis is present, then one is faced with the choice of crossing system and its components to maximize the heterotic effect. This involves the testing of strains (perhaps natural strains, such as breeds of cattle, or artificial lines as are used in poultry), and of mating systems. This could conceivably be the end point of a breeding programme, with perhaps nothing further to do but continue strain testing and replacement where indicated. However, the more usual situation is that heterotic effects are important only for part of breeding goal, and that the exploitation of heterosis for some traits is accompanied by continued selection within strains for other traits. The construction of a selection programme to exploit additive variation is now supported by a great deal of theoretical and experimental work. The first question that arises within the selection scheme is whether it contains inherent antagonisms. If not, that is if there are no serious negative genetic correlations between the elements of the selection goal, then it merely remains to optimize the selection scheme. In the strictly technical sense of maximizing the total economic value of genetic gain using given information this is achieved by using a selection index. However, when the cost of the information used in the index is taken into account, a new dimension is added. Most of the work on cost effectiveness in cattle breeding seems to concern a particular case of this problemreconciling costs and selection gains for dairy bulls used in AI. If there are unfavourable genetic correlations, two courses of action may be possible. The first is to proceed with index selection. A case in point is the problem of improving milk yield and composition. Here one is forced to select in the face of difficult correlations, because the yield of a cow cannot be separated from the composition of her milk. The alternative course of action is to make two groups of the components of the total genotype and to pursue each group separately by selection in males or in females, in other words, to begin separate sire and dam line development. This idea has come to be fairly well accepted in pig breeding where it is now common to pursue heterotic effects in a hybrid female line, while improving the mainly additive performance and carcass traits by selection in a male line. It offers considerable possibilities in cattle populations also, and I would to like develop this idea in a later section. If the choice is for separate sire and dam lines, then the operational problems are first to choose and evaluate the potential lines, and secondly to optimize selection within lines. This outline presents in broad terms the full spectrum of possibilities that our current knowledge of inheritance offers to livestock improvement. One can distinguish the strategic decisions that it containschoice of breeding goal, choice of selection or crossing systems, choice of breeds, strains, or indeed populations. At a lower level are the tactical decisions concerned mainly with the optimization of operations within a given breeding structure. I believe that in cattle breeding, as in many other areas of human activity, we have tended to concentrate effort on the tactical choices, to the neglect of the strategic ones. I would like first to review some work on cost effectiveness in the only area of cattle breeding where it has been studied to any extent -AI bull selection. I also wish to discuss the way in which the breeding programme interacts with the population structure and to indicate what appear to me to be some strategic options that have not been greatly appreciated. COST EFFECTIVENESS IN AI BREEDING SCHEMES Genetic optimization for dairy traits The optimization of decision-making in dairy-cattle breeding, using Mendelian inheritance as a basis, can be said to have begun with the simple indexes described by LusH (zg 45 ). The spread of artificial insemination in the 1940 's introduced a new phase of interest. The wide-spread use of a bull in AI made it both possible and imperative to estimate his genetic merit with increased accuracy. This immediately concentrated attention on the efficient use of progeny testing. Early work was concerned mainly with balancing the accuracy and intensity of progeny test selection of bulls so as to maximize the rate of genetic gain for milk yield (RoB!xTSOrr, 1954 ). With a limited pool of recorded cows in the population progeny group sizes, and therefore progeny test accuracy, can be increased only by reducing the number of bulls tested. The main factors affecting this balance are the heritability, number of progeny records available (N), number in each progeny test (n), number of bulls tested (N/n), number required (S) , and proportion selected (!cS/N). R OBERTSON ( 1957 ) simplified the relationships between these factors by defining a testing ratio K = N/S. He was then able to describe algebraically population structures that maximized genetic gain largely as functions of SKJ!RVO!,D ( 19 64 b) developed these ideas further, and mapped the approaches to maximum gain for varying selection intensities, population sizes, her it abil iti es and inbreeding effect. Ros!R'rsow ( 1954 ), S KJERvo LD (i 9 6 3 ), and S K jE RVOLD and LANGHO!,z (1964 a) looked at the way in which selected bulls affect the population via their sons and daughters, and at the relative use of young and proven bulls. Their general conclusions were : 1 0 The main agency for population change is selection intensity among young bulls. To keep this high, a large proportion of services should be carried out by young bulls. 20 Very high selection intensity should be used for sires of bulls. 3 o Approches to maximum rates of genetic gain are fairly gradual, so that considerable variation in breeding structure is possible, while still maintaining a high proportion of maximum gain. These studies provided an adequate basis for rational decisions on the physical structure of AI breeding operations. Since they were published, two developments have reinforced their general conclusions. The first is that as selection schemes became more effective, particularly in the choice of sires and dams of bulls, the genetic merit of young bulls began to be as good as that of the previous generation of proven bulls, so that the emphasis on use of young bulls carries even greater weight. The second is the development of semen freezing and dilution techniques which have greatly extended the potential use of individual bulls, thereby making possible high selection intensity without a corresponding increase in the scale of the operation as a whole. Genetic and economic optimization for dairy traits The first systematic extension fo these ideas to include financial considerations was by PouTous and V I SSAC ( 19 62). They set out to find the breeding structure that would maximize the total return on expenditure. They succeeded in writing a formula ( 25 ) for the cost-effectiveness of the programme as a whole. This formula contains sixteen parameters describing the physical, economic and genetic structure of the population. They chose to concentrate on progeny group size and level of bull selection, and found that the values of these variables which maximized cost-effect iveness depended in turn on the costs per progeny recorded and per bull maintained. In general, their results suggested milder intensities of bull selection than the previous studies. Their paper introduced several new elements into this area of work: consideration of the multi-generation effects of an insemination, of population structure and of interest rates and discounting procedures to allow for the spread in time of costs and returns. Varr V!,!Cx ( 19 6 4 ) constructed an algorithm which traced the cumulative gain in genetic merit in young and selected bulls from the start of a dairy progeny testing and selection programme. From this, it was possible to calculate the total gain from testing for any particular population. C UNNINGHAM and CLEAVES ( 19 65) used this method to calculate the annual genetic gains over 20 years for a range of programmes in which number of insemination per bull, proven versus young bull usage, level of progeny recording and bull selection intensity were varied. CurrrrtrrGHAM (1966! added costs and calculated returns, demonstrating that the same programme is seldom optimum in both genetic and economic terms. Indeed in several situations, genetically optimum schemes cost more than the predicted return. For many combinations of structure however, the return on expenditure exceeded 10 : i. B RAS -CAMP ( 1972 ) used discounting procedures to standardize the long-term effects of genetic improvement in milk yield through the four possible paths linking parents and offspring. His results showed that the path sire-son is less important from an economic than from a purely genetic point of view. Genetic and economic optimization for beef and dairy traits The extension of the basic model to include a measure of meat production in addition to milk immediately introduces several new dimensions to the problem of both genetic and economic optimization. S OLLFR et al. ( 19 66) adopted a selection index approach to the calculation of genetic gain in milk yeild and liveweight from various combinations of performance and progeny testing. To allow for the differential expression of bull merit for milk and beed traits, they introduced the idea of the composition of his average offspring, i. e. the proportion of his progeny in which the different products are obtained. They used quite detailed calculations to arrive at the net discounted value of gains in liveweight. They also studied variation in the relative economic values of milk and liveweight. Their general conclusions were that this ratio is relatively stable, and that under reasonably general conditions, the returns from dairy progeny testing are much greater than from beef testing. Beef performance testing is likely to be quite cost-effective, but beef progeny testing less so. L INDHE ( 19 68) studied the genetic and economic gains from beef performance testing followed by dairy progeny test selection of bulls. He introduced a new variable variable : the usage level for each bull selected, which becomes more a manageable factor with frozen semen. He looked first at the effect of five levels of prior selection on beef performance test on gains in milk. The effect was negligible, since the traits were assumed uncorrelated and the only interaction is in the need to use a larger number of bull dams to allow for culling fo their sons on performance test. He ordered the more than 10 ooo alternative programmes possible by limiting consideration to those with a marginal return on capital exceeding io p. 100 . Among these, five alternatives covering the range of 20 00 o to 40 ooo doses of semen per bull were studied in detail. The higher levels of bull usage were more cost effective, but not greatly so. Average returns on capital for the programme as a whole were about 130 p. 100 . Return on capital for the beef performance test alone was about 30 p. 100. ' H INKS ( 1970 a, b) looked at costs and returns in bull testing and selection, first for dairy traits alone, and then with a beef performance test included, with the requirements of the British Milk Marketing Board's AI service particularly in view. He concluded that on the dairy programme, greater use of selected bulls, through frozen semen, would give substantial benefits, and that the costs involved in a policy of slaughtering bulls after io-i 5 ooo semen doses had been collected were too high for it to be recommended. His results also suggested that moderate selection intensities (up to I in 7 ) were desirable. He found that performance, but not progeny testing for beef traits in dairy bulls could be justified in terms of return on capital. However, even performance testing gave a lower return than did marginal investment in the dairy progeny testing programme. It would require an increase in the relative values of meat and milk from 7 : 1 to 12 : 1 to justify the diversion of investment to performance testing in these circumstances. In a further paper (H INKS , 1971 ) he looked at the longer-term consequences of alternative dairy programmes, using as a measure of cost-effectiveness the discount rate at which a particular programme recovered its total costs a specified number of years the conclusion was that the main factors of importance were the bull selection intensity and the marginal profitability of milk production. HILL ( 1971 ) treated in detail the financial appraisal of alternative breeding programmes for meat production from a dairy population. In particular, he compared the returns from performance test selection in the dairy population and in a purebred beef population producing bulls for crossing on dairy cows. He gave particular attention to the use of discounted cash flow techniques and to the effect of varying discount rates. He concluded that investment in the production of beef breed bulls is better than investment in dairy bull performance testing, though the latter would also be profitable at interest rates up to 15 p. 100 . M C C LIN T O C K and C UNNIN G HAM ( 1972 ) pointed out that since the unit of use of a selected bull is an insemination, and since the costs of the breeding programme are usually carried on the insemination charge, both costs and returns should be calculated per insemination. They developed a « discounted gene flow » method which adjusts the genetic effects of an insemination for the number of descendants it involves, their relationship to the animal being evaluated, and the time interval separating the insemination from its economic consequences. Using this method, they were able to define a milk plus beef breeding objective which maximizes the economic value of the genetic merit conferred by an insemination. Using an index selection of bulls for this aggregate genotype, they demonstrated that for a wide range of conditions the most cost-effective element in an integrated breeding scheme is the dairy progeny test, with beef performance testing on a lower level and beef progeny testing even less valuable. ' The discounted gene flow method depends on the probability that an insemination will result in a dairy cow. This probability in turn depends on several parameters of population structure : reproductive rate, cow turnover rate and rate of crossing with beef breed bulls. CurrrnNGrrnM and M C C LIN TOC K ( 1972 ) pursued the effects of variation in these parameters, since as they vary, so does the selection objective for which dairy or dual-purpose bulls should be chosen, and with it the whole breeding strategy that should be followed. It emerged that the rate of beef crossing in particular had a large effect. As beef crossing increases, the balance in the breeding objective moves towards milk, and so the relative cost-effectiveness of dairy and beef testing and selection also moves in favour of milk. With high beef crossing, the probability that a dairy insemination will lead to a dairy cow is greatly increased. As a result, the total genetic and economic consequences of the insemination are increased. This means that for a given expenditure on testing and selection the return can be increased by promoting beef crossing. They concluded therefore that the cost-effectiveness of dairy breeding programmes is greatly improved by the parallel use of beef breed bulls. This conclusion is dealt with again in the next section. Genetic and economic optimization for beef traits Few studies have been done on the cost-effectiveness of schemes for beef alone. M OCQUO T and F OULL E Y ( 1973 ) compared selection on performance test, on progeny test, and on a two-stage use of both types of test for a beef breed used as a sire line for suckled veal production. They discounted costs and returns and made allowance for the reduction of variation due to prior selection in the two-stage case. They considered discount rates, duration of pay-off period, usage of selected bulls and variation in genetic parameters. They concluded that two-stage selection was best. Other studies on economic optimization for beef cattle include that of L EIGH et al. (ig!2) who used linear programming methods to compare crossbreeding strategies. Their criterion was overall financial benefit of each programme, and this was influence by breed combination, size of cow, level of reproduction performance and farm size. The british M. L. C. SCIENTIFIC STUDY GROUP REPORT ( 197 1) includes cost-effectiveness considerations of beef breed substitution, selection for beef in dairy cattle and selection in beef breeds. POPULATION STRUCTURE AND GENETIC EFFICIENCY Basic structures Consider first the simplest of structures in which we have a single population with a common breeding goal. Such might be the Dutch Friesian, New Zealand Jersey, American Hereford or French Charotais populations. The goal may be beef, dairy or dual purpose. In each case the population structure is such that both the bulls and cows used for breeding are produced within the population. Because of the large number of new females needed each year to maintien population, size, these replacement heifers will be bred throughout the population. In a population of traditional structure, the males used for breeding are produced by a relatively small subsection of the population. These bull-breeding herds usually, through not always, consist of pedigree cattle. In some countries this traditional structure is given a legal basis, with laws prohibiting the use of any but pedigree bulls. This type of structure undoubtelly was useful a century ago when the problem of improvement was seen as one of extending the use of improved strains through a largely nondescript unimproved population. It may even have merit to-day in some populations, since the elite nucleus of herds may be the only ones in which recording is carried on, and therefore the only ones in which objective selection can be practised. However, in a modern population this kind of structure does impose a restraint on the rate of genetic improvement, since it automatically excludes from the breeding programme males from most of the population. Since outstanding females can make a substantial mark on the population only through widely used sons, it also eliminates many of them. This kind of structure was practically universal in European cattle populations until recent years. The spread of AI has levelled out genetic differences between herds. and in several countries the bulls used for breeding are now produced throughout the population. This may require that formal breed boundaries be disregarded, as in the Norwegian dairy population (SxJ!RVOr,D, 1972 ) or in a French beef population (RONDEAU, zg 7 2). A modern breeding structure of this kind does not support an elite of breeders, and so in general needs to be pioneered and promoted by an AI organization. Its most necessary feature is a means of identifying potential bull mothers in the population at large. Probably the most extensive such enterprise is the American cow indexing system (D ICKINSON C t !/., 1971 ), through the methods are quite well developed elsewhere (g. e. BuxrtsiD!, 1970 ). Considering the substantial gains possible from evevery simple bull dam selection (H!ND!xsorr, zg6!), it seems that all populations served by AI will eventually tend to this type of structure. Beef crossing in dairy populations Whether the basic structure of a dairy cattle population is traditional or modern, it may be altered in another direction by the use of a certain amount of beef bulls, Beef crossing superimposed on such populations is illustrated in figure 2 . The beef bulls have no permanent effect on the population since all their progeny are slaughtered. The extent of beef crossing possible is determined mainly by the rate of cow turnover : the more lactations per cow, the fewer replacements needed annually and the more beef crossing can be practised. The limits to beef crossing, assuming that 35 animals of breeding age are produced per ioD cows per year, are : In fact beef crossing is practised to any considerable extent only in Britain and Ireland, where the percentages of dairy cows mated to beef bulls on 1970 were 3 8 p. 100 and 54 p. 100 , and in France where on 19 65 1 6 p. 100 of Friesian, 21 p. 100 of Normandy and 2 6 p. 100 of M ontbéliarde cows were lated to beef bulls. The advantages of a certain amount of beef crossing are as follows : . It allows for specialized selection for beef traits in the beef line and dairy traits in the female line, thus permitting faster genetic improvement in each set of traits. o It increases the possibility that a dairy insemination will result ultimately in a milking cow, and thus increases the net return to the farmer from that insemina-tion. This effect also increases (up to double) the return to the industry as a whole for money spent on dairy testing and selection. Since fewer dairy bulls are required, it allows the scale of th 2 dairy testing to be reduced, while maintaining a given level of quality in the bulls selected. It forces the farmer to exercise more selection in the dams of this replacement heifers than might otherwise be the case. Each time he breeds a cow, he must decide if she is good enough for a dairy insemination. It provides a supply of beef X dairy heifers which can be used as replacement cow for beef herds. o If dairy heifers are mated to bulls of a specially selected beef breed calving problems can be minimized, and dairy inseminations can be concentrated on those cows which have completed at least one lactation. It allows the exploitation of whatever direct heterosis there is for beef traits. For growth alone, this can amount to 8. 5 p. 100 between beef breeds (GREGORY, 1970 ) and at least 6 p. 100 in beef X dairy crosses (M. I,. C., 1971 ). There are some disadvantages to beef crossing : * It reduces the amount of culling that the farmer can practise among both incoming heifers and cows already in the herd. However, S y RSZ nD ( 1972 ) has shown that while the maximum gain in current herd mean production from cow culling is 7 p. 100 , the genetic effect on the herd is negligible. Few farmers can practise theoretically maximum rates of culling, and the selective mating of good cows to dairy bulls will tend to offset the effect of reduced culling levels. It appears therefore that the total adverse effect of beef crossing through reduced opportunities for cow culling will be small. A contrary view is taken by V AN V!,!cK (r 97 a). . If beef crossing near the limit is practised some herds may not have enough heifers for replacement in some years. This problem will be most acute in small herds, where variation in the sex ratio will be greatest. In these circumstances, a reduced level of beef crossing may be called for (L INDSTR 6 M , r 97 o). The alternative is to develop a method for the exchange of heifer calves between farms. The balance of advantage and disadvantage therefore seems to favour maximizing beef crossing. For a dairy population not already practising beef crossing, such a change in strategy will have widespread effects on the breeding programme. In the first place, some effort must be devoted to the testing and improvement of potential beef lines or breeds. Secondly, the balance of dairy and beef traits which are expressed I through each insemination by a dairy bull becomes such that bull testing and selection programme should overwhelmingly emphasize dairy traits. This means that large investments, and intensive culling, for beef traits in dairy bulls are not worthwhile. The rising demand for the more extreme dairy types of HoLstean-F y iesian in some European countries whose populations have in the past been firmly dualpurpose, indicates that milk producers are already seeking more specialized cattle in any case. Cyossbreeding structures for single-goal populations The advantages of systematic breed crossing in populations of specialized beef cattle have been very adequately demonstrated. Literature summaries by M A soN ( 19 66) and C UNDIFFE ( 1970 ) and the report of a recent large trial by GREGORY (i 97 o) all indicate that the cumulative advantages of crossbreeding can amount to 20 -25 p. 100 in terms of weight of calf weaned per cow per year. Despite this, little systematic stable breef crossing is done in the major beef producing countries. No doubt are there physical deterrents to crossbreeding under ranching conditions. However, it seems likely that the tend will be to greater use of crossbreeding in these countrie (C AR T WRI G H T, ig 7 o). In European countries, beef production from pure beef breeds is found on a substantial scale only in France. While crossing among the French beef breeds is little practised, the large experiment at Bourges (L EF È VR E and D ARD E, rg 7 i) may lead to developments in this direction. The advantages of crossbreeding among dairy or dual purpose breeds is less well documental. However, the review by P EARSON and M C D OWELL ( 19 68) of seven large experiments indicated heterosis for survival rates, growth rates and milk production. Again, despite apparently worthwhile advantages, little systematic breed crossing is to be found in practice. Such crossing as does take place is largely in the form of cc croisement d'absorption » in which males of one breed are used continuously on another breed so that a breed change takes place. Once the change is complete, the structure reverts to one of those described in figure 2 . Since most European cattle populations are dairy or dual purp3se, we can discuss possible crossbreeding structures for this kind of situation, through much of what follows could also relate to a specialized beef or indeed any single-goal population. The main questions which arise are : -Whether a crossbreeding structure is preferable to a simple within-population selection structure. -What kind of crossbreding structure is best. On the first question, generalization is difficult because of the scarcity and variability of experimental results to-date, and because of the complexity of any measure of dairy merit. However, the review of P EARSON and McDANiEi.1. ( 19 68) indicated heterotic gains averaging over 5 p. 100 for milk yield. McDowE!.!, and McDANiEL (1969) showed a crossbred advantage for total economic dairy merit, and Ho2rr (1971) found crossbred cows more productive than purebreds in a dual-purpose situation. These results must be qualified by the statement that in general the crosses involving Hotstein-Friesian do not outproduce the pure Holstein-Friesian. However, the evidence does suggest that the cumulative heterotic advantages of crossbreeding are worth pursuing. Assuming that this is the case, what structure best serves this end? HILL (1971) has thoroughly discussed the theoretical aspects of alternative crossbreeding structures. In cattle populations, certain practical factors limit the range of possibilities. To be useful, the structure must, i. Be stable, i. e. maintain a constant genetic composition in the population. 2 . Allow female replacements to be generated throughout the population. -3. Effectively exploit heterosis. 4 . Not interfere with selection for additive traits. Of the alternatives listed by HILL ( 1971 ), only rotational crossbreeding fulfills these requirements. The options can be further narrowed to a comparison of two-and three-breed rotational crossing, since anything more complex is likely to be impractical in cattle. Five of the experiments reviewed by PE ARSON and M C D OWELL included 3 -way crosses. In two of them the 3 -way crosses showed more, and in the other three, less, heterosis than the 2 -way crosses for milk production. So in general one might suppose equal heterosis effect. This, of course, refers only to the first generation. After about 3 generations of either twoor three-breed rotational crossing, the genetic composition of the population stabilizes with 86 p. 100 of maximum heterozygosity in the case of the 3 -breed, and 66 p. 100 of maximum heterozigosity for the 2 -breed structure. The question therefore is whether these two structure are likely to differ in heterotic effect. We have no experimental evidence on this in cattle. However, S KA xMArr'S ( 19 6 5 ) experiments with pigs show that backcrosses (with only 5 0 p. 100 of maximum heterozygosity) showed as much heterotic effect as the initial crosses. It is reasonable to theorize that the effects of heterosis lay not be a linear function of the degree of the heterozygosity and that the increase from 66 p. 100 to 86 p. 100 of maximum heterozygosity is unlikely to be worth pursing in cattle. Two-breed crossing has some practical advantages over the use of 3 breeds. It is simpler to operate. Each herd contains just two types of cow : those to be mated to sires of line A and those to be mated to line B. Paralled selection in two lines is likely to be more affective than in three. The cost to the population of maintaining two sire lines is less than for three. In general, therefore, it appears that the choice of a crossbreeding structure for a dairy or dual-purpose cattle population should involve just two breeds. This kind of structure is illustrated in figure 3 . The mating plan can be called reciprocal back crossing (RBC). The main cow population consists entirely of crossbred cows. After the first few generations, every cow has a genetic constitution which is either AAB or ABB, i. e. on average two-thirds of her loci contain genes from both A and B lines, while the remaining third contain genes of either the A or the B line. The mating rule is simple: if a cow's sire was from line A, she is mated to a bull of line B, and vice versa. A simple ear-notching convention could make this foolproof in practice. The approximate conditions which are necessary for such a crossbreeding structure to be worthwhile can be summarized follows : The first condition says that the sire lines must be of roughly equal merit. If they are not, producers will continue crossing to the better line, as usually happens at present where one of the lines is Holstein or Friesian. In dairy cattle, therefore, a prerequisite for this kind of structure is the development of a strain genetically distinct form, but of equal additive merit to the Friesian. At least one attempt is currently being made !TI;V!RSON, 1970 ) to produce such a line based on the red dairy breeds of Europe. The second condition says that two-thirds of maximum heterozygosity should give approximately the same heterotic effect as full heterozygosity. The third and fourth conditions say that the 2/3 crossbreeds should not be inferior to the purebreds for additive traits, and that the must be some heterosis worth pursuing in the first place. In our present cattle populations, these seem reasonable, if not yet fully supported by experimental evidence. Such a structure is operable only in a population served by AI. In one sense, it reverts to the traditional structure, since bull production is now confined to two small purebred sub-populations. However, if their absolute size is reasonable (say 10 ooo cows each) then bull dam selection efficiency might be kept at much the same level as in a population of modern structure. Sire selection would, of course, be based on progeny testing carried out as before in the main population. One of the side advantages of such a system is that inbreeding in the main population need no longer be of any concern. However, it is concentrated in the sire lines, so that, depending on their size, some degree of planed mating to minimize its effects might be necessary. There is a further, if somewhat hypothetical advantage to such a structure. Bulls in lines A and B would be selected largely on additive merit based on their progeny test on crossbred daughters. If non-additive effects contributed to their progeny test, then the bulls selected in line A would be chosen in part for their genetic dissimilarity from line B. Thus the long-term effects of paralled selection in the two lines would be for genetic divergence, and hence the enhancement of heterosis between them. Beef crossing can of course be superimposed on a two-breed crossbreding dairy population. This structure is also shown in figure 3 . The use of a beef line here could be even more advantageous than in a conventional dairy population, since it would reduce the number of bulls to be supplied by lines A and B and thus reduce the minimum size that these lines must achieve to keep inbreeding under control. Crossbreeding structures for two-goal populations In Ireland, 2 8 p. 100 of the cow population consists of beef cows. In Scotland the figure is 57 p. 100 , in England 19 p. 100 , and in France 21 p. IOO . In other European countries, numbers of specialized beef cows are small, but growing. The two populationsof beef and dairy cowsmay occupy geographically distinct areas, as in France, or they may also be intermingled, as is common in Britain and Ireland. In either case, their physical proximity to one another is close enough for an exchange of stock. This contrasts with the situation in most of the other major beef producing countries, whose dairy and beef populations may be very far removed form each other. Whatever the structure in the dairy cow population, a certain amount of crossing with beef on dairy cows offers the opportunity for a very efficient link between the breeding structures of the two populations. All the evidence (e. g. C UNDI FF E , 19 6 0 ) suggests that beef cows should be crossbreds. British figures (M. I,. C., 1971 ) show that the growth of beef calves out of beef X dairy cows exceeds that of progeny of traditional beef type cows, with the advantage being greatest in good farming conditions. This suggests that the cows in the beef population could be bred in part 2 of the dairy population. This, in fact, in dairly commonly the practice in the British Isles. Using this source of replacement females for the best population has the following advantages : -They should be available at a lower cost than females from within the beef population. -If the beef crossing line used on the dairy herd is of an early maturing type (Hereford or Ansug), the mature bodyweight of cows in the beef herd can be kept down. This can be quite important, since, for example, it has been calculated that in an Angus cow herd producing calves for sale at 6 months, 8 7 p. 100 of the total feed imput to the herd is required for the maintenance and growth of the breeding animals (LONG and F I T ZHU G H , i 9 6 9 ). -It permits the exploitation of the full effects of crossbreeding in the female : complementarity of beef and dairy characteristics, together with the full effects of heterosis for maternal traits. -The choice of terminal size line can be made to give further complementarity (large size in the male, moderate in the female) together with whatever further heterosis is achieved by the use of a third breed. In figure q I have illustrated this structure linking a dairy and a beef population. The dairy population is shown as having basically a two-line reciprocal back crossing structure, though it could equally have any of the other structures already described. One consequence of this general kind of population structure is that it puts great importance on the choice of beef sire lines. In populations served by AI, and this is likely to be feasible in beef populations before long, the purebred nuclei from which these lines are drawn become extremely important sources of genetic improvement. A great deal of investment will therefore be justified in the formation and testing oi specialized beef sire lines. Un schema precis avec la succession des decisions a prendre pour un programme d'amélioration génétique est trait6 systématiquement. On examine aussi les travaux en cours concernant la planification genetique et économique de la selection bovine. On parle également. de façon assez détaillée, de 1'interaction entre de tels programmes d'amélioration génétique et la structure de la population.

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2018-04-03T02:21:03.430Z

1973-04-01T00:00:00.000Z

33046529

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Factors influencing detection of salmonellae in rendered animal by-products. Detection of salmonellae in animal by-products is influenced by the enrichment and plating media and by quantity of product tested, and is related to the total plate count. A linear relationship exists between detection of salmonellae and total plate counts from 10(4) through 10(7) per gram. Detection of salmonellae in contaminated food and feed ingredients is dependent upon many factors. No single method has been developed that satisfactorily recovers all Salmonella serotypes from all types of foods (1). The purpose of this study was to develop a better understanding of the factors influencing the detection of salmonellae in naturally contaminated animal by-products. The influence of media and the relationship between total counts and isolation of salmonellae were examined. MATERIALS AND METHODS During 1967 and 1968 samples were collected from plants throughout the industry for analysis in our laboratory. Total plate counts were determined by using plate count agar (Difco) at 37 C for 48 h. Two 20-g samples were analyzed for Salmonella. One 20-g sample was added to 80 ml of tetrathionate-Brilliant Green-iodine (TB) broth and the other 20 g was added to 80 ml of selenite-cystine broth (SCB). Both broths contained 0.6% Tergitol no. 7 (Union Carbide Chemical Co.) and were incubated 24 h at 37 C. The enrichment broths were streaked onto plates of Brilliant Green-sulfa agar (BGS; Difco) and Salmonella-Shigella agar (SS; Difco) plates and incubated 24 h at 37 C. Plates showing less than three suspect colonies were incubated an additional 24 h at room temperature and reexamined. Three Salmonella-like colonies were picked from each plating medium to triple sugar iron (TSI) agar slants. Cultures with Salmonella-like reactions on TSI after 18 to 24 h were tested serologically by using polyvalent 0 and H antisera. Cultures reacting postively in either or both serological tests were confirmed biochemically. RESULTS Of 183 Salmonella-positive samples, 139 and 148 samples were found positive in TB and SCB, respectively ( Table 1). The combination of SCB and SS agar plates gave the highest number of positive samples (127). The combination of SCB with BGS agar plates gave the lowest number of positive samples (92). From 3 to 9% of the samples were positive in only one of the four possible media combinations. Table 2 shows a grouping of 405 samples according to total plate count results. The rate of salmonellae isolations increased from 12.7 to 70.6% as the total plate counts increased from less than 10'/g to 107/g. Samples with total plate counts in excess of 107/g were associated with a decreasing rate of salmonellae isolations. Linear regression analysis confirmed a positive relationship between the total plate count and detection of salmonellae (P = 0.05). However, examination of the regression line by chi-square analysis revealed a lack of fit, for samples having total plate counts below 104 and at 108/g and above. This supports the conclusion that the relationship is linear only for samples having total plate counts from 104 through 107/g. The probability of detecting salmonellae in animal by-products was developed from further statistical analysis of the data. For example, if the product has a total plate count in the range of 104 to 105/g, then the probability of detecting salmonellae in a single sample would be 0.40 (Table 3) by using the methods described. Comparing the data for samples positive from only one enrichment (20 g) versus both enrichments (40 g), we found that 40 g significantly (P = 0.05) increased the detection of salmonellae. This conclusion was dependent upon the level of contamination and the assumption that the concentration of salmonellae would be higher in samples having higher total plate counts. The data in Table 2 assumption. For example, a higher percentage (64%) of the positive samples was found positive in only one enrichment (20 g of product) when the total counts were lower (104/g). At 108 total counts per g, 25% of the positive samples were positive in one enrichment. DISCUSSION No practical difference was found between TB and SCB in terms of the number of samples found positive. Both TB and SCB failed to detect salmonellae in approximately one out of five of the total 183 positive samples ( Table 1). The best combination of media (SCB-SS) found only 69% of the 183 to be positive. This failure to detect salmonellae could be due to inhibition of certain serotypes by the enrichment media, a low level of contamination, or uneven distribution of salmonellae through the product, or all three (5,9). Using both TB and SCB results in a higher rate of salmonellae isolations from animal feed ingredients (2,5,9). However, using both enrichments also doubles the quantity of product analyzed. The degree to which sample size influences the isolation rate of salmonellae in animal by-products deserves more consideration. Huhtanen et al. (3) analyzed 16 samples of meat and bone meal by using ten 3-g samples and ten 30-g samples for each. They found only 38 individual 3-g samples positive as compared to 86 to 89 individual 10-g samples positive. Laramore and Moritz (4) analyzed 73 samples of meat meal by subdividing each into two 30-g samples. They found results from the samples to agree only 86.2% of the time. Adding 20 g of meat and bone meal to both tetrathionate and selenite broths, Leistner et al. (5) found 12 samples positive. By using 10 g from the same samples, they found only seven samples positive. They then reported 37 samples positive from tetrathionate or selenite broths, or both. However, 13 (35%) of the 37 samples were positive in only one or the other enrichment broths. They state that this could be due to a low or nonuniform level of contamination (or both) as well as inhibition of certain serotypes. We did not conduct quantitative determinations to learn the concentration of salmonellae. However, assuming the concentration of salmonellae to increase relative to the total plate counts, it was possible to statistically determine that quantity of product (20 versus 40 g) is a significant factor. We conclude that the difference we observed between enrichment media for detecting sal-SALMONELLAE IN ANIMAL BY-PRODUCTS monellae is more apparent than real. Others have clearly established that an inhibitory effect exists due to the failure of certain serotypes to grow in the enrichment media. The data which is becoming available for naturally contaminated animal by-products suggests that the quantity of product being tested, at least in the range of 3 to 40 g, may be the more important factor. Our data confirms earlier reports (6, 7) that a relationship exists between total plate counts and the detection of salmonellae in animal by-products. However, it was learned that the linearity of the relationship exists only in the total count range of 104 through 107/g. There is a decrease in the percentage of Salmonellapositive product having total plate counts greater than 107/g. This is probably due to overgrowth of salmonellae by other bacterial species during enrichment or plating, or both. It is less likely that the product is actually less contaminated with salmonellae. The use of total counts as a measure of the microbiological safety of foods must be assessed in terms of the particular situation presented (8). Animal by-products appear to be one of the few materials where a linear relationship exists between total counts and the incidence of salmonellae. This is of practical value for in-plant control purposes and for evaluating improvements in manufacturing and sanitation. It is important that the limitations of the total plate count be recognized and it is sug-gested that total counts be used to supplement salmonellae testing programs.

v3-fos

2014-10-01T00:00:00.000Z

1973-10-15T00:00:00.000Z

2258700

s2

On methods of estimation of maternal heterosis and recombination effects from a specific three breeds crossing system SUMMARY The present note is an extension of D ICKERSON ' S (A. B. A., 19 6 9 , 191-202) paper. Three different methods for the estimation of maternal heterosis from a specific 3-breed crossing system were evaluated for possible biases. Method II was found to provide most nearly an unbiased estimate of maternal heterosis (h M). For example, It was further suggested that the same data could be used to estimate epistatic recombi-nation effects. D ICKERSON (19 6 9) stated, « Near maximum performance is expected in the best 3-breed cross of superior sire breed with crossbred females of 2 other breeds having the best economic combinations of F, maternal and transmitted performance characteristics n. This will be generally true for those offspring characteristics that are influenced by indirect maternal effects such as, growth. Thus, to maximize and to accurately predict immediate gains from a specific 3-breed crossing system a knowledge of the magnitude of maternal heterosis and epistatic recombination effects is essential. The present note is an extension of D ICKERSON ' S paper and here we briefly review different methods used to estimate maternal heterosis for their merits and limitations. It is further suggested that the same data can also be used to arrive at estimates of epistatic recombination effects. D ICKERSON ( 19 6 9 ) stated, « Near maximum performance is expected in the best 3 -breed cross of superior sire breed with crossbred females of 2 other breeds having the best economic combinations of F, maternal and transmitted performance characteristics n. This will be generally true for those offspring characteristics that are influenced by indirect maternal effects such as, growth. Thus, to maximize and to accurately predict immediate gains from a specific 3 -breed crossing system a knowledge of the magnitude of maternal heterosis and epistatic recombination effects is essential. The present note is an extension of D ICKERSON ' S paper and here we briefly review different methods used to estimate maternal heterosis for their merits and limitations. It is further suggested that the same data can also be used to arrive at estimates of epistatic recombination effects. DEFINITIONS Let mean performance of three breeds be represented by letters A, B and C, respectively. Further assume that all possible pure breeds, 2 -and 3 -breed crosses are produced contemporaneously. Then, A i = individual heterosis and is the mean deviation in the performance of 2 -breed crosses from the average performance of pure breeds due to increased average heterozygosity of F l s, plus any epistatic interaction between purebred parental gametes. h M = maternal heterosis and is the same as h I but for indirect maternal effects of F! crossbred dams. It is a measure of average dominance interaction deviations in maternal effect (m) of F, dams relative to that of purebred dams, for example h P = paternal hetevosis and is analogous to h M but for indirect paternal effects. r ! = epistatic recombination effects and is the deviation due to change in epistatic gene interaction effects in 3 -breed crosses, relative to those in 2 -breed crosses, from recombinations between gametes derived from parent breeds of F, crossbred sires or dams. The parameters-h M , h P and Y ' are specific effects of crossbred dam's or sire's genotype but are measured as environmental effects from analyses of offspring data. EVALUATION OP' VARIOUS METHODS Various comparisons used to estimate maternal heterosis and epistatic recombination effects have been summarized in the following table along with their genetic expectations. No attempt has been made to explicitely derive these expectations. However, it can be easily accomplished in closely following DicxExsorr (ig6g). To arrive at these expectations and to obtain unbiased estimates certain assumptions must be made ; the critical ones are : a) linkage equilibrium and randomness of mating. b) interaction effects involving three or more loci are negligible. c) additive (no interaction) combination of genetic and heterotic contribution of different breeds in various crosses, for example : that is, environmental effect is the same for all breed groups (purebred, 2 -and 3 -breed cross). d) no interaction between genetic contribution of the sire and the maternal ability of the dam. Such interaction might arise if a sire gave his offspring genetic growth potential that was incompatible with the milking ability of the dam. e) no confounding with differences in proportion of multiple births ; sex ; year, season and date of birth ; heterosis in male (h a) and female (h?) reproductive performance. f) r I = o (applicable only to method I). g) hP = o. In species like swine, sheep and rabbit, use of individual observations will lead to less precise estimates of h M due to confounding of differences in proportion of multiple births between crossbred and purebred dams. The estimates of h M and r I will be further less precise if h 3 and h! was of significance. In mathematical sense, these estimates will be unbiased but will not necessarily have minimum variance. If only three breeds were involved, method III could not provide specific estimates of h M for each kind of crossbred dam and method II should be the choice if unbiased estimates were desired. However, if four breeds were involved in the production of 3 -breed crosses it should be possible to obtain specific estimates of h M , for example. where D represents the fourth breed. For this comparison to be true one must further assume that there is no interaction between the genotype of the offspring (C(AB), D(AB)) and that of the crossbred dam (AB). This is an extension of method II utilizing information on both possible 3 -breed crosses.

v3-fos

2020-12-10T09:04:13.110Z

1973-11-01T00:00:00.000Z

237229951

s2

Influence of Milk Components on the Injury, Repair of Injury, and Death of Salmonella anatum Cells Subjected to Freezing and Thawing Fast freezing and slow thawing Salmonella anatum cells in various milk components inactivated from 20 to 98% of the cells and damaged 40 to 90% of the cells surviving the treatments. Injured cells failed to form colonies on a selective medium (xylose-lysine-peptone agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective plating medium (xylose-lysine-peptone agar). The major milk components—lactose, milk salts, casein, and whey proteins—influenced the extent of injury, repair of injury, and death. The percentages of cells injured and inactivated were decreased by the presence of any milk components except whey proteins. Also, repair of injury was promoted by the presence of each milk component except whey proteins, which in contrast inhibited repair. Phosphate was the most influential milk salts component that protected the cells and promoted repair of injury. These individual milk components may have decreased the extent of freezing-induced death and cellular damage by stabilizing the S. anatum cell envelope. Several environmental stresses are known to induce bacterial death and injury. Recent reports in the literature have indicated that exposure of bacteria to irradiation (8,9), sanitizing compounds (23,24), ethylenediaminetetraacetic acid (7), aerosolization (5), heating (15,16), freezing (4,10), and freeze-drying (20,21) resulted in sublethal injury. Two types of injury were observed: (i) injury as measured by increased nutritional requirements for recovery, and (ii) injury as measured by increased sensitivity to selective agent in the recovery medium. In both types of injury, the damage was found to be repairable under specific conditions. The extent of bacterial injury and death was influenced by the suspending medium used for freezing and freeze-drying. A number of compounds including sugar, milk proteins, peptone, gelatin, meat extract, glycerol, and phosphate protected bacteria from freezing or freeze-drying. These compounds apparently protected the cellular membranes from damage (4,10). The purpose of this investigation was to ' determine the influence of individual milk components and their interactions on the injury, repair of injury, and death of Salmonella anatum cells after freezing and thawing. A preliminary report of these findings has been made (D. W. Janssen and F. F. Busta, Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 18,1973). MATERIALS AND METHODS Freezing and thawing of cell suspensions. S. anatum MF3 cells were propagated and maintained in reconstituted nonfat dry milk (10% solids not fat) as described previously (20). A 1-ml portion of this culture was inoculated in 100 ml of tryptic soy broth containing 0.3% yeast extract (Difco) and grown for 16 to 20 h at 35 C. The cells were centrifuged, washed with sterile water, and suspended in the test solutions as described previously (19). Portions of 10 ml each were placed in test tubes (150 by 25 mm) and frozen rapidly in a dry ice-acetone bath for 10 min. The contents were thawed in a water bath at 4 C for about 45 min and tested for cell injury, death, and repair of injury. Determination of cell injury, death, and repair. The thawed test solutions were incubated in a water bath at 25 C. At indicated intervals, cells from the test solutions were counted on xylose-lysine-peptone agar (XLP) and XLP with 0.2% sodium deoxycholate 725 added. A 0.1-ml quantity was surface plated in triplicate on each medium. Death, injury, and repair were determined as described previously (20). Composition and preparation of the freezing and repair menstrua. To examine the effect of milk proteins in the suspending menstruum, 9-ml test solutions of several concentrations (wt/vol) of sodium caseinate (Fisher Scientific Co.), beta lactoglobulin (NBC Co.), alpha lactalbumin (NBC Co.), and bovine serum albumin (Sigma Chemical Co.) were prepared in sterile water. The pH was adjusted to 6.6 with sterile 0.1 N NaOH or HCl. Sodium caseinate was dissolved by a mild heat treatment and pH adjustment. Lactose (J. T. Baker Chemical Co.) was added in crystalline form to the test solutions to achieve the desired concentrations. A solution containing the salts present in milk was also prepared according to the following concentrations: KH2PO4, 0.158%; K2SO4, 0.018%; K2CO3, 0.03%; KCl, 0.10%; potassium citrate, 0.05%; ammonium citrate, 0.18%; magnesium citrate, 0.05%; CaCl2, 0.13%. The milk salts were prepared in sterile water by the suggested method of Jenness and Koops (6). Concentrations of test media approximated those found in fluid milk. Experimental design to determine the influence of milk components on injury, repair, and death. Full factorial designs, response surface designs, analysis of variance, and Yates analysis were used as described by Davies (3). All statistical computations were made utilizing programs provided by the Pillsbury Co., Minneapolis, Minn. RESULTS The influence of milk components on injury and death was examined. S. anatum cells were suspended and frozen in whole milk, skim milk, reconstituted nonfat dry milk (10% solids not fat), whey, sodium caseinate (2.5%), whey proteins (0.7%), beta lactoglobulin (0.4%), milk salts, and distilled water. The test solutions were thawed and incubated at 25 C for testing at 0, 1, and 2 h. The data are presented in Table 1. Whole milk, skim milk, and reconstituted nonfat dry milk (10% nonfat solids) afforded protection to the cells from injury and death and promoted repair. Whey or whey proteins also facilitated repair and protected the cells from freezing injury, but the number of damaged cells increased. The presence of casein (sodium caseinate) in the freezing and repair menstrua reduced the percentage of death to 26%, which was comparable to whole milk. Beta lactoglobulin in pure form was more protective than water alone, but did not promote repair to the extent that was observed with milk salts. To investigate the importance of whey proteins on injury, repair, and death, S. anatum cells were frozen and thawed in 0.07% alpha lactalbumin, 0.03% bovine serum albumin (BSA), or 0.4% beta lactoglobulin. The data are presented in Table 2. Individual whey proteins showed similar effects on repair and death. The percentage of injury did not decrease with incubation at 25 C. Freeze-injured cells thus apparently could not repair in solutions of alpha lactalbumin, BSA, or beta lactoglobulin. The individual whey proteins also did not protect the cells from death by freezing and thawing. However, differences existed in the extent to which whey proteins protected the S. anatum cells from initial injury. All the proteins were more protective than water alone, but BSA was less effective than beta lactoglobulin or alpha lactalbumin. The combination of the three whey proteins reduced the percentage of death. The data indicating the effects of whey proteins in combination with milk salts are pre- sented in Table 3. All the test solutions were protective and promoted repair in comparison to the water control. Phosphate alone, at the concentration found in milk salts, appeared to be the important ingredient in the milk salts that was required for protection and repair. However, less death occurred in milk salts than in phosphate. The combination of milk salts and whey protein decreased death and promoted the repair of injury. To determine the effect of milk components and their interaction on the injury, repair of injury, and death of S. anatum cells subjected to freezing and thawing, a two-level, full-factorial experimental design was used. The factors examined were milk salts, lactose, whey proteins, and casein. This design was used to determine the effects of these major components when they were either absent or present at normal milk concentrations (Table 4). A positive effect indicates greater death or injury when the factor was at the high level (i.e., present at the concentration levels in milk) in the test solution. The data indicated that the single factors of lactose and whey proteins significantly influenced the injury of S. anatum cells. The significant interactions were: AD (milk salts and casein), BC (lactose and whey proteins), ABC (milk salts, lactose, and whey proteins), and ACD (milk salts, whey proteins, and casein). The presence of whey proteins increased the percentage of injury, whereas lactose decreased the percentage of injury. However, there TABsi 3. Effect of whey proteins and milk salts on the injury, repair, and death of Salmonella anatum NF3 subjected to freezing and thawing was an interaction such that the effect of whey proteins was greater in the presence of lactose. Also, the effect of lactose was reduced in the presence of casem. The effects of casein, lactose, whey proteins, and milk salts on the injury of frozen and thawed S. anatum cells after incubation for 2 h at 25 C (i.e., a measure of repair) also are evident in Table 4. The data indicated that all single factors except whey proteins significantly influenced the repair of injury. The negative effects of milk salts, lactose, and casein indicated that these compounds reduced the percentage of injury or facilitated repair. The significant interactions were: AB (milk salts and lactose), AD (milk salts and casein), BD (lactose and casein), and ABCD (all milk components). An analysis of two-factor interactions indicated that casein greatly enhanced the repair process and milk salts also aided repair, but the effect was not as great when in combination with casein. The effects of the major milk components on the death of frozen and thawed S. anatum cells are also shown in Table 4. The data suggested that the single factors of milk salts, lactose, and casein influenced the percentage of death. These factors had negative effects which indicated that their presence in the freezing menstrua decreased death. The significant interactions were: AB (milk salts and lactose), AD (milk salts and casein), and CD (whey proteins and casein). An analysis of the two-factor interactions suggested that casein greatly reduced death and milk salts also reduced death; however, the effect was greater when casein was absent. Also, the presence of lactose in combination with casein resulted in less death. Figure 1 shows the results of a response surface experiment that demonstrated the influence of concentration of milk salts, lactose, whey proteins, casein and their interactions on the percent injury of S. anatum cells immediately after freezing and thawing. The experiments using five different concentrations of each variable were designed for the determination of quadratic equations which were then used to generate the response surfaces. Only sections of the response surfaces are shown. The axis for the dependent variable extends out from the page. Percentage of injury ranged from <40 to >65% under the test conditions. The differences in the surfaces indicated that high concentrations of lactose (8.54%) reduced the percentage of injury. Each section had curved response contours that suggested a casein-milk salts interaction. Lactose and whey protein concentrations influenced the percentage of injury. With high concentrations of lactose, injury was independent of casein and dependent on milk salts. High levels of whey proteins decreased the percentage of injury along with a high concentration of milk salts and a low concentration of casein. The different effects of increasing levels of lactose and whey proteins demonstrated a four-factor interaction among milk components. Figure 2 shows the results of a response surface experiment to demonstrate the influence of the concentration of the major milk components and their interactions on the percentage of injury after 2 h at 25 C (i.e., repair). Effect of the freezing and thawing menstruum on the injury of Salmonella anatum NF3 cells. Responses are expressed as percentage of injury of the survivors immediately after thawing. Injured cells were capable of forming colonies on a nonselective plating medium, xylose-lysine-peptone agar (XLP), but were unable to form colonies on a selective plating medium, XLP with 0.2% sodium deoxycholate. The three surfaces in each horizontal row correspond to a fixed percentage of whey proteins (e.g., the three surfaces in the top row were obtained with 0.44% whey proteins). The three surfaces in each vertical column correspond to a fixed percentage of lactose (e.g., the three surfaces in the left vertical column were obtained at 1.46% lactose). F test of significance for model, significant at P = 0.10. Responses indicating repair are expressed as percentage of injury of the survivors after 2 h at 25 C. Injury was defined as the inability to form colonies on a plating medium containing deoxycholate as the selective agent; therefore, less injury indicates more repair. As in Fig. 1, the three surfaces in each horizontal row correspond to a fixed percentage of whey proteins (e.g., the three surfaces in the top row were obtained at 0.44% whey proteins). The three surfaces in each vertical column correspond to a fixed percentage of lactose (e.g., the three surfaces in the left column were obtained at 1.46% lactose). F test of significance for model, significant at P = 0.025. The percentage of injury after 2 h at 25 C ranged from 0 to 35%. A low percentage of injury after the incubation period indicated substantial repair of injury. The differences in surfaces suggested that the repair process was greatly influenced by lactose and whey proteins. Also, the curved response contours in each section indicated a milk salts-casein interaction with the greatest repair at low levels of lactose and whey proteins. With a high level of casein, the repair process was less dependent on milk salts. The least repair occurred at low levels of casein (0.59 to 1.0%) and milk salts (0 to 20%), with whey proteins at a high concentration (2.56%) and lactose at lower levels (1.46 and 5.0%). Repair was inhibited at high levels of whey proteins as indicated by the steepness of the surfaces. Figure 3 shows the results of a similar response surface experiment that demonstrated the effects of concentrations of milk salts, lactose, whey proteins, and casein on the percentage of death of frozen and thawed S. anatum cells. The percentage of death was from 33 to >51% under the test conditions. The curved response contours in each section indicated a milk salts-casein interaction. In all cases, low levels of casein and milk salts resulted in greater death at the various concentrations of whey proteins and lactose. The greater steepness of the surfaces with increased levels of whey proteins suggested that these proteins increased death. Lactose appeared to decrease death especially at higher levels of whey proteins. DISCUSSION Several investigators have reported the influence of the suspending medium on the recovery of bacteria after freezing or freeze-drying. Also, a great deal of information exists on the ability of specific nutrients to promote the repair of cells sublethally injured by freezing, irradiation, freeze-drying, and heating. However, the literature is lacking on the influence of constitu- Responses are expressed as percentage of death after freezing and thawing. Death is measured by the reduction in numbers on xylose-lysine-peptone agar measured after freezing. As in Fig. 1 and 2, the three surfaces in each horizontal row correspond to a fixed percentage of whey proteins and the three surfaces in each vertical column correspond to a fixed percentage of lactose. F test of significance for model, significant at P = 0.10. ents of the suspending medium on injury, repair of injury, and death of bacteria. Therefore, this paper presents the effects of milk components and their interactions on injury, subsequent repair of injury, and death of S. anatum. Janssen and Busta (manuscript submitted for publication) previously showed that milk solids had a protective influence on S. anatum cells subjected to freezing and thawing. In this study, the protective influence of milk systems was verified. Whole milk, skim milk, and reconstituted nonfat dry milk (10% nonfat solids) protected cells from injury and death and promoted repair. The presence of 2.5% sodium caseinate in the freezing and repair menstrua gave results comparable to whole milk. Whey was shown to protect the cells from freezing injury, but the extent of death in whey was greater than in whole or skim milk. The major whey proteins (beta lactoglobulin, alpha lactalbumin, and BSA) were more protective than water alone, and BSA was less protective than beta lactoglobulin or alpha lactalbumin. Whey proteins did not promote repair. Milk salts were the important ingredient of whey for protection and repair. In milk salts, phosphate was the most important ingredient for protection and repair. However, a combination of milk salts and whey proteins decreased death and enhanced the repair process. The effects of milk salts, lactose, whey proteins, casein, and their interactions were evaluated by full-factorial and response surface designs. All of these factors affected the extent of S. anatum injury after freezing and thawing. The presence of lactose in the freezing menstrua decreased the percentage of injury, especially at a high level (8.54%). Several investigators have found that sugars were protective against immediate freezing injury (1,10,11,17,18). The mechanism of action remains obscure, but Mazur (10) suggested that sugars prevented injury to the cell membrane. The protective action of many compounds, including sugars, may be related to their ability to hydrogen bond (10). Mazur concluded that the ability of hydrogen bonding substances to protect cells from freezing injury must be related to their influence on the structure of water. Milk salts alone or in combination with other milk components influenced injury. Phosphate was found to be the major protective component of milk salts. Moss and Speck (14) showed that Escherichia coli cells frozen in phosphate buffer were more resistant to freezing damage than those frozen in distilled water. They reported that protective peptides leaked from the cells suspended in phosphate buffer. Davies (2) suggested that the phosphate ion was capable of preventing the conformational destabilization of macromolecules. The major milk protein, casein, also influenced injury of S. anatum cells. A significant casein-milk salts interaction was observed. Casein is a large protein with numerous exposed functional groups. The protective ability of this protein might be related to its association with phosphate, its hydrogen bonding properties, or its capacity to regulate cellular rehydration upon thawing. Several investigators (2,4,10,12,13) have reported the protective nature of milk proteins and other complex colloidal macromolecules, but the mechanism of protection remains obscure. Mazur (11) suggested that proteins might protect the cell from injury by interacting at specific membrane sites. The effects of milk salts, lactose, whey proteins, and casein on the repair of freezing and thawing-induced injury of S. anatum were evaluated. At normal milk concentrations, all single factors except whey proteins promoted the repair process. However, as concentrations were increased, lactose and whey proteins slowed or reduced repair. The repair process was dependent on high levels of casein or milk salts. The least repair was observed at low levels of casein, milk salts, and lactose, with a high level of whey proteins. The repair inhibition by whey proteins might be related to an ability to bind the essential components of milk salts such as Mg2+ and phosphate. It had been established previously that the repair of S. anatum or E. coli cells frozen and thawed in water required phosphate and MgSO4 (19,22). The influence of milk components on the death of frozen and thawed S. anatum cells was evaluated. The results suggested that milk salts, lactose, and casein protected S. anatum cells from death induced by freezing and thawing. These same factors also protected the cells from injury. The mechanism of protection afforded by these compounds is only speculative. As mentioned previously, these components might regulate rehydration after thawing, stabilize the cell envelope by hydrogen bonding, or alter cell permeability.

v3-fos

2018-04-03T03:56:19.795Z

1973-06-01T00:00:00.000Z

37080055

s2

Identification of the volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas fragi. Volatile compounds produced by Pseudomonas fragi strain 18 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. The fruity odor that developed in fish muscle during incipient spoilage was attributed to a synergistic flavor interaction involving the ethyl esters of acetate, butyrate, and hexanoate. Castell and Greenough (3) described a fruity or ester-like odor that commonly developed in chilled fish muscle during the early stages of spoilage. This distinctive odor, encountered more often on commercially prepared fillets rather than round or eviscerated fish, was reproduced in sterile fish tissue and fish media by bacterial cultures isolated from fish. The causative bacterial species was identified as Pseudomonas fragi, a psychrophilic organism which utilizes a variety of amino acids for odor production (4,5). This study was initiated to identify the volatile compounds produced in sterile fish muscle (Sebastes melanops) by P. fragi. Particular emphasis was placed on the identification of the compounds responsible for the characteristic fruity odor. MATERIALS AND METHODS Sterile muscle tissue. Sterile fish muscle was obtained from black rockfish (S. melanops) by a modified method of Lobben and Lee (10) plates for 48 h at 25 C, were collected and suspended in sterile, distilled water. Sterile muscle tissue (pH 6.4-6.7) was homogenized, inoculated, and dispensed in screw-capped vials as reported previously (13). An additional muscle homogenate, adjusted to pH 7.3 with sterile NaOH, was treated as described above. Duplicate samples (pH 6.4-6.7 and pH 7.3), supplemented with 0.2% ethyl alcohol, were also prepared. Sterile homogenized milk and milk fortified with 0.2% ethyl alcohol were prepared by autoclaving at 121 C for 10 min. All sample vials and appropriate controls were incubated at 5, 15, and 25 C and checked periodically for odor production and microbial counts. When the fruity odor was pronounced, the contents of selected vials were analyzed by combined gas-liquid chromatography and mass spectrometry. Gas-liquid chromatography and mass spectral analyses. The gas chromatographs, mass spectrometer, chromatographic columns, and methods of sample preparation and analysis used for the separation and identification of aliphatic methylamines and other low-boiling compounds were reported previously (11)(12)(13). RESULTS AND DISCUSSION An ester-like or fruity odor was produced by P. fragi strain 18 in sterile fish muscle (S. melanops) incubated at 5, 15, and 25 C. The characteristic odor developed more rapidly at the higher temperatures and was gradually superceded by a distinct sulfide odor. Since strain 18 did not reduce trimethylamine oxide to trimethylamine, no typical amine odor was apparent. Preliminary gas chromatographic analyses of the volatiles produced by strain 18 in sterile muscle tissue at pH 6.4 to 6.7 and pH 7.3 indicated limited concentrations of several components that were suggestive of ethyl esters on the basis of past experience. In an attempt to enhance ester production, the sterile fish homogenates were supplemented with 0.2% ethyl alcohol, and the volatile compounds produced by strain 18 were identified by gas-liquid chromatography and mass spectrometry after 4, 8, and 12 days incubation at 15 C. Compounds positively identified and listed in Table 1 included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. Limited amounts of acetone were detected in the distilled water used for all analyses, and methylene chloride was considered a persistent contaminant in the atmosphere in which the samples were prepared and analyzed. The microbial count in fish homogenates (pH 6.4-6.7), supplemented with 0.2% ethyl alcohol, increased from 4.0 x 106 cells/g at 0 days to 1.1 x 1010 cells/g at 8 days. The olfactory evaluation of each component, eluting from the column, was facilitated by a splitter which was attached to the effluent end of the column (13). The compound that eluted with a retention time almost identical to that of ethyl butyrate had a strong, fruity odor but, because of the limited concentration, the mass spectrum was weak. Although the parent ion for ethyl butyrate, m/e 116, was not discernible, the relative intensities observed for mle 29, 60, 71, and 88 were strongly suggestive of an ethyl ester. Ethyl hexanoate was tentatively identified by relative retention time. A retention time of 99.7 cm for the authentic compound compared reasonably well with the retention time of 98.6 cm recorded for the peak in question. The fruity aroma produced by strain 18 in fish during the early stages of spoilage or incubation was attributed to a synergistic flavor interaction involving ethyl acetate, ethyl butyrate, and ethyl hexanoate (17). The strong sulfide odor that persisted during continued incubation was the result of marked increases in methyl mercaptan (tentative identification), dimethyl sulfide, and dimethyl disulfide. Castell et al. (4) reported that lipolytic and nonlipolytic strains of P. fragi isolated from fish produced fruity odors primarily from monoamino monocarboxylic acids. It was suggested that the fatty acids were produced by a number of different reactions involving deamination, methyl mercaptan and dimethyl disulfide could be formed as reported previously (13). Repeated attempts to produce fruity odors in sterile fish muscle inoculated with the strain of P. fragi obtained from the Department of Microbiology at this institution were unsuccessful. Apparently, this strain lost its ability to produce esters. Strong, fruity odors are characteristic of new isolates of P. fragi, but the ability to form esters is easily lost by continued subculturing of the organism under laboratory conditions. However, in some cases, ester production can be restored by growing the organism on a medium containing the necessary or suitable substrates. Since authentic strains of P. fragi characteristically produce a fruity aroma in many dairy products (7,14,17), P. fragi strain 18 was also cultured in sterile, homogenized milk supplemented with 0.2% ethyl alcohol. A typical flame-ionization detector chromatogram of the volatiles produced by strain 18 in milk incubated at 15 C for 4 days is illustrated in Fig. 1. Compounds identified are listed as follows with respective peak numbers: (1) methyl mercaptan (tentative identification), (2) dimethyl sulfide, (3) acetaldehyde, (4) ethyl acetate and acetone, (5) ethyl alcohol, (6) not identified, (7) ethyl butyrate, (8) dimethyl disulfide, (9) not identified, (10) ethyl hexanoate, and (11) heptanone. The small peak immediately after peak 6 had a retention time identical to that recorded for 2-butanone. Bills and Day (2) reported that acetaldehyde, dimethyl sulfide, butanone, and acetone are usually present in milk. Although the concentrations of the latter three compounds remained relatively constant throughout the 4-day incubation period, acetaldehyde increased between 12 to 24 h and then decreased substantially with continued incubation. An increase and subsequent decrease in acetaldehyde content during the early phases of incubation were also observed in fish homogenates inoculated with strain 18. Keenan et al. (9) previously observed a very active reduction of acetaldehyde to ethyl alcohol by several pseudomonads, including P. fragi. Marked increases in the concentrations of ethyl acetate, ethyl butyrate, and ethyl hexanoate were noted between 1 and 4 days of incubation at 15 C, and the ratio of peak areas of the esters at 4 days was approximately 4:2: 1, respectively (Fig. 1). The microbial count in homogenized milk increased from 4.9 x 105 cells/g at 0 days to 2.4 x 108 cells/g at 5 days, and a reasonable correlation with the production of esters was observed. In addition, there was no evidence of bacterial growth or ester formation in the uninoculated controls. The fruity aroma produced by P. fragi strain 18 in milk was due primarily to the production of ethyl butyrate and ethyl hexanoate. These results correlated well with data previously reported for recognized strains of P. fragi cultured in milk (16) and, therefore, further substantiated the reclassification of Pseudomonas type III no. 18 to a type II pseudomonad, P. fragi. Although P. fragi strain 18 produced a fruity aroma in homogenized milk and sterile fish muscle, quantitative differences in the resultant ethyl esters were quite apparent. Appreciable amounts of ethyl acetate, ethyl butyrate, and ethyl hexanoate were produced in homogenized milk (Fig. 1). In contrast, ethyl acetate was the major ester produced in sterile fish muscle, and only limited concentrations of ethyl butyrate and ethyl hexanoate were detected. Therefore, it is quite apparent that ester production can be influenced considerably by the medium or available substrates (15). The data presented above indicate that P. fragi, the cause of the fruity defect in dairy products, apparently plays a similar role in the spoilage of chilled fish muscle. Although sterile fish muscle homogenates were used in this investigation, the fruity and sulfide odors produced by P. fragi have been associated with naturally spoiling fish (6) and were also reproduced in sterile muscle blocks (6) as well as on heat-sterilized muscle and autoclaved fish media (3).

v3-fos

2020-12-10T09:04:12.685Z

1973-04-01T00:00:00.000Z

237235025

s2

Factors Influencing Detection of Salmonellae in Rendered Animal By-Products Detection of salmonellae in animal by-products is influenced by the enrichment and plating media and by quantity of product tested, and is related to the total plate count. A linear relationship exists between detection of salmonellae and total plate counts from 104 through 107 per gram. Detection of salmonellae in contaminated food and feed ingredients is dependent upon many factors. No single method has been developed that satisfactorily recovers all Salmonella serotypes from all types of foods (1). The purpose of this study was to develop a better understanding of the factors influencing the detection of salmonellae in naturally contaminated animal by-products. The influence of media and the relationship between total counts and isolation of salmonellae were examined. MATERIALS AND METHODS During 1967 and 1968 samples were collected from plants throughout the industry for analysis in our laboratory. Total plate counts were determined by using plate count agar (Difco) at 37 C for 48 h. Two 20-g samples were analyzed for Salmonella. One 20-g sample was added to 80 ml of tetrathionate-Brilliant Green-iodine (TB) broth and the other 20 g was added to 80 ml of selenite-cystine broth (SCB). Both broths contained 0.6% Tergitol no. 7 (Union Carbide Chemical Co.) and were incubated 24 h at 37 C. The enrichment broths were streaked onto plates of Brilliant Green-sulfa agar (BGS; Difco) and Salmonella-Shigella agar (SS; Difco) plates and incubated 24 h at 37 C. Plates showing less than three suspect colonies were incubated an additional 24 h at room temperature and reexamined. Three Salmonella-like colonies were picked from each plating medium to triple sugar iron (TSI) agar slants. Cultures with Salmonella-like reactions on TSI after 18 to 24 h were tested serologically by using polyvalent 0 and H antisera. Cultures reacting postively in either or both serological tests were confirmed biochemically. RESULTS Of 183 Salmonella-positive samples, 139 and 148 samples were found positive in TB and SCB, respectively ( Table 1). The combination of SCB and SS agar plates gave the highest number of positive samples (127). The combination of SCB with BGS agar plates gave the lowest number of positive samples (92). From 3 to 9% of the samples were positive in only one of the four possible media combinations. Table 2 shows a grouping of 405 samples according to total plate count results. The rate of salmonellae isolations increased from 12.7 to 70.6% as the total plate counts increased from less than 10'/g to 107/g. Samples with total plate counts in excess of 107/g were associated with a decreasing rate of salmonellae isolations. Linear regression analysis confirmed a positive relationship between the total plate count and detection of salmonellae (P = 0.05). However, examination of the regression line by chi-square analysis revealed a lack of fit, for samples having total plate counts below 104 and at 108/g and above. This supports the conclusion that the relationship is linear only for samples having total plate counts from 104 through 107/g. The probability of detecting salmonellae in animal by-products was developed from further statistical analysis of the data. For example, if the product has a total plate count in the range of 104 to 105/g, then the probability of detecting salmonellae in a single sample would be 0.40 (Table 3) by using the methods described. Comparing the data for samples positive from only one enrichment (20 g) versus both enrichments (40 g), we found that 40 g significantly (P = 0.05) increased the detection of salmonellae. This conclusion was dependent upon the level of contamination and the assumption that the concentration of salmonellae would be higher in samples having higher total plate counts. The data in Table 2 support this assumption. For example, a higher percentage (64%) of the positive samples was found positive in only one enrichment (20 g of product) when the total counts were lower (104/g). At 108 total counts per g, 25% of the positive samples were positive in one enrichment. DISCUSSION No practical difference was found between TB and SCB in terms of the number of samples found positive. Both TB and SCB failed to detect salmonellae in approximately one out of five of the total 183 positive samples ( Table 1). The best combination of media (SCB-SS) found only 69% of the 183 to be positive. This failure to detect salmonellae could be due to inhibition of certain serotypes by the enrichment media, a low level of contamination, or uneven distribution of salmonellae through the product, or all three (5,9). Using both TB and SCB results in a higher rate of salmonellae isolations from animal feed ingredients (2,5,9). However, using both enrichments also doubles the quantity of product analyzed. The degree to which sample size influences the isolation rate of salmonellae in animal by-products deserves more consideration. Huhtanen et al. (3) analyzed 16 samples of meat and bone meal by using ten 3-g samples and ten 30-g samples for each. They found only 38 individual 3-g samples positive as compared to 86 to 89 individual 10-g samples positive. Laramore and Moritz (4) analyzed 73 samples of meat meal by subdividing each into two 30-g samples. They found results from the samples to agree only 86.2% of the time. Adding 20 g of meat and bone meal to both tetrathionate and selenite broths, Leistner et al. (5) found 12 samples positive. By using 10 g from the same samples, they found only seven samples positive. They then reported 37 samples positive from tetrathionate or selenite broths, or both. However, 13 (35%) of the 37 samples were positive in only one or the other enrichment broths. They state that this could be due to a low or nonuniform level of contamination (or both) as well as inhibition of certain serotypes. We did not conduct quantitative determinations to learn the concentration of salmonellae. However, assuming the concentration of salmonellae to increase relative to the total plate counts, it was possible to statistically determine that quantity of product (20 versus 40 g) is a significant factor. We conclude that the difference we observed between enrichment media for detecting sal-monellae is more apparent than real. Others have clearly established that an inhibitory effect exists due to the failure of certain serotypes to grow in the enrichment media. The data which is becoming available for naturally contaminated animal by-products suggests that the quantity of product being tested, at least in the range of 3 to 40 g, may be the more important factor. Our data confirms earlier reports (6, 7) that a relationship exists between total plate counts and the detection of salmonellae in animal by-products. However, it was learned that the linearity of the relationship exists only in the total count range of 104 through 107/g. There is a decrease in the percentage of Salmonellapositive product having total plate counts greater than 107/g. This is probably due to overgrowth of salmonellae by other bacterial species during enrichment or plating, or both. It is less likely that the product is actually less contaminated with salmonellae. The use of total counts as a measure of the microbiological safety of foods must be assessed in terms of the particular situation presented (8). Animal by-products appear to be one of the few materials where a linear relationship exists between total counts and the incidence of salmonellae. This is of practical value for in-plant control purposes and for evaluating improvements in manufacturing and sanitation. It is important that the limitations of the total plate count be recognized and it is sug-gested that total counts be used to supplement salmonellae testing programs.

v3-fos

2018-04-03T03:03:23.625Z

1973-09-01T00:00:00.000Z

36020885

s2

Viability of Actinomycetales Stored in Soil' About 1,800 Actinomycetales stored in soil for up to 20 years were checked for viability. About one-half were viable. lyophilization for preservation of all strains of aerobic Actinomycetales, Since the early 1950s, we have used soil culture, agar slant culture, and lyophilization concomitantly for preservation of all strains of aerobic Actinomycetales, mostly streptomycetes and streptoverticillia. The large number of strains accumulated over the years has forced abandonment of routine transfer on agar slants and, more recently, the use of soil cultures. The decision to abandon preservation by soil culture prompted examination of these materials to determine how many had survived storage at room temperature over periods up to about 20 years. The information obtained is presented herein, along with some miscellaneous observations relating to this collection. Three different kinds of soil cultures had been prepared. An initial lot had been made by adding suspensions of spores to sterilized loamy soil. These preparations, stored in a refrigerator under humid conditions, became contaminated with molds and, for the most part, had to be destroyed. A second lot of soil cultures was prepared by adding broth cultures to sterilized prairie black loam soil and allowing them to dry at room temperature (2) with subsequent storage at room temperature. In 1959, a third lot of cultures was prepared in a silica sand-CaCO, amended soil (screened, air-dried prairie black loam soil, 10,000 g; white silica sand, 7,500 g; and CaCO,, 25 g; mixed by hand and screened). Because of the possibility of mite infestation, the cotton stoppers of one lot of tubes were treated with a miticide (0.1% HgCl, in 95% ethanol containing identification dye). Unfortunately, this lot was additionally sealed with rubber stoppers over the cotton plugs to facilitate numbering of tubes for rapid retrieval from large storage racks. Many of these cultures died, possibly because of the exclusion of air or permeation of the soil with ethanol, or both. IThis study was presented, in part, at the 73rd annual meeting of the American Society for Microbiology, Miami Beach, Fla., 6-11 May 1973. New soils had to be prepared for each of these after only short periods of storage (up to 1 year). With the passage of time, much dust accumulated on the cotton stoppers. Despite this, few cultures were found to be contaminated (Table 1). About one-half of the tested soil cultures contained viable Actinomycetales ( Table 1). Age of storage seemed to have little effect on the number of strains viable. The results suggest some other reason for death of the cultures, e.g., the method of preparation, the drying out process, or exposure to fluctuating room temperature. Our experiences with these soil cultures and with maintenance on agar slants is an argument in favor of lyophilization as a means of preservation. Lyophilized preparations have shown much higher percentage of survival and less risk of contamination of the cultures based on the experiences of the several curators of the Agricultural Research Service Culture Collection over a 25to 30-year period. B. P. was an Agency for International Development scholarship trainee, Department of Health, Bangkok, Thailand.

v3-fos

2020-12-10T09:04:12.365Z

1973-12-01T00:00:00.000Z

237233555

s2

Population Changes in Enteric Bacteria and Other Microorganisms During Aerobic Thermophilic Windrow Composting Composting of wastes from swine feeding operations was studied. The effects of the frequency of turning the wastes and addition of straw to improve the physical structure were studied to determine the most effective technique to rapidly increase the temperature and, consequently, destroy coliforms and Salmonella. Four different treatments were studied; the results showed that, with addition of 5% (wt/wt) straw and mechanical turning of the compost 20 times per week, the temperature reached 60 C within 3 days and enteric bacteria were destroyed within 14 days. According to Wadleigh (12), animal farm waste production in the United States amounts to about two billion tons annually. Traditional methods for utilizing these wastes as fertilizers are not being employed widely, and the vast amounts that are accumulating present serious threats from both environmental and public health standpoints. Solutions to the problems of disposal of animal solid waste require complex environmental control. Methods must be utilized that do not cause contamination of water, pollution of the atmosphere, or desecration of the land. Burning of waste in open dumps or poorly designed incinerators is a major source of air pollution. Other disposal methods, such as sanitary landfill, aerobic and anaerobic lagoons, and spreading of liquid and solid on soil, may cause odors and chemical and microbial pollution of surface and ground waters. Many of these problems can be avoided by composting; with proper control of this method, wastes are incorporated into the biogeochemical cycle without serious detriment to the ecosystem. Composting can be defined as the decomposition of organic wastes under semi-dry conditions by aerobic, thermophilic organisms. The products of composting are carbon dioxide, water, heat, and stable humus-like organic material. Several investigators (2, 5, 10) have concluded that since relatively high temperatures are attained during the composting process any I Paper of the Journal Series, New Jersey Agricultural Experiment Station. pathogens present should be destroyed. Recently, Wiley and Westerberg (13) selected representatives from four groups of pathogens as indicators and related the temperature of a laboratory composter to survival of these organisms. Few studies of a similar nature have been reported on a large-scale field operation. This paper describes the survival of Salmonella, other enteric bacteria, and fecal streptococci during the composting of swine waste in windrows that were turned mechanically with a composting machine. Changes in the levels of actinomycetes, filamentous fungi, and cellulolytic microorganisms occurring during composting are also discussed. MATERIALS AND METHODS Preparation of compost. Swine waste was obtained from a hog feeding operation in southern New Jersey. The swine were fed hotel and restaurant garbage, which was cooked with live steam and dumped outdoors in concrete-floored pens. The swine waste-a combination of uneaten garbage, bottles, cans, plastic, paper, bones, other inedible material from the garbage, and swine feces-was scraped up daily with a front-loading tractor and removed. The material was trucked to the compost site and stockpiled over a 5-week period until a sufficient amount of waste accumulated for construction of four 40-ft (about 12.2 m) windrows, which were installed on a concrete floor. The swine waste was turned by a self-propelled composting machine (Roto-Shredder, Roto-Shredder Co., Division of Imco, Crestline, Ohio) which straddled the row and traveled its length, shredding, grinding, and pulverizing the material (Fig. 1). As the machine moved forward, the waste fell behind the machine, reforming the windrow. Through this mixing action (turning), oxygen (air) was incor- porated into the mass. After turning, the windrow was less compact, and the particle size of materials such as paper, cardboard, vegetable matter, and bones was greatly reduced. Windrow 1 contained approximately 40 tons (36 metric tons) of unsupplemented swine waste and was turned twice a week. Windrows 2, 3, and 4 were turned 20 times per week. Windrow 2 contained approximately 40 tons of swine waste; windrow 3 contained approximately 23 tons (21 metric tons) of waste and 18 tons (16 metric tons) of old compost that has been previously composted for 180 days with turning twice weekly. Windrow 4 contained 40 tons of stockpiled waste and 1.5 tons (1.36 metric tons) of straw. Sampling procedure. Samples of compost to be assayed were collected from the windrow surface immediately after turning on the days indicated in Fig. 5 to 9. Samples weighing approximately 3 g were taken randomly along the windrow and combined in a sterile quart Mason jar. The total composite sample weighed about 1 kg. In the laboratory, the sample was mixed thoroughly, and duplicate portions (10 g) were taken for testing. These were suspended in 90 ml of sterile water, and 1-ml volumes of the suspension were diluted serially (wide-mouthed pipette). Samples (100 ml) of run-off water were collected from seepage at the foot of the pile; 1-ml volumes of these samples were diluted serially. Determination of microorganism numbers. The standard plate count method (1) was used for estimating the numbers of microorganisms in the compost. Coliforms were counted on Levine eosin methylene blue agar (Difco) plates, as justified by Poelma (8) and used by the Food and Drug Administration. The identification methods used for Salmonella were, in general, those of Poelma (8). Plates for counting Salmonella were poured with brilliant green agar, Salmonella-Shigella agar, or bismuth sulfite agar (all Difco products), and incubated at 37 C for 2 days. Proportions of the colonies appearing on these plates were cultured and characterized by biochemical tests (fermentation of glucose, lactose, dulcitol, and mannitol; decarboxylation of lysine, ornithine, and asparagine; production of H2S, indole, acid [methyl red], acetoin [Voges-Proskauer]; and utilization of citrate, urea, malonate, and triple sugar-iron). About 75% were indicated to be Salmonella and 25% were Proteus types (urease positive). Serological tests were not performed. Counts in tables are based on presumed Salmonella from plate counts. Fecal streptococci were counted on KF agar plates (1); M-enterococcus agar was also used and gave similar results. For total bacterial counts, plates were poured with nutrient agar (Difco) and incubated at 37 C for 2 days. Only typical bacterial colonies were included in the counts; these were generally mucous or small, yeflow, lenticular colonies. Total counts were also investigated on compost agar (9), but fewer colonies were observed. Fungi were enumerated on acid-agar or potato-glucose-novobiocin agar plates (9) incubated at 28 C for 5 days. Colonies counted as fungi were typically filamentous or large, round, mucous colonies with yeast-like appearance. Actinomycetes were enumerated on caseinate agar and Czapek-Dox agar (Difco). These plates were incubated for 7 days at 37 C, and only the small, powdery, wrinkled, or pasty colonies were counted as actinomycetes. When these colonies were isolated, their plates had the typical earthy odor associated with actinomycetes. Cellulolytic bacteria associated with compost were enumerated by plating the compost homogenate on a cellulose-mineral salts agar medium (6). The medium contained per liter: (NH4)2S04, 0.5 g; K2HPO4, 0.5 g; KH2PO4, 0.2 g; CaCl2, 0.5 g; MgSO4, 0.5 g; NaCl, 1.0 g; agar, 15 g; and 150 ml of a slurry of cotton-fiber cellulose, ball milled for 24 h in 4% HCl and washed with distilled water. The colonies which produced clearing zones on this medium were recorded as cellulolytic microorganisms. The plates were incubated aerobically for 7 days at 37 C. Determination of windrow conditions. Temperatures of the composting windrows were determined by a thermocouple potentiometer and recorded on a battery-operated, 12-point Brown recorder (Minneapolis-Honeywell Regulator Co., Industrial Division, Philadelphia, Pa.). The thermocouples were mounted on a probe that could be inserted in the piles (one is visible at the lower left in Fig. 1). This probe had three parallel rods of :'4-inch (about 1.89 cm) diameter galvanized pipe with an aluminum point on one end and a junction box on the other. Each rod had two openings-one each at 6 and 24 inches (about 15.24 and 60.96 cm, respectively) from the tip of the aluminum point. At each opening was a thermocouple (electrically insulated from the metal rod). The rods were 24 inches apart. They were inserted vertically into the top of the windrow and forced down until the tip touched the concrete base, so the readings were made 6 and 24 inches from the bottom of the piles. The three thermocouples at each of the two depths were connected in parallel, thus yielding an average reading. The thermocouples were connected to the recorder, which printed the average temperature. The printout was controlled by a timer set to record the temperature at 6-h intervals. The pH of compost samples was measured on three replicate samples, each containing 10 g/500 ml of distilled water. The suspensions were stirred with a magnetic mixer for 5 min, and the pH was determined with a Beckman Expandomatic pH meter, model 7600. The oxidation-reduction potential (Eh) of the material in the windrows was determined with a redox probe constructed by Starkey and Wight (11). This 4-ft-long (about 121.9 cm) pointed probe was composed of inner and outer tubes; after the probe was inserted in the windrow, the inner tube could be rotated to make the openings in the two tubes coincide, exposing the electrodes (two platinum and one calomel half-cell) to the windrow material. Readings were made by connecting the calomel half-cell to the lower terminal on a Beckman model G pH meter and by connecting one of the two platinum electrodes to the 700 terminal. After the electrodes had stabilized and a reading was taken, the other platinum electrode was connected to the 700 terminal and a duplicate reading was taken. The electrodes were cleaned before and after each reading by being washed successively with 5% acetic acid, 5% nacconol, water, 10% H202, and distilled H2O. The electrodes were standardized with solutions of known E, value; the meter was standardized by adjustment to zero H+ ion and checked by connecting the electrodes-in standard solution-to an Electronic Associates Inc. 6200 digital volt meter. The moisture content was determined by weight loss of 25-g samples which were dried for 3 days at 103 C or until a constant weight was reached. RESULTS Windrow temperature, pH, and Eh. Changes in temperature within the windrows during composting are shown in Fig. 2. Windrow 1 showed a lag period of 38 days before a rapid rise in temperature occurred. The temperature in the center of windrow 2 (swine waste turned 20 times per week) reached a thermophilic range of 55 to 65 C in 25 days. The temperature in windrow 3 (approximately 50% old compost) reached this range in 15 days, whereas windrow 4 (waste and straw mixture) had reached 60 C within 3 days of composting. The highest temperature recorded at any position in any windrow was 72 C in windrow 4. After reaching thermophilic temperature, windrows 2 and 3 remained thermophilic until they were removed from the concrete floor on day 80. Windrow 4 cooled to ambient temperature (20 to 30 C) by day 38. Changes in the pH of the waste during composting are shown in Fig. 3. Windrow 4 reached a pH of 8.0 in only four days-indicative of rapid decomposition in this windrowwhereas windrow 3 took 16 days to reach this pH; windrow 2 reached pH 7.5 in 25 days but did not reach pH 8.0; and windrow 1 took approximately 80 days to reach pH 8.0. The initial Eh of the windrows was -450 mV (Fig. 4). In windrow 1, the Eh rose gradually to -200 to -250 mV over the first 18 days of composting and then remained constant for the next 20 days; active thermophilic composting, indicated by pH and temperature rise, began only after this period. The Eb rose to +50 to Influence of composition on the progress of decomposition, as indicated by pH pattern. The symbols are: 0, windrow I (unsupplemented swine waste turned twice a week); 0, windrow 2 (unsupplemented waste turned 20 times a week); 0, windrow 3 (waste plus 180-day-old compost); A, windrow 4 (waste plus 5% straw). + 100 mV immediately after the windrow was turned but decreased within 60 min to the level observed before the turning. In windrows 2, 3, and 4 during the active composting (pH >7, temperature > 60 C), the Eh was -50 to -100 mV before the turning. Windrow 4 reached this level (from the initial -450 mV) within 3 days. These more aerobic conditions presumably result from greater porosity of the pile, especially in windrow 4. Active composting (pH > 7, temperature >50 C) began in windrow 1 only after the moisture content fell below 40%, 40 days after the composting process was begun; however, since windrows 2, 3, and 4 reached the active composting stage when moisture content was still 45 to 55%, dryness of the material was not solely responsible for active composting. Effects of thermophilic composting on intestinal microorganisms. Enumeration of Salmonella in windrow 1 and its run-off water at various times indicated the number of organisms that could survive and possibly pollute water supplies. Salmonella numbers, after an initial drop, increased in the windrow (Table 1, 40 days). When the temperature rose above 48 C, the population of presumptive Salmonella decreased sharply and continued to decrease as temperature increased. Similarly, the numbers of Salmonella, coliforms, and streptococci in the run-off water increased initially (after a drop in coliforms), then decreased as the temperature in the center of the windrow passed 52 C ( Table 2). The count of fecal coliforms, shown in Table 2, appeared to remain higher than fecal streptococci or Salmonella. The coliform count decreased rapidly as composting proceeded in windrows 2, 3, and 4 (Fig. 5). The coliform test was negative in windrow 4 at day 14 of composting when the temperature was 71 C. When windrow 4 was removed on day 40, presumptive Salmonella colonies could not be detected. The number of coliforms was reduced by 104-fold in windrows 2 and 3. In 40 days of composting, the number of mesophilic bacteria decreased between 104-and 105-fold in windrows 3 and 4 (Fig. 6). The bacterial population in windrow 2 increased during the first 15 days and then started to decline. The decline in mesophilic bacteria occurred at the time when the temperature began to rise rapidly in these windrows. The number of mesophilic fungi did not drop as much as the number of bacteria (Fig. 7). In windrow 4, fungi were reduced 103-fold, but in windrows 2 and 3 the decrease was less than 100-fold. The population of mesophilic actinomycetes responded differently from either bacteria or fungi. This population increased by factors of over 103 in windrow 4, 103 in windrow 3, and less than 102 in windrow 2 (Fig. 8). The increase in windrow 3 followed a decrease in the first week from the high initial numbers present as a result of the addition of old compost. The number of cellulolytic organisms showed a re- sponse similar to that of the population of actinomycetes. Again, the population in windrow 4 developed more rapidly and attained a much higher number (Fig. 9). DISCUSSION Although considerable use is made of composting in the disposal of municipal wastes, garbage, sewage sludge, etc. (4,5), little information is available on the numbers of enteric bacteria actually resulting from such operations. Typically, thermal death points of common pathogens present have been determined, and the assumption was made that attainment of these temperatures during the composting process would destroy the pathogens (2,5). In one study (13), introduced indicator organisms (poliovirus, Candida albicans, Ascaris lumbricoides, Salmonella newport) were shown to disappear within 43 h from a pilot-scale composter maintained at 60 to 70 C. The possibility of survival of pathogens at the cool surface of a windrow operation, however, has not been disproven. Our results indicate clearly a marked decrease in coliforms, salmonellae, and enterococci during the thermophilic stage of com-posting. Before the thermophilic stage, there was presumably anaerobic decomposition of carbohydrates, proteins, and fats to form organic acids and other intermediate compounds that could be used by Salmonella, coliforms, and enterococci for growth under partially anaerobic conditions (7). Therefore, an increase in these organisms during the first stages of composting was expected. From a public health standpoint, it is desirable that this early increase be minimized and that the windrows maintain a temperature above 48 C long enough to destroy pathogens like Salmonella. These results indicate that, for maximal sanitary safety, the thermophilic stage of composting should be reached as soon as possible. The noteworthy practical observation is that inclusion of straw (windrow 4) fostered more aerobic conditions and thereby facilitated very rapid attainment of thermophilic conditions and destruction of enteric bacteria. Frequent turning of the windrows was much less effective. The original rationale for addition of straw was increase of the carbon-nitrogen ratio, since excess nitrogen is liable to be eliminated as ammonia and other malodorous amines. However, the primary effect of addition of straw -_ t appeared to be improvement of the physical structure of the windrow, allowing more natural aeration (compare Fig. 4) and a rapid rise in temperature consequent upon intense aerobic microbial activity. This had the beneficial effect of rapid destruction of pathogens. Waste materials with similar structural properties, such as cornstalks, chipped wood, shredded municipal refuse, etc., should also have this effect. The possibility of use of vegetable wastes such as cornstalks, which are also becoming a disposal problem as open air burning is banned, to improve the composting of animal wastes is particularly attractive. A succession of microbial populations was observed during the composting process. The bacteria increased in number before the temperature of the windrows rose and then declined, whereas cellulolytic organisms and actinomycetes in general increased in the thermophilic stage. Presumably, the mesophilic bacteria rapidly attack the more readily available organic constituents, resulting in a temperature increase. The increased temperature favors the cellulolytic organisms, and the mesophilic bacteria largely disappear. The actinomycetes appeared in the final stage to such an extent that the surfaces of the compost piles were white or gray. These organisms are known to play a role in the humification of organic matter, which results in a stabilized product (3).

v3-fos

2014-10-01T00:00:00.000Z

1973-07-15T00:00:00.000Z

16239703

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Field testing of young breeding pigs. II. – The accuracy of field testing The accuracy of the field test, for the individual selection of gilts and for the progeny testing of boars, was investigated. Data from Ioz 7 gilts, measured in a farm testing scheme, and being all offspring of AIboars, were analyzed. The relative contributions of additive genetic effects (h 2), litter environment effects and farm effects to the total variation were estimated for a performance index, a score for weight, a score for backfat thickness, average daily gain and some conformation traits. The index was a linear combination of both scores. It was found that the heritability of the index was .22 and that litter environment effects and farm effects contributed 2i p. ioo and 9 p. 100 respectively to the total variation. It was concluded that this heritability value was high enough to apply individual selection in gilts by means of the field test and that selection within farms would not increase very much the accuracy of the test. The repeatability of the progeny test of boars, based on data of the field test of their offspring, was estimated empirically as well as theoretically. In an AI breeding population this repeatability was in the order of .6 when about 64 offspring per boar were measured. INTRODUCTION Farm testing has some advantages over station testing. Its costs are low and the selection capacity in the field is almost unlimited. For these reasons it is also possible that farm testing may become a substitute for station testing of young boars, the more so as the risks of spreading diseases are much lower with field testing than with performance testing boars at the stations. A serious drawback of farm testing is of course its lower accuracy. In particular, farm effects may bias the breeding value estimation of animals. The aim of this study was to investigate the accuracy of field testing for individual selection as well as for progeny testing. MATERIAL AND METHODS 0 Data of young Dutch Landrace gilts, measured in the field testing programme in the provinces Limburg and Noord-Brabant, were analyzed. About 2/3 of the gilts were sired by AIboars and i/ 3 by natural service boars. The numbers of animals measured and their distribution over farms, sires and dams, are listed in table i. The gilts were weighed and their backfat thickness was measured at an age between 150 and 295 days. Since January 1970 the animals were given an index, which was a linear combination of a score for weight and a score for backfat thickness. This index was not perfect since older animals systematically got a higher index vaiue. The reason for this was that the score for weight was based on an inaccurate correction for age. For the analysis however all animals were given the new scores and the new index as described in Part I. In each province farm testing was carried out by one special technician (inspector) of the herdbook. They also judged the animals on conformation. The following characteristics were taken into consideration : a) muscularity of back and loin, b) form and shape of hams, c) legs, d) size and development. For each trait a point scale from I (poor) to 5 (excellent) was used. Finally total points for conformation were calculated. Each province used a different system. Limburg : total points L = 3a + 3 6 + 3c -! d (maximum 50 points) Noord-Brabant : total points NB = a + b + c + d (maximum 20 points) Estimation of components of variance Estimation of components of variance was performed for the traits : index, score for weight, score for backfat thickness, average daily gain = weight/age, the four conformation traits and the total points for conformation. The model chosen to describe the performance traits, was : where : Yijkl is the observation on the I th individual of the k th dam and the j th sire on the ith farm ; y is a constant ; xt is the contribution of the i th farm ; (3p is the contribution of the j th sire ; yij is the contribution of the interaction of the i th farm with the j th sire ; 8 ijk is the contribution of the k th dam mated to the j th sire on the i th farm ; Õ :¡j kl is the contribution of the I th individual of the k!h dam and thej th sire on the i th farm. All contributions (except f .I. )were supposed to be mutually independent and distributed with mean o and standard deviations a a , a a , ay, a 8 and o s respectively. Farms and sires form cross classifications ; sires, dams and individuals (daughters) are nested. Since there are many empty subclasses, the data are unbalanced. Therefore the different variance components cr2, al, al, 0 82 and G2 were estimated according to Henderson's method i (e. g. Ssnxr.E, 1971 ). The sum of these 5 variance components yielded the total phenotypic variance aT. The following relative measures of variation, assuming random mating, were calculated for each trait : h 2 = heritability = relative additive genetic variation = 4!!s (e. g. B ECKER , 19 6 7 ) aT !2 !! el = relative variation, attributable to common litter environment = 6s z 2 a 6T -a C2 2 = relative variation, attributable to common farm environment -a!2T aT Approximate standard errors were calculated for the heritabilities, according to the for mula where : SE( h 2 ) : standard error of heritability ; a' T total phenotypic variance ; k : a weighted If average number of progeny per sire ; MS 13 mean square for sires ; MS s : mean square for dams ; s : number of sires ; d : number of dams (litters) ; i : degrees of freedom for farm X sire interaction term. The formula is a rough approximation of a formula given by L E Ro y ( 19 66), which was developed for data from a nested classification. The estimates were derived for each province separately and within the provinces for data of progeny of all boars as well as for data of progeny of AI-boars only. For the traits : index, scores and average daily gain the estimates were obtained also for both provinces combined. For the conformation traits a combined estimate did not seem to be meaningful, since the subjective judgement could be different for each of both inspectors. Estimates of components of covariance were obtained in an analogous manner to obtain phenotypic (r P ) and genetic (rg) correlations between each pair of traits x and y, where : where a ( x y) stands for the covariance of traits x and y, and the subscripts refer to the various effects as outlined in the description of the model. For the computation of the standard errors of the correlations the following approximations were used : where n = number of animals measured Daughtev-dam regression A heritability estimate of the index also could be obtained from the regression of offspring (0) on dams (D), since in the province Limburg 127 tested gilts had 45 8 daughters, that entered the farm test later on. The heritability, estimated as twice the regression coefficient b oD , was calculated over farms as well as within farms. In this regression analysis the number of offspring per dam was weighted according to the method described by FALCONER (i 9 6 3 ). RESULTS For each province the means (x) and overall standard deviations (s!) of the traits are listed in table 2 . First of all a possible influence of season was investigated by plotting the means against the months of measuring. No systematic season effect could be found on any trait. However, in both provinces a sudden shift in the average index, scores and daily gain was observed. In Limburg the means of these traits were much better after March 1970 , i. e. about 3 months after the introduction of the index tables in that province, and in Noord-Brabant after March 1971 , that means about 3 months after the beginning of farm testing in that province. The cause of this sudden change was not clear. A possible explanation could be that the technicians as well as the farmers got accustomed to the use of the index after about 3 months, so after this period they were able to apply some pre-selection in order to save costs. So may be the slowest growing animals would not be offered for measuring. Another possibility could be that the farmers found out that they could get a higher index for their pigs by feeding their pigs to a heavier weight. This would raise the index of their animals since the score for weight in the old index was not correct. Before performing an analysis of variance a correction for this sudden shift was applied for all traits. In each province the observations in the first period were increased by the average difference between the two periods in the respective province. The method of correction applied is debatable if the sudden change was caused by a pre-selection of the animals. However, it turned out that the statistical analysis of the corrected data yielded about the same relative estimates of components of variance as analyses of the data for each period separately. The estimation of components of variance showed that the contribution of the interaction between farms and sires to the total variance was in most cases low or even negative. So this interaction does not seem to be of much importance. From the variance components heritability estimates were derived. It turned out that the h 2 -values in the total material (progeny of AI and natural service boars) were much highter than the h 2 -values in the AI-population. In natural service there is a strong confounding of farms and sires, so the AI-population provides more reliable estimators of the different variance components. For this reason only the results of the analysis of data from AI-progeny will be given. _ In table 3 the heritabilities and the relative contributions of litter environment (c 2 ) and farm environment (c2) are listed. _ The h 2 -value of 224 of the index is about the average of the h 2 -values of both scores. The h 2 of score for backfat thickness is about 3 times as high as the h 2 of score for weight. Average daily gain yields about the same values as score for weight, as would be expected. From the conformation traits the judgement of muscularity of the back and loin and of form and shape of ham have the highest heritabilities. They are of the same order as the h 2 of the index. Low values are found for legs and for size and development. From the total conformation scores the method used in Limburg yields the highest h 2 -value. This is explained by the fact that in the « I,imbu_rg total » the first three conformation traitsfrom which two have a reasonable h 2 -valueare given three times as much weight as in the « Noord-Brabant total ». The proportions of variance due to litter environment and due to farm environment are rather high, especially for the score for weight and for average daily gain. About 21 p. 100 of the total variation of the index is due to litter effects. This is about the average of the corresponding values for both scores. Farm effects contribute about 9 p. 100 to the variation of the index and this is much lower than the average of the corresponding values for both scores. The h 2 of . 224 , derived from the paternal half-sib analysis of the AI-data, is in reasonable agreement with the estimate, derived from the regression of daughters on dams, as is listed in table q . This regression was done on the index values, corrected for the sudden shift between periods. The calculation within farms did not change the h 2 -value. Often in the analysis of field data of farm animals the within farms regression of daughters on dams is yielding lower but more reliable heritability-estimates than the total regression, which usually is biassed upwards by a correlation between daughters and dams due to their common farm environment. This correlation is eliminated in the within farms regression. However, as in our analysis daughters are measured at least one year later than their dams, the environment within farms may have changed considerably during this time lag, and consequently may have broken down the environmental correlation between daughters and dams in the total regression calculation. The phenotypic and genetic correlations between the traits are listed in table 5. The estimation of components of covariance was based on the AI-data, corrected for the shift differences. In the cells with two values, the upper value refers to Limburg, the lower to Noord-Brabant. In the interpretation of the correlations one has to realize that a negative score for backfat thickness is desirable. The phenotypic correlations between index and both scores are almost of the same size, the genetic correlation between index and score for backfat thickness, however, is much higher than the genetic correlation between index and score for weight. Both scores are phenotypically slightly unfavourable correlated ; the genetic relationship has the same sign but is somewhat stronger. Score for weight and average daily gain are highly correlated and can be considered as same traits. The phenotypic correlations between the index and the conformation traits are all positive. The same holds true for the correlation between the score for weight (or average daily gain) and the conformation traits. The score for backfat thickness is only slightly correlated with the conformation traits ; the correlation with muscularity of back and loins is slightly unfavourable, and with form and shape of hams slightly desirable. The points for legs are almost uncorrelated with the other conformation traits. The genetic correlations usually have the same sign as the phenotypic correlations, taking into account their high standard errors. FARM TESTING USED FOR PROGENY TESTING OF BOARS Farm testing could also be used for the progeny testing of AI-boars in the field. A criterion for the accuracy of the progeny test is its repeatability. An empirical estimation of this repeatability was derived from the AI-data. From the progeny of each AI-boar 2 or more samples of the same size N were drawn. This sampling was done according to the date of measuring of the animals. The first sample comprised the first N measured progeny, the second one the second N measured animals, etc. The sampling was done for various sample sizes : N = 8, N = 1 6, N = 24 , N = 3 2. The following analysis of variance was carried out on the sample means : The repeatability b of the progeny test was estimated as : The sampling was done in two ways : A. Sampling per boar within provinces and periods (before and after the sudden shift) and pooling of the respective sum of squares over provinces and periods. B. Sampling per boar over both provinces, but restricted to the period after March 1971 . The results are listed in table 6. For larger sample sizes the repeatability estimates are based on less boars, and so are less reliable. In order to evaluate these findings also a theoretical estimate of the repeatability was derived from a model population, assuming random distribution of the progeny of v boars over farms and litters. Each boar has ! offspring per litter, with I litters on each of f farms, so the total number N of offspring per boar is flfi. The analysis of variance of such a hierarchical classification can be written as follows : Genetic interpretation of relative components of variance : where : h 2 , Cl and c2 are as defined earlier. c' = relative proportion of variance due to non genetic differences between progeny groups of boars (e. g. area differences, season differences) . where : e 2 = relative proportion of variance due to random environmental differences. Now the repeatability of the progeny test can be defined as : With this formula the theoretical repeatability of the progeny test was calculated for various combinations of p, 1 and f, substituting !2 = 22, ê! i = . az, c = . 09 and ?' = o (and also ca = . 05 ). The results are presented in figure i (for 'e,2 = o) and figure 2 (for ê; = . 05 ). In both figures the empirically derived values from table 6 are also plotted. It shows that in most cases these values are within the range of the theoretical possibilities. DISCUSSION A 9 2 -value of 22 for the index is rather low, but still high enough to be useful for individual selection of young breeding animals. In fact the value is of about the same magnitude as the h 2 of milk yield in cows. The litter environment contribution to the variation of the index is very high ( 21 p. ioo), much higher than the farm contribution. In practice the selection mainly will be carried out within farms, at least for gilts. However, this increases the accuracy of the breeding value estimation only very little, since the heritability within farms ( 9)) can be estimated as: Selection within litters eliminates the variation due to differences in litter environment, but it also halves the genetic variation, so it does not increase the effectiveness of selection. Our parameters are based on gilts, and so strictly speaking only applicable to the selection of gilts. Extending of the findings to the selection of boars must be done with reservation. Keeping this in mind it seems to us that farm testing of young boars will not be a complete alternative to the performance test of the boars on central stations, since the latter has a much higher accuracy and also the food intake can be measured. The heritability of a selection index for boars, tested on stations, usually is in the order of .5-.6. In a breeding structure, where much AI is applied on the breeding farms, as is the case in the most important pig provinces in the Netherlands, the field test can also be used for progeny-testing AI-boars. If precautions are made to avoid as far as possible environmental differences between progeny groups, the repeatability of the progeny test is .6, when about 6 4 offspring per boar are tested. This holds true for practical circumstances where on average 2 litters per boar per farm and 4 progeny per litter are measured (see figure i).

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Microbiology of Lebanon Bologna Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 104/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also. fibrous approximately RH four days either an incubator or house. end the fermentation period, the bolognas were mel- lowed intervals during the processing, samples of meat or bolognas were removed deter- mination of Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 104/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also. Lebanon bologna is a semidry, fermented, all-beef sausage which is smoked but not cooked. The sausage originated among the Pennsylvania Germans in the Lebanon, Pa., region. There is a paucity of literature concerning Lebanon bologna technology and microbiology; however, federal specifications for "Lebanon" style bologna have been published (3) and a nontechnical discussion of the processing of Seltzer brand Lebanon bologna has appeared (1). A few spice formulations for Lebanon bologna are known (8,9,13). Various aspects of the manufacture of Lebanon bologna have been presented elsewhere (Palumbo et al., manuscript in preparation). This paper is concerned with the microbiology of Lebanon bologna technology. MATERIALS AND METHODS Viable counts on meat or Lebanon bolognas were performed as follows: 50 g of beef cubes or 50 g of material from the center of a bologna were removed aseptically and ground at high speed in a Waring blendor with 200 ml of peptone water, and appropriate dilutions were then surface plated in triplicate. The types of media employed, temperatures, and times of incubation were: (i) total aerobic count on APT agar, 3 days at 25 C; (ii) micrococci on phenol red mannitol salt agar (MSA), 3 days at 32 C; (iii) lactic acid bacteria on acidified Rogosa SL agar (RSL), 3 days at 25 C; (iv) yeast on acidified potato dextrose agar (PDA), 3 days at 25 C; and (v) gramnegative bacteria on eosin methylene blue agar (EMB), 1 day at 37 C. All media were obtained from Difco Laboratories, Detroit, Michigan. Dilutions were made in 0.1% peptone (Difco) water. Gram stains of all colony types found on the various media were examined; in addition, the catalase test was performed on isolated colonies. RESULTS Viable counts were determined on 14 commercial brands of sausages, including 10 Lebanon bologna types. The viable counts are presented in Table 1 and types of microorganisms are presented in Table 2. The fermented sausages had catalase-negative, gram-positive rods (characteristic of lactobacilli) on APT and RSL except for the products produced by companies D and H, which contained catalase-negative, gram-positive cocci. Company D adds a pediococcus starter culture to their Lebanon bologna, and the predominant flora of thuringer has been shown to be pediococci (2). A number of the sausages contained lactic acid bacteria that produced gum on RSL plates. Lactics were not found in the sweet bologna produced by company C, and this is reflected in the high pH a The pH was determined by inserting the electrode into the mass of meat or into the center of a bologna. b A "sweet" Lebanon is similar to the regular type except that more sugar is present. The pH is low but the sweetness masks the acid "tang." c The sweet bologna produced by company C was not a Lebanon variety nor was it a fermented product. of 5.6. Typical coliforms were not observed in any of the sausages. Micrococci were present in company A's sweet Lebanon and in both types of Lebanon bologna produced by company G as well as in the cervelat and dry Italian salami; micrococci were not found in the other sausages. The individual steps in Lebanon bologna manufacture were investigated to determine the microbial flora involved and the factors responsible for the development of the flora that brings about the desired acid formation and nitrate reduction. The data in Fig. 1 represent the course, with time, of the viable count in aging salted beef. The viable counts on all five types of media used show a gradual increase in numbers during the aging period. The colony types found on APT consisted of catalase-positive, gram-positive, and gram-negative rods; catalase-positive, gram-positive cocci were found on MSA. The organisms on EMB were catalase-positive, gram-negative rods, but the colonies were not typical coliforms in appearance. Yeasts were present on PDA and catalase-negative, grampositive rods characteristic of lactobacilli were found on RSL. The viable counts for the bolognas made from meat aged for varying periods of time are given in Table 3. The only bologna that had a low pH (4.5) was made from meat aged 14 days, at which time the lactic count on RSL was approximately 104/g ( Fig. 1). In general, if the lactic acid bacteria count of the salted beef was not in the 104/g range, then a low pH was not achieved in the bolognas. Both APT and RSL plates show catalase-negative, gram-positive rods, with a large number of gum producers on RSL; yeasts tend to disappear in the bolognas (Table 3). Typical coliforms were not observed; MSA contained catalase-positive, gram-positive cocci in all of the bolognas except the one in which the pH was 4.5; at the low pH, the cocci were replaced by catalase-positive, gram-negative rods. In aging beef for Lebanon bologna manufacture, the level of NaCl used is critical. As the salt concentration is increased, the total aerobic count (APT), lactic acid bacteria count (RSL), and the gram-negative bacterial count (EMB) decreased (Fig. 2). The counts on MSA and PDA were less affected by increasing salt concentrations. In the absence of NaCl, catalase-positive, gram-negative short rods were the predominant flora on APT, and at 10 days the meat had the strong fruity odor that is associ- Yeast was present on PDA only with company A regular Lebanon, G sweet Lebanon, and I dry Italian salami. ' There was no growth on RSL at 102 dilution. ated with pseudomonads. A few catalase-positive, gram-positive rods were found at 1% salt, and at 2% there were about equal numbers of catalase-positive, gram-negative and catalasepositive, gram-positive organisms (APT). At 3 and 4% NaCl, very few gram-negative orga- Influence of time on the viable count of salted beef aging at 5 C. Beef chuck was ground through a 3/4-inch plate, and NaCI was added to the ground meat to make the concentration of salt to 3%o. One kilogram of beef cubes was placed into individual plastic bags, and the meat was allowed to age at 5 C. At intervals, a bag was removed from the cooler; 50 g was used to determine the viable count, and the remainder was used to make bolognas. The starting pH of the meat was 5.8 and did not change during the aging period. a Spices, sugar, and KNO3 were added to the salted beef cubes and the mixture was ground through a %4-inch plate. The mixture was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The sausages were then mellowed at 5 C for 4 days. At the end of the mellowing period, a 50-g sample was removed aseptically from the bologna, and bacterial determinations were made. The viable counts for the meat are given in Fig. 1. b Number of days meat was aged at 5 C with 3% NaCl before making bolognas. Effect of varying salt concentrations on the viable count of beef aged for 10 days. Beef chuck was ground through a 3/4-inch plate, and 0, 1, 2, 3, or 4% NaCI was added. Each lot of beef was placed into individual plastic bags, and the meat was allowed to age at 5 C for 10 days. At the end of the aging period, 50 g was used to determine the viable count, and the remainder was used to make bolognas. The starting pH of the meat was 5.6 and did not change during the aging period. nisms were present on APT. The microorganisms found on RSL were catalase-negative, gram-positive rods; catalase-positive, grampositive cocci were present on MSA, and yeasts were found on PDA. In Table 4, the viable counts of bolognas prepared from meat aged with varying concentrations of NaCl are presented. The counts on APT and RSL were similar, and the organisms found on both media were catalase-negative, gram-positive rods. Micrococci were not found in the bolognas; the organisms found on MSA were catalase-positive, gram-negative and gram-positive rods. Bolognas prepared from meat aged with low salt had a suitably low pH but they were defective in odor and taste. The bolognas made from meat aged with 4% salt did not reach a low pH because the numbers of the lactic acid bacteria were low (Fig. 2). Under normal conditions of the manufacture of Lebanon bologna, the fermentation occurs PALUMBO APPL. MICROBIOL. during the smoking process; however, a satisfactory bologna with low pH and good color can be obtained by incubation of the stuffed sausages in a constant temperature-constant humidity cabinet (35 C and 85% relative humidity [RH]). Some studies were done by utilizing the incubator rather than the smokehouse because of the restrictions associated with the use of the smokehouse. Although bolognas of similar pH, color, and texture were obtained by incubating in the smokehouse or incubator, the sequence of the microbial flora observed differed between the smoked and incubated bolognas. The effect of the presence or absence of smoke on the viable count is illustrated in Fig. 3. The counts on APT, RSL, and MSA decreased markedly during smoking at 35 C and during the subsequent mellowing period at 5 C (Fig. 3A). Both APT and RSL agars contained catalase-negative, gram-positive rods. MSA had colonies that consisted of catalase-positive, gram-positive cocci at the beginning of the fermentation, but by the second day of fermentation the number of micrococci decreased. The cocci were replaced on MSA by catalase-positive, gram-positive and gram-negative rods; by 10 days of mellowing, no micrococci could be detected (< 1 x 102/g). With nonsmoked bolognas (Fig. 3B), the MSA count decreased rapidly during the fermentation period, but the APT and RSL counts were not as severely reduced as under smoked conditions. APT and RSL contained catalase- a Spices, sugar, KNO., and salt (to make the concentration to 3% except for the 4%) were added to the beef cubes, and the mixture was ground through a 5/4-inch plate. The material was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The sausages were mellowed at 5 C for 1 day and then a 50-g sample from each bologna was removed aseptically for bacterial determinations. The viable count for the meat is shown in Fig. 2 Beef chuck was ground through a 3/4-inch plate, and NaCI was added to make the concentration of salt to 3%. The salted meat was packed into a wooden barrel and allowed to age at 5 C for 10 days. Spices, sugar, and KNO, were added to the meat, and the mixture was negative, gram-positive rods; MSA showed a rapid die-off of micrococci which were replaced by catalase-positive, gram-negative and grampositive rods (the microbial pattern on MSA was quite similar in both the smoked and nonsmoked bolognas). Experiments were performed to determine if Lebanon bologna could be prepared from frozen beef. Viable counts on thawed meat and the bolognas prepared from thawed meat are presented in Table 5. Thawed meat, before the addition of salt, contained catalase-positive, gram-negative rods on APT, yeast on PDA, and catalase-positive, gram-positive cocci on MSA. After 10 days of aging in the presence of salt, the viable count and the types of organisms found in the thawed, salted, aged meat appeared to be similar to that of unfrozen, aged, salted meat: 10 20 30 40 DAYS ground through a 524-inch plate. The material was stuffed into fibrous casings, and the sausages were fermented at 35 C at approximately 90% RH for four days either in an incubator or smoke house. At the end of the fermentation period, the bolognas were mellowed at 5 C. At intervals during the processing, 50-g samples of meat or bolognas were removed for determination of bacterial numbers and pH. catalase-positive, gram-positive rods were found on APT, yeast was found on PDA, catalase-negative, gram-positive rods were found on RSL, and catalase-positive, gram-positive cocci were found on MSA. EMB plates had catalasepositive, gram-negative rods which were not typical coliforms. The bolognas made from the thawed beef had a low pH of 4.6 with good color and texture. The organisms found on APT and RSL were catalase-negative, gram-positive rods; catalase-positive, gram-positive and gram-negative rods were found on EMB; MSA had catalase-positive, gram-positive rods and cocci; and no yeast was present at 102 dilution on PDA. Unaged beef or beef aged at 5 C (in the presence of 3% NaCl) for short periods of time did not give Lebanon bologna with a normal low (Table 3). The fermentation period was lengthened to determine whether bolognas made from fresh meat could attain the desired low pH. The data presented in Table 6 indicate that bolognas made from fresh meat did not reach a normal product pH after 12 days of incubation at 35 C and 80% RH. The microbiology also was different in some respects from the properly fermented product. At days 0, 1, and 2, catalase-positive, gram-positive cocci were the predominant organism on all plates that showed growth except for RSL, which had gum-producing catalase-negative, gram-positive rods. From day 3 on, APT and RSL had catalase-negative, gram-positive rods (gum producing on RSL); catalase-positive, gram-positive cocci were present on EMB and MSA. A portion of the same beef chuck that was used for the above experiment was ground through a 3/4-inch plate, salted (final concentration of NaCl was 3%), and then aged at 5 C for 12 days. The aged meat was made into bolognas in the usual way and incubated at 35 C and 80% RH. The pH of the meat going into the fermentation was 5.3; after 1 day, the pH was 5.3, at 2 days, the pH was 4.7, and at 3 days, the pH was 4.6. Thus, Lebanon bologna could be made from aged beef but not from fresh beef. However, if Lactacel MC (Merck & Co., Rahway, N.J.) starter culture were used with fresh beef and bolognas prepared in the usual manner, a fermented sausage with a pH of approximately 4.5 was produced within 24 h. DISCUSSION Semidry and dry sausages are meat products that have been fermented by lactic acid bacteria (2, 10). Our work (Table 1) and the work of others (2; L. B. Jensen and L. S. Paddock, U.S. Patent 2,225,783,1921) show that lactic acid bacteria are isolated in large numbers from Lebanon bologna. In the manufacture of Lebanon bologna, cubed beef plus salt is allowed to age at refrigerated temperatures for several days. Pseudomonads and lactic acid bacteria have been shown to be the predominant flora of ground beef during refrigerated storage in the absence of salt (4). At low concentrations, NaCl prevents the growth of pseudomonads (12) but permits the growth of the more salt-tolerant lactic acid bacteria (11). The role of salt as an inhibitor of bacterial growth has been reviewed by Ingram and Kitchell (6). Therefore, the primary reason for salting the cubed beef during the aging Fresh beef chuck was ground through a 3/4-inch plate, NaCl, spices, sugar, KNO, were added, and the mixture was ground through a %4-inch plate. The sausage mix was stuffed into casings 55 mm in diameter, and the bolognas were incubated at 35 C and 80% RH. At each time interval, a bologna was removed and bacterial numbers were determined by utilizing a 50-g sample. " No growth occurred at 10' dilution on PDA. c At 12 days, the pH of the bolognas was still 5.6. a Beef chuck was ground through a 3/4-inch plate, placed in plastic bags, and frozen for approximately 4 months. The meat was thawed for approximately 24 h at 5 C and a 50 g sample was removed to determine bacterial numbers. NaCl was added to make the concentration to 3% salt and a kilogram of salted meat was placed into individual plastic bags and was allowed to age at 5 C. b Viable counts were determined after 10 days of aging at 5 C. c Spices, sugar, and KNO, were added to the salted beef, and the mixture was ground through a W-inch plate. The mixture was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The bolognas were mellowed for 4 days at 5 C; bacterial numbers were determined on a 50-g sample. During the aging or holding period used in these studies, microbial populations changed considerably. Deibel et al. (2), in the manufacture of summer sausage, found little or no change in the microbial flora during a short, 2to 4-day aging period. We found that such a short holding period was not sufficient to permit development of the lactic acid-producing flora. During the long aging period, at least 10 days in our studies, the lactic acid bacteria increased to about 104/g, and this concentration appears to be critical for adequate decrease in pH in the bolognas ( Fig. 1; Table 3). Therefore, a long aging period is necessary to allow the development of sufficient numbers of lactic acid bacteria. Another important function of aging is to allow the micrococci to develop. The micrococci reduce nitrate to nitrite and thus give bolognas a good cured meat color (F. W. Kurk, U.S. Patent 1,380,068, 1921; reference 10). The concentration of micrococci does not seem to be as critical as the concentration of lactic acid bacteria; good color was generally found in all bolognas regardless of the micrococcal count. Micrococci are more halotolerant than lactic acid bacteria and pseudomonads (12). With too much salt, the increase in the lactic acid bacterial population was very slow and the numbers at the end of the aging period were not high enough to produce bolognas of desirable low pH (data for 4% salt in Fig. 2 and Table 4). When meat was aged with low concentrations of salt (1-2%), pH decreased satisfactorily during processing but the excessive development of pseudomonads in the meat gave the bologna inferior flavor. Salt at the 3% level appears to be a good compromise between the concentration that inhibits the pseudomonads and one that is not too inhibitory to the lactic acid bacteria. Our results indicated that smoking is inhibitory to the lactic acid bacteria because the viable count of the lactic flora decreased drastically on both APT and RSL (compare A and B, Fig. 3). Apparently some component of smoke and not acid caused the killing because the pH decrease was similar under smoked and nonsmoked conditions. Handford and Gibbs (5), using liquid smoke, found that certain lactic acid bacteria and micrococci were inhibited by smoke constituents. The MSA count rapidly decreased during fermentation and mellowing under both smoke and nonsmoke conditions (compare A and B, Fig. 3). The loss of micrococci probably was due to the acid content of the sausage rather than to smoke. Also, the organisms might be sensitive to the nitrite formed by their reduction of nitrate. By day 20, few if any micrococci were found (Fig. 3); they apparently were superseded by the outgrowth of rod forms. The counts on PDA and EMB also decreased during fermentation and mellowing regardless of smoking or nonsmoking. The yeast and gram-negative bacteria are probably sensitive to the acid environment of the sausages. Sausage makers generally believe that the use of frozen meat does not produce a high-quality product (7). However, our experiments indicate that frozen beef chuck, when thawed, salted, and aged, yields a satisfactory Lebanon bologna. Prolonged incubation under fermentation conditions did not give a bologna of low pH when fresh rather than aged meat was used (Table 6). However, a portion of the same batch of meat that had been salted and aged at 5 C for 12 days did yield bolognas that reached a low pH in 2 to 3 days. One possible explanation for the failure of the fresh meat bolognas to ferment is that the lactic acid bacteria count never reached a critical level to initiate sufficient acid production. Aging at low temperatures may allow the selection and growth of salt-tolerant organisms that are active acid producers. In a typical Lebanon bologna process, the micrococci die off quickly once the fermentation leads to acid conditions, but in the bolognas made from fresh beef, the micrococcal count remained quite high (Table 6). When the number of lactic acid bacteria going into the fermentation is low, the continued high density of micrococci might interfere with lactobacilli proliferation by competing for nutrients. Apparently, whatever the reasons, Lebanon bologna of consistently good quality cannot be made from unaged meat. However, fresh meat can be used for Lebanon bologna manufacture if a suitable starter culture is used.

v3-fos

2018-04-03T00:48:17.934Z

1973-01-01T00:00:00.000Z

26866008

s2

Effects of Homologous Bacteriophage on Growth of Pseudomonas fragi WY in Milk' Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml. Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml. (2) isolated two strains of bacteriophages and their bacterial hosts from raw skim milk. They isolated 36 other strains from additional refrigerated foods. This created speculation that the growth of bacteria within refrigerated foods might be slowed by homologous bacteriophage, thus extending shelf life of the food. The present report describes conditions which had to exist if a homologous bacteriophage of Pseudomonas fragi WY was to significantly affect growth of the host bacterium in refrigerated milk. MATERIALS AND METHODS Bacteriophage and host. P. fragi WY and its homologous bacteriophage, wy, were isolated from ground beef, and their characteristics have been reported (3). A suspension (160 ml) of bacteriophage particles containing 2 x 1012 plaque-forming units (PFU) per ml was prepared by propagation on 25 large petri plates (150 mm) by the double-layer method (1). The semisolid layer of each confluently lysed plate was macerated in 6 ml of tryptic soy broth (TSB) and combined in a beaker. After 5 hr of incubation at 4 C to complete lysis, the slurry was centrifuged for 15 min at 10,000 x g, and the supernatant fluid was sterilized by consecutive filtration through membranes with pore sizes of 1. Anti-phage serum. Four adult rabbits were subcutaneously inoculated twice weekly for 3 weeks with 5 ml of the high-titer phage suspension. One week after the last injection, rabbits were bled by cardiac puncture. The blood was allowed to coagulate at 37 C in vaseline-lined centrifuge tubes before overnight storage at 4 C. After centrifuging for 10 min at 5,000 x g, the serum was drawn off, filtered through a membrane filter (0.45 Am diameter pore size), stored at 4 C, and assayed for anti-phage activity. After assay, the sera were pooled and stored frozen. Assay of anti-phage activity. Serum was diluted 1:100 and 1: 1,000 in TSB, and high-titer phage stock was diluted to 107 PFU/ml. Phage suspension (0.1 ml) was added to 0.9 ml of each dilution of antiserum at room temperature. At 5-min intervals, 0.1-ml samples of the phage-serum mixture were added to 9.9 ml of TSB to stop the antibody reaction, and 0.1-ml samples of this dilution were plated by the agar layer method (1). If phages were not inactivated, about 1,000 plaques appeared after incubation; 90% inactivation resulted in about 100 plaques, and 99% inactivation (the desired level) resulted in about 10 plaques. Effect of phage on its host in skim milk. Skim milk, obtained from the University of Missouri Dairy, was sterilized by heating at 121 C for 15 min. The milk was divided into 400-ml lots and then inoculated with P. fragi WY in two concentrations: 1,000 and 100,000 cells/ml. (Previous observation showed that a 24-hr TSB culture contained approximately 2 x 108 cells/ml; this figure was used as a basis for dilutions prior to inoculation.) The host cells were mixed thoroughly with the milk by stirring 5 min with a Teflon spinbar driven by a magnetic stirrer. Each 400-ml lot of milk containing either 10' or 10' host cells per ml was subdivided into four 100-ml samples; these samples were inoculated with 0, 0.1, 10, or 1,000 PFU/ml. These ratios of phage to host were consid-P. FRAGI WY ered to be those which might occur in milk normally. Controls consisting of milk containing no phage particles or bacteria and one containing 100 phages per ml but no bacteria were also incubated. All samples were incubated at 7 C for 72 hr (a time and temperature at which grade A milk might be held in practice). Samples were then plated in triplicate to determine bacteria and phage counts. Plates were incubated at 21 C; phage counts were made after 15 hr, and bacteria were counted after 48 hr. Antiserum was added at a 1: 1,000 concentration to the first dilution bottle used in making bacterial counts to prevent additional infections of bacteria. These experiments were replicated three times. RESULTS AND DISCUSSION Anti-phage serum was used to preclude lysis of host cells during plating of samples to determine bacterial counts. Our preliminary experiments had suggested this as a possibility. Our serum inactivated 99% of the bacteriophages at room temperature in 5 min when diluted 1: 100 and in 20 min when diluted 1: 1,000. No bacteria were found in controls with only added phage, and no phage were found in controls with only added bacteria. When the ititial concentration of P. fragi WY was 100,000/ml, addition of 10 or 1,000 PFU/ml of homologous phages caused significantly lower bacterial counts after 72 hr at 7 C (Fig. 1). The count appeared to be lower when only 0.1 PFU/ml was added, but the difference was not statistically significant. Bacteriophage titers were significantly different at the end of incubation, with the lowest and highest counts corresponding to the lowest and highest inputs of phage, respectively. Effects of added phages were much less pronounced when the initial bacterial concentration was 1,000/ml (Fig. 2). In fact, the bacterial count of the control was lower (insignificantly) than the average counts of each sample to which phage was added. Numbers of phages increased markedly in samples to which 10 and 1,000 PFU/ml were added. There was no detectable multiplication of phage in the samples to which only 0.1 PFU/ml was added. These results indicate that bacteriophage can have an influence on shelf life of refrigerated milk, but the conditions necessary for significant effect are improbable. These conditions are (i) a relatively high population of the host bacterium, (ii) presence of its homologous

v3-fos

2018-04-03T00:58:31.527Z

1973-10-01T00:00:00.000Z

19502775

s2

Bacillus sp. ATCC 27380: a Spore with Extreme Resistance to Dry Heat An unusual mesophilic Bacillus sp. was isolated from heated soil, and a cleaned spore preparation showed extraordinary resistance to dry heat (D,2", = 139 h) and relative sensitivity to moist heat (D.0 c = 61 min). Biochemical tests and morphology fit no described species. For the past several years, we have been involved in the study of dry-heat inactivation kinetics of bacterial spores as related to terminal sterilization of spacecraft destined to detect extraterrestrial life. These investigations have included spores in their naturally occurring environments (soils and dusts) as well as environmental isolates cultured with various types of media (2,3). Prior to 1960, relatively little was known about inactivation by dry heat at temperatures below 160 C, and workers initially involved in the problem of sterilizing a spacecraft at approximately 125 C began using spores of Bacillus subtilis var. niger to study changes in dry-heat resistance levels due to pressure, gases, moisture, and combinations of these and other physical parameters. The spores of this organism, now commonly used as a biological indicator for dry-heat cycles, came to be considered as a representative of "average" resistance (4). In earlier studies of naturally occurring spore populations in 25 soil samples collected in various parts of the United States, we observed D125 c values (length of time necessary to effect 90% kill at 125 C) ranging from 16 to 126 min (2). Pure Portions (0.1 ml) of the suspension were applied to stainless steel strips (0.5 inch by 0.5 inch), and the dry heat resistance level was determined by a method reported earlier (2) using Trypticase soy agar (TSA; BBL) supplemented with soluble starch and yeast extract (3) as the recovery medium. Figure 1 shows the survivor curve when four strips were heated at each interval and the D125C value of 24 h was calculated from a best-fit regression line of the mean data points (ignoring N., the unheated controls) by using a least squares method. During an end-point determination with this naturally occurring spore population, in which survivors at the 48-h interval were recovered in broth (3), an unusual, slow-growing sporeformer was isolated after 1 month of incubation at 32 C. Positive broth tubes were recognized only when vigorously agitated, thereby showing a clear, compact, mucoid sediment with no turbidity in the supernatant broth. When subcultured on supplemented TSA at 32 C, growth and sporulation were also slow, requiring 10 ical to slightly oval, central to subterminal spores having rough, stainable walls. After leaving the swollen sporangium, the spores appeared to increase in size from 1.2 gm to approximately 1.7 gm while assuming a more oval shape. Vegetative cells became pleomorphic in older cultures, and the highly sculptured spore surfaces were discernible using light microscopy; the less dense forms showing surface texture were most likely spore wall debris left after germination. Figure 2 is a scanning electron photomicrograph of a single spore showing the surface morphology in detail with suggestion of exosporium remnants. The honeycomb pattern of polygonal depressions surrounded by straight ridges is similar to that seen in electron photomicrographs of "B. megaterium 350" described by Robinow (10) and the freeze-etched preparations of B. polymyxa and B. fastidiosus shown by Holt and Leadbetter (6). Attempts at identification of this isolate by standard biochemical methods (12; R. E. Gordon, W. C. Haynes and C. H-N. Pang, The Genus Bacillus, U.S.D.A., Agricultural Handbook no. 427, in press) have shown that it fits no described pattern. Positive tests were obtained with (i) growth in 2, 4, and 10% NaCl broth, and (ii) production of catalase. Negative tests were (i) motility, (ii) utilization of citrate, (iii) hydrolysis of starch, gelatin, or casein, (iv) production of acetylmethylcarbinol or indole, (v) reduction of nitrate or methylene blue, (vi) growth at 45 C (growth was poor at 37 C), and (vii) utilization of carbohydrates (arabinose, glucose, lactose, sorbitol, rhamnose, mannitol, or xylose). Growth on supplemented TSA in a Brewer Anaerobic Jar (BBL) was negative in 14 days at 32 C. The Bacillus sp. isolate was submitted to the American Type Culture Collection and was given an accession number of 27380. An actively growing culture was streaked on thickly poured 100-by 15-mm plates of AK #2 sporulation agar (Difco) supplemented with 20 ,gg of magnesium sulfate per ml and 80 Aig of calcium chloride per ml. After incubation for 20 days at 32 C, growth was harvested and cleaned by a method described previously (2), and spores were resuspended in phosphate-buffered distilled water (BDW; reference 1) held at 4 C. Survival of the spores when exposed to dry heat at 125 C was determined by the stainless steel strip assay method (3) modified by insonating for 60 s with a Biosonic III Ultrasonic Probe (2) at 60% maximum intensity prior to plating the rinse solution from each strip. The moist heat D value was determined by placing inoculated stainless steel strips in tubes containing 10 ml of BDW and heating the tubes at 80 C for appropriate intervals in an oil bath (Blue M Electric Co., Blue Island, Ill., Mod. MW1115A). Assay subsequent to cooling of the tubes was identical to the dry-heat determination. All heating and assay manipulations were conducted in a vertical laminar flow clean room (3). Plates were incubated at 32 C and counted at 2-day intervals for 2 weeks, and Fig. 3 shows the results when five test units were heated at each interval. The Survival of spores of Bacillus sp. ATCC 27380 exposed to 80 C moist heat or 125 C dry heat. concave downward (8) shapes of both survivor curves indicate that Bacillus sp. ATCC 27380 spores require either dryor moist-heat activation (5); it is emphasized that the maximum level of germination in the dry-heat system was reached only after 24 h at 125 C. The gentle slope of the dry-heat curve (D = 139 h) indicates that 125 C is barely the threshold temperature of lethality. The extreme magnitude of the dry-heat resistance (5, 7-9, 11) and also the difference in resistance levels between dry and moist heat may make this organism a valuable tool in the elucidation of spore germination mechanisms, the biophysical nature of spores, mechanisms of heat inactivation, and possibly as a more stringent biological indicator for dry-heat sterilization cycles. Services were provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration under Contract W-13,062. We are indebted to Ruth Gordon of Rutgers University for her helpful comments and assistance with identification schemes for Bacillus spp. Sincere appreciation is also expressed to H. Orin Halvorson and to personnel of the Applied Science Division (Div. 5252), Sandia Laboratories, Albuquerque, New Mexico, for their cooperation.

v3-fos

2020-12-10T09:04:12.908Z

1973-10-01T00:00:00.000Z

237234519

s2

Use of Coating to Protect Lyophilized Bacillus popilliae from Moisture Lyophilized cells of Bacillus popilliae were protected from moisture when suspended in pellets of tung oil polymer which were then coated with paraffin wax. The survival of the protected cells at various levels of relative humidity (RH) and under various storage conditions was determined. During 6 months of storage, moisture appeared to have little effect on survival of the cells when the RH level was 22% or less; but, at higher RH levels, survival declined upon storage. Viable cells were recovered when pellets were stored for 3 months at 33% RH, 2 months at 42% RH, 1 month at 50% RH, and 4 days in distilled water. Under field conditions, some cells survived at least 1 week of storage. Spores of Bacillus popilliae Dutky have been used successfully in biological control of the Japanese beetle, Popilliajaponica Newman (2). However, attempts to develop a method for the economical production of infective spores in vitro have had only limited success, and interest has developed in the possibility of using stabilized vegetative cells for control of the Japanese beetle. Vegetative cells of B. popilliae have been preserved by lyophilization (1,3,5). The lyophilized cells maintain their morphological and cultural characteristics and ability to initiate disease in susceptible hosts (3). In a previous report (4), it was shown that lyophilized cells of B. popilliae survived for at least 1 year in dry soil, but viability was lost in less than 1 month when the relative humidity above the soil was 42% or higher. For lyophilized cells to be useful for control of the Japanese beetle, they must be protected from the destructive effects of moisture. In this study (portion of a thesis presented by the senior author in partial fulfillment of the requirements for the M.S. degree in bacteriology at North Dakota State University), lyophilized cells of B. popilliae were incorporated into pellets of tung oil polymer and coated with paraffin wax to protect the cells from moisture. MATERIALS AND METHODS Organism and medium. The strain of B. popilliae used and the medium for growth have been described 1 Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article no. 413. (4). Actively growing cultures were maintained by daily transfer of a 5% inoculum of brothcultures. Flasks were shaken on a rotary shaker at 200 rpm. All incubations were at 25 to 28 C. Lyophilization. After 16 h of growth, cells were harvested from the liquid medium by centrifugation and resuspended to a concentration of 4.4 x 109 cells per ml in a solution of 5.0% monosodium glutamate plus 0.5% gum tragacanth. Samples (0.5 ml) of the suspended cells were placed in 10-ml screw-cap vials fitted with split rubber stoppers (VirTis Co., Gardiner, N.Y.). Cells were frozen by rotating the vials for 1 min in methanol maintained at -60 to -70 C. Upon removal from the freezing bath, vials were attached to a VirTis freeze dryer (model no. 10-147 MR-BA), and a vacuum of 5 to 10 um of Hg was maintained throughout a 3-to 5-h drying period. The vials were sealed uider vacuum by forcing the rubber stoppers into place. Vials not immediately opened were sealed with plastic sealing bands and stored at room temperature. Coating of lyophilized cells. To prepare the tung oil polymer, tri-n-octylaluminum (Texas Alkyls, Inc., Houston) was used as a drier to polymerize raw tung oil. The drier was handled under nitrogen and injected beneath the surface of the oil. The polymer prepared was an 80:20 (wt/wt) tung oil and drier mixture. Samples (3 ml) of the mixture were placed into capped vials to prevent complete polymerization of the oil prior to its use. Recently lyophilized cells having a viable count of 2.4 x 10' cells per ml were used. The contents of two vials were ground to a fine powder and added to 3 ml of the unpolymerized oil-drier mixture. After thorough mixing, drops of the oil were placed on talc powder to form spherical pellets 2 to 3 mm in diameter. To allow complete polymerization, pellets were stored for 3 days at room temperature in a desiccator containing CaSO.. The polymerized pellets were then dipped individually into paraffin wax PROTECTION OF LYOPHILIZED B. POPILLLAE maintained at 55 to 60 C in a water bath, removed immediately by means of a small wire loop, and placed in a sterile petri dish for several minutes to allow the coating to harden. Storage conditions. The various relative humidity (RH) levels were maintained with saturated salt solutions as described by Robinson and Stokes (7). Dry air (0% RH) was maintained in a desiccator containing CaSO.. For exposure to the various moisture levels, the wax-coated pellets were placed in 50 g of sterile soil contained in pint jars with loosened screw-cap lids and placed in desiccators containing the saturated salt solutions. Pellets stored in water were placed in screw-cap dilution bottles containing 100 ml of sterile, distilled water and stored at room temperature. For exposure to field conditions, the pellets were buried approximately 2 inches (5.08 cm) below the soil surface of an outdoor garden. The climatological statistics for the period of storage under field conditions during the month of June were as follows. During the month, the average temperature was 67.8 F, with a range of 38 to 91 F; the average RH was 59.9% and ranged from 43 to 97%; the total precipitation amounted to 0.99 inches (2.51 cm). Viable counts of protected cells. Control and experimental counts were obtained by grinding the pellets individually in 10 ml of 0.1% tryptone solution in a Sorvall Omni-Mixer (Ivan Sorvall, Inc., Norwalk, Conn.), speed setting five, for 1 min. Serial dilutions were prepared in 0.1% tryptone, and appropriate dilutions were plated in replicate on five standard medium plates. Pellets removed from garden soil were rinsed for 2 min in a 0.1% solution of Roccal (Winthrop Lab., N.Y.) to remove surface contaminants prior to determination of viable cell counts. Plates were incubated for 4 days at 25 to 28 C. Three pellets were used to determine each viable count, and recorded counts are log averages of the replicate counts. RESULTS Incorporation of lyophilized cells of B. popilliae into tung oil polymer pellets coated with paraffin wax increased the survival of the cells upon storage in the presence of moisture. Fig. 1 shows the survival of lyophilized cells contained in wax-coated pellets that were stored at various moisture levels. Immediately after application of the wax coating, three pellets gave an average count of 1.2 x 106 viable cells per pellet. After 6 months of storage at 0, 11, and 22% RH, survival ranged between 0.3 and 1.3% of the original cells per pellet. At the higher RH values, viability of the protected cells decreased more rapidly. Some cells survived for 3 months at 33% RH, 2 months at 42% RH, and 1 month at 50% RH. Pellets stored in distilled water showed an average cell survival of 11.5% after 4 days but no survival after 2 weeks. Under field conditions, 25.7% of the cells survived 1 week, but no cells were recovered after 4 weeks. DISCUSSION The results indicate that the survival of ayophilized B. popilliae in the presence of moisture was increased when the cells were incorporated into pellets of tung oil and coated with paraffin wax. Lingg and McMahon (4) found that lyophilized cells of B. popilliae could not survive in soil for even 1 month when the RH above the soil was 42% or higher. Although some protection was given to the cells, the moisture-proofing ability of the paraffin wax coating was limited. Paraffin wax is a moistureproof coating only when maintained as a continuous film. The thin layer of wax that was applied to the pellets was brittle and easily cracked. Once the coating had cracked, moisture was able to enter the pellets and destroy the cells. It may be possible to increase the durability of the wax coating by incorporating certain modifiers into the paraffin wax. Recent experiments indicate that the addition of a small percentage of rubber may cause a significant increase in the protective ability of the coating and thereby improve cell survival in the pellets. Cells incorporated into tung oil pellets coated with a 97:3 (wt/wt) mixture of paraffin and rubber cement have remained viable for at least 10 weeks when stored in distilled water. The tung oil polymer proved to be a useful primary coating for the cells. Tung oil, or Chinawood oil, is a natural drying vegetable oil that absorbs oxygen from the air and forms a solid, water-resistant polymer. The drier, tri-noctylaluminum, increased the polymerization rate of the oil and enabled the formation of uniform pellets from drops of the quickly polymerizing oil. The results show that lyophilized cells can be coated to protect them from adverse conditions. If the wax coating can be improved, the use of vegetative cells of B. popilliae for control of the Japanese beetle may be possible. The coated cells must be infective upon ingestion by beetle larvae. The coatings used, a natural plant product and a wax, have potential as substances that larvae will be able to ingest. If some of the lyophilized cells are released into the gut of the larvae, by mechanical disruption or digestion of the pellet, infection may occur. When injected, vegetative cells produce infection at much lower dosages than spores. According to St. Julian et al. (8), the injection of 300 to 1,000 viable vegetative cells into the hemolymph of larvae causes 50 to 80% of the larvae to become grossly infected. By comparison, 104 to 106 spores must be injected to gain a comparable percentage of infection. These workers believe that a relatively large number of spores, compared to vegetative cells, are needed for optimum infectivity because only a small number of spores germinate in the larvae hemolymph. Studies to determine infectivity of pelleted cells are planned and will be the subject of a further report. Although the purpose of this study was to develop a method of preserving vegetative cells for use in biological control, results obtained suggest other uses for such stabilized cells. Cells lyophilized by the procedure used in this study and stored in vacuo at room temperature for 6 months showed a survival rate of 2.8% of the original cells (J. A. Cloran, M.S. thesis, North Dakota State Univ., Fargo, 1973). When cells were incorporated into tung oil pellets coated with paraffin wax and stored at 0% RH for 6 months, 1.3% of the cells per pellet retained viability. These results suggest that the technique may be useful for preserving stock cultures. A culture collection of pelleted, lyophilized cells would be compact and easily stored, handled, and maintained. One small vial could contain enough pellets to start over 100 cultures. If the surface of a pellet became contaminated during handling or storage, it could be disinfected by rinsing the pellet in a common disinfectant prior to crushing of the pellet for initiation of a new culture. This method of preserving stock cultures is somewhat similar to the technique described by Nagel and Kunz (6). Their method involves the coating of glass beads with a mixture of broth culture and horse blood. Beads are then stored in a freezer at -70 C. Pelleted, lyophilized cells may be stored in the refrigerator or at room temperature.

v3-fos

2020-12-10T09:04:16.713Z

1973-12-01T00:00:00.000Z

237229763

s2

Microbiological Profiles of Four Apollo Spacecraft Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (104 per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch2. A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft. Microbiological profiles of automated and manned spacecraft are being determined on a continuous basis due to national and international agreements which stipulate that microorganisms which have a potential of being transported in a viable state to the surface of the moon be enumerated and identified, and an inventory of the levels of contamination at each landing site be maintained (13). In addition, because the existence of life on other planets is possible, scientific investigations for determining the possibility of extraterrestrial life forms must not be jeopardized. Contamination of Mars and other planets of biological interest with terrestrial microorganisms will be controlled to the extent that the probability of contamination will be 1 in 1,000 (10-3) (5,10,11,15). Dry-heat sterilization of the spacecraft will be employed to achieve these objectives. The assessment of microbial contamination levels, especially bacterial spores on space hardware, will be one of the essential controlling parameters in the sterilization of interplanetary spacecraft because the number of bacterial spores present on the spacecraft, as determined by microbiological assays, will determine the extent and character of the dry-heat sterilization cycle (2,3,10,11). The objective of this study was to determine and compare the levels and types of microorganisms on various components of four Apollo spacecraft. MATERIALS AND METHODS Microbiological assays were conducted on Apollo spacecraft during assembly and testing, and sampling locations were selected on the interior and exterior surfaces of various spacecraft components. A prerequisite for sites was that they be representative surfaces of the entire spacecraft and be accessible throughout the sampling periods. The Command Module (CM), Lunar Module ascent stage, Instrument Unit (IU), Saturn *S-4B stage (S-4B), and Spacecraft Lunar Module Adapter (SLA) were interior surfaces studied. Exterior surfaces included the ascent and descent stages of the Lunar Module, and, for Apollo 15, the first Lunar Roving Vehicle (LRV). The various spacecraft components were studied at three periods during assembly and testing. The CM was sampled at 14 days, 7 days, and 24 h, and other spacecraft components were sampled at 14 days, 7 days, and 57 h, respectively, before launch. At each interval, 15 locations on each spacecraft component were sampled. Sterile cotton swabs, moistened in sterile distilled water, were rubbed over the surfaces to be sampled which were outlined with a sterile paper or aluminum template (4 square inches). Surface areas smaller 8:38 than 4 square inches were determined by direct solution of Tween 80 (polyoxyethylene sorbitan monomeasurement. Five swabs were returned to a sterile oleate, Hilltop Research, Inc., Miamiville, Ohio). The screw-cap test tube (25 by 150 mm) containing 25 ml swab heads were broken off below the portion of the of sterile buffered rinse solution with 0.02% (vol/vol) handles touched by the sampler. Tubes were taken immediately to the laboratory, agitated on a Vortex mixer for 5 to 10 s, placed in an ultrasonic bath (tank LTH60-3; generator, A-300; Branson Instruments, Inc., Stamford, Conn.) containing a 0.3% (vol/vol) Tween 80, and insonated for 2 min at 25 kHz (14,18,19). Randomly, sterile swabs were moistened in sterile distilled water and then returned to sterile screw-cap tubes containing sterile buffered rinse solution with 0.02% (vol/vol) solution of Tween 80. These swabs were then assayed as described above and served as controls. After insonation, replicate portions from each tube were plated with Trypticase soy agar (TSA; BBL). For Apollo 12, portions also were spread over the surface of blood agar (TSA plus 5% defibrinated sheep blood), MacConkey agar (BBL), and Mycophil agar (BBL). Spore assays were performed by heat shocking the remaining rinse fluid in each tube at 80 C for 15 min and plating with TSA. Brewer jars for anaerobic incubation were flushed three times with a gas mixture of nitrogen (80%), carbon dioxide (10%), and hydrogen (10%), filled a fourth time with the gas mixture, and connected to an electrical source for 45 min for catalytic removal of oxygen. The CM interior surfaces of Apollo 15 were sampled at approximately 9 h (preflight) prior to launch and also after the mission (postflight) when the CM was taken on board the recovery vessel. The sampling procedures were similar to the above, except that each cotton swab was placed into 10 ml of sterile veal infusion broth. The postflight samples were kept at 4 C, transported to the Planetary Quarantine Laboratory at Cape Kennedy, Fla., and assayed within 30 h after being taken. In addition to plating on TSA, portions were spread over the surfaces of blood agar and blood agar enriched with vitamin K and hemin. All media except TSA were incubated at 37 C under aerobic, anaerobic, and CO2 conditions. The TSA culture plates were incubated at 32 C under aerobic conditions. All laboratory procedures were performed in a horizontal laminar flow clean bench (7) to eliminate background contamination. Other details of the sampling procedure have been described previously (14). Plates were incubated at 32 C and colony counts were performed after 48 and 72 h. For each Apollo mission, 1,000 to 2,000 colonies were picked randomly from culture plates, gram stained, and identified. All isolates were subsequently lyophilized and stored for future reference. Micrococcaceae were classified by the scheme of Baird-Parker (1), aerobic sporeformers (Bacillus spp.) were classified by the method of Smith et al. (24), Enterobacteriaceae were classified by the schemes of Edwards and Ewing (4), and the Pseudomonas-Achromobacter-Flavobacterium group and related gram-negative bacteria were classified by the method described by Shewan et al. (23). Bergey's Manual (7th ed.) was used for classifying other groups of bacteria. RESULTS AND DISCUSSION Comparison of the levels of microbial contamination detected on the four Apollo spacecraft is shown in Table 1. Aerobic mesophilic Unidentified ascomycete 1 microorganisms per square foot of surface for each of the component parts were relatively consistent for the four Apollo spacecraft. Although the levels of total microorganisms were similar for all CM, IU, and S-4B, the concentrations of bacterial spores and molds on the latter two components were higher than on the CM, which was consistent with what was found on previous Apollo spacecraft (21). The highest percentage of bacterial spores was detected on the surfaces of the SLA, although this component had the lowest number of microorganisms. The constant flushing of the SLA with high volumes of filtered air might have reduced the vegetative microbial population due to physical removal or desiccation, resulting in a relatively A total of 5,862 microbial colonies were picked and identified from the four Apollo spacecraft. Table 2 shows the types of aerobic mesophilic microorganisms isolated from each of the Apollo spacecraft by using TSA. The distribution by types of microorganisms on the four Apollo spacecraft was remarkably similar. Vegetative microorganisms of human origin such as Staphylococcus spp., Micrococcus spp., and the Corynebacterium-Brevibacterium group accounted for the vast majority of microbial contamination detected. This pattern is consistent with previous Apollo spacecraft (17,20,21,22). The percentage of these microbial types (i.e., indigenous to humans) as detected on the various components of the Apollo spacecraft is shown in Table 3. The highest percentages were found on the interior surfaces of the Command and Lunar Modules. The levels of microorganisms associated with soil and dust (bacterial sporeformers, molds, and actinomycetes) have increased with each Apollo spacecraft. Normally, these types of microorganisms reflect the degree of environmental and personnel controls employed, and when environmental controls are relaxed there is a marked increase in the types of microorganisms originating from soil and dust. Nineteen genera of molds were isolated from Apollo spacecraft (Table 4), with Aspergillus, Bipolaris, Curvularia, and Penicillium being the predominant. The elimination of the 21-day back-contamination quarantine period of the Apollo 15 mission made possible the first opportunity to take postflight microbiological samples on the interior surfaces of the CM. In addition to the 14-day, 7-day, and 24-h sampling of the CM, a 9-h (preflight) sample was taken by a member of the astronaut back-up crew. The postflight samplin'g was conducted by the flight surgeon on board the recovery vessel. Samples were taken from the same locations as for preflight, and a comparison of the quantitative results is shown in Table 5. The levels of microorganisms increased in some areas by 3 logs per square inch. A total of 1,682 microorganisms were isolated and identified from the Apollo 15 spacecraft. Of these isolates, 183 and 327 were obtained from the interior surfaces of the CM at preflight and postflight sampling periods, respectively. Tables 6 and 7 list the types of microorganisms detected in the CM from pre-and postflight samples employing various media and incubation methods. These microorganisms were isolated from TSA, blood agar, and enriched blood agar. All media except TSA were incubated at 37 C under aerobic, anaerobic, and CO2 conditions as requested by the Manned Space Center (MSC). Three types of microorganisms (Streptococcus-Viridans group, Peptostreptococcus spp., and Lactobacillus spp.) were detected only on postflight samples. For the identification data to be meaningful to MSC, colonies resulting from postflight samples were selected by the MSC protocol, i.e., every different colonial type on every culture plate was picked. The standard method used in the Planetary Quarantine Laboratory employs a template with randomly selected points for picking colonies only from aerobic TSA plates. With the exception of the three types of microorganisms detected from postflight sample plates incubated under special conditions (CO2 or anaerobic), all other types were detected on aerobic TSA plates. Nine types of microorganisms were detected on TSA which were not detected on the other media. No gram-negative microorganisms were isolated from preor postflight samples. This was not surprising since this group of bacteria is very sensitive to drying and normally is not found on spacecraft surfaces. Analysis of the types of microorganisms isolated from the various sites on the surfaces of the CM of Apollo 15 from pre-and postflight samples revealed that no apparent recovery pattern existed. Any microorganism which was isolated was equally likely to be found on any given site. It is evident from the results obtained that the levels and types of microorganisms on surfaces of Apollo spacecraft remain relatively constant among spacecraft (20-22) but greater than some of the automated (6,9,16) spacecraft. This was to be expected since Apollo spacecraft are assembled and tested in environmental areas (20) which do not have the same degrees of environmental and personnel controls exerted in those areas used for automated spacecraft (25). If the total number of microorganisms was used as a criterion for evaluating spacecraft microbial cleanliness, the Apollo 14 spacecraft was clearly the most contaminated Apollo spacecraft flown to date. However, if numbers of microorganisms indigenous to soil were used as the standard, the Apollo 15 would be the most contaminated. It has been shown that man is the chief source of microbial contamination to which spacecraft are exposed when assembled and tested in rigidly controlled environments such as high-quality conventional clean rooms (8,12) and, to a greater extent, in laminar flow clean rooms. When environmental constraints are non-existent or nominal, the percentage of soil microorganisms in the total microbial population increases. This might explain the increase of these microorganisms on the Apollo spacecraft with each mission.

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2020-12-10T09:03:01.283Z

1973-12-01T00:00:00.000Z

237232759

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Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O2. Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects. The ability of microorganisms to utilize methanol as their sole carbon source is well established (6). Most of the work in this area has been concerned with bacteria; however, in recent years, studies by Ogata et al. (11)(12)(13), Asthana (16), and Hazeu et al. (9) have dealt with the growth of yeast on methanol. An examination of this literature, however, shows that quantative data relating to methanol utilization by yeasts are still quite sparse. The purpose of this study was to isolate and examine the growth properties of a yeast able to use methanol as its sole carbon-energy source in a defined mineral salts medium at temperatures greater than 35 C. This system was chosen for the following reasons. (i) Recovery of yeast by either centrifugation or filtration is significantly easier and cheaper than recovery of bacteria due to the larger cell diameter. (ii) Yeast, as single-cell protein, is psychologically more palatable for human consumption. In addition, a precedent for yeast as a food supplement has already been set. (iii) This system with high temperature (35 to 45 C) and low pH (4.0 to 5.0) has a lower tendency toward contamination. (iv) Higher growth temperatures would mean less expense for fermentor cooling. These criteria evolve from the desire to assess methanol utilization of yeast as it applies to single-cell protein production and, more generally, to any methanol-based fermentation. MATERIALS AND METHODS Isolation of organism. The organism used in this study was isolated from soil by means of a continuous enrichment technique. Soil samples were collected and incubated at 37 C as a slurry in a methanol-water solution. After approximately 1 week, these slurries were used as inocula for a nonseptic, continuous culture. The medium was the simple mineral salts medium described below, except without vitamins. Methanol concentration in the feed was 10 ml/liter. The temperature was maintained at 37 C, and the pH was set initially to 4.5 but was allowed to settle with culture growth to 3.5. The pH was set low to select preferentially for yeast over bacteria. The dilution rate of the continuous enrichment was set at 0.07 per h to insure selection of an organism capable of doubling its mass in at least 10 h. After 2 to 3 weeks, this selection technique yielded an unstable, mixed population growing on methanol. From this mixed culture, a yeast (DL-1) was isolated by streaking on agar plates. The plates were made with Difco yeast nitrogen base medium, containing 15 ml of methanol/liter. The isolate was maintained on agar slants of either the Difco yeast nitrogen base with methanol or the mineral salts-methanol medium with biotin and thiamine. Media. Two growth media were used during this 982 investigation: a mineral salts medium and the prepared Difco yeast nitrogen base. The mineral salts medium has the following composition (grams per liter): (NH4)2.SO4, 5.0; CaCl2 2H2O, 0.1; NaCl, 0.1; MgSo4, 0.5; biotin, 2.0 x 10-6; thiamine-hydrochloride, 400 x 10-6; and trace salts solution, 10 ml/liter stock solution. Stock trace salts solution used has the following composition (grams per liter): MgSO4, 6.0; CaCl2 2H20, 0.015; FeSO4 7H2O, 0.028; ZnSO4 7H20, 0.140; CuSO4-5H20, 0.025; Na2MoO4-2H20, 0.024; CoCl2*6H20, 0.024; and MnSO4 1 20, 0.084. The phosphate concentration was maintained at 0.07 M for all experiments. For shake-flask experiments, pH was set by varying the ratio of KH2PO4 to Na2HPO4. In chemostat experiments, 0.07 M KH2PO4 was used to give the broth a slight buffering capacity to minimize the "overshoot" effect of the pH control system. The concentration of methanol in the chemostat media was 5 ml/liter and in most shake flask experiments it was 10 ml/liter. Cell dry weight analysis. The cell pellet from a centrifuged volume of fermentation broth was washed twice and dried in a predried, pretared aluminum weighing dish for 8 h at 110 C. The weight of the dried pellet determined the cell dry weight per liter of broth. Total nucleic acids analysis. The total nucleic acids content of actively growing cells was estimated by extracting a cell sample with perchloric acid (PCA). A 2-ml volume of cell suspension (about 1 g of dry cell weight/liter) was centrifuged, washed once with 3 ml of cold, distilled water, and centrifuged again. The supernatant from the wash was saved, and the pellet was extracted with 3 ml of 0.5 N PCA at 0 C for 30 min. This suspension was centrifuged and the supernatant was saved. The pellet was then extracted with 3 ml of 0.5 N PCA at 70 C for 20 min. Again the suspension was centrifuged and the supernatant was saved. The 260-nm absorbance of the wash and two PCS extractions was measured on a Gilford 240 spectrophotometer, and total nucleic acids content was determined by calculation, assuming a gram extinction coefficient of 32. Cellular protein analysis. The protein content of isolate DL-1 was estimated by the biuret method. A 1-ml volume of washed cell suspension was contacted with 4 ml of 1 N NaOH at 100 C for 5 min. The suspension was then cooled to room temperature, and 0.15 ml of 25% CUSO4 5H2O was added. The precipitate was broken up with a Vortex mixer, and the suspension was allowed to stand at room temperature for 30 min. After being centrifuged, the supernatant was examined for 540-nm absorbance on a Gilford spectrophotometer. Protein concentration was determined by comparison with a standard curve prepared from known concentrations of bovine albumin, fraction V (Sigma). Amino acids analysis. The amino acid content of isolate DL-1 was analyzed for two duplicate samples: samples I and II taken from steady state continuous culture, and III and IV taken from a batch, 14-liter fermentation. All samples were acid hydrolyzed prior to analysis on a Beckman model 121 amino acid analyzer (samples I, II) and a Beckman 120C amino acid analyzer (samples III, IV). The procedure used for acid hydrolysis is the following. A known amount of washed cells was suspended in cold 6 N HCl. This suspension was then sealed under vacuum and incubated for 20 h at 110 C. After incubation, the vial was cooled to room temperature and opened, and the suspension was flash evaporated'at 50 C. The residue was dissolved in sodium citrate buffer, pH 2.2, and filtered through a membrane filter (Millipore Corp.) to remove solids. The initial cell concentrations varied with the individual requirements of each amino acid analyzer. Model 121 required protein concentrations of less than 500 mg/ml and model 120C required concentrates less than 150 mg/ml. Dilutions were made before hydrolysis. Methanol analysis. Methanol concentration was analyzed in a Varian Aerograph 1200 gas chromatograph with a stainless-steel column (3 ft by 1'18 inch [about 91.44 by 0.32 cm]) packed with 80to 100-mesh Porapak T. The detector was of the flame ionization type. An injector temperature of 215 C, column temperature of 110 C, and a carrier gas (N2) flow rate of 25 ml/min were the operating conditions. An internal standard of ethanol was used, and the sample size was 1 Aliter. When the methanol concentration in the fermentor broth was to be analyzed, the cells were removed when chilled broth was passed through a membrane filter (Millipore Corp.) under positive pressure. All samples were held frozen until analyzed and were kept at 0 C during analysis. Identification of the isolate. The yeast isolate was identified by the typing service of Centraalbureau voor Schimmelcultures (CBS), Delft, the Netherlands. Confirming tests were carried out in our laboratory by following the procedures and identification keys of Lodder (10). Shake flask experiments. Growth characteristics of isolate DL-1 were found to be sensitive to culture conditions prior to inoculation; therefore, a standard procedure was followed for all shake flask experiments. All growth experiments were performed with mineral salts-methanol medium with vitamins, at a methanol concentration of 10 ml/liter. Methanol and vitamins were added aseptically to autoclaved growth medium. Baffled, 500-ml side arm flasks containing 50 ml of media were inoculated either from a slant, or serially with a 1% log-phase inoculum. All flasks were brought to growth temperatures before inoculation. Reproducibility of data was examined over at least two serially inoculated flasks at identical conditions. Growth rate was determined by measuring broth optical density on a Klett-Summerson colorimetor with a red filter over a short interval of log-phase growth. This was done to insure constant pH of the medium. For pH 4.5, broth pH was found to be highly unstable with increasing cell growth; consequently, the more sensitive Beckman DU spectrophotometer was used to follow cell growth over a smaller range of cell densities, before significant pH changes occurred. Chemostat studies. Continuous culture studies were carried out aseptically in a 1-liter fermentor. A magnetic stirrer provided agitation. Temperature was controlled by a water bath, and pH was monitored and controlled by an Ingold pH probe connected to a Leeds and Northrup controller. The controlled addition of 1 M KOH maintained a constant pH. Fermen-tor volume was set at 375 ml by means of a level controller, rather than by the conventional overflow tube, since discrepancies were found between exit cell concentration and reactor volume cell concentration with the overflow system. A vibrator pump provided aeration by pumping room air at approximately 1 liter/min. All continuous culture experiments were carried out with the mineral salts-methanol medium with vitamins. Feed methanol concentration was set at 5 ml/liter. In all cases, vitamins and methanol were added aseptically to the medium reservoir after it was autoclaved. The cellular yield on methanol (YM.OH, grams of dry cell weight per grams of methanol consumed) of isolate DL-1 was determined for a number of steady states at various reactor conditions. Steady states were defined by stability of cell concentration over a time period equal to at least four turnover volumes of the fermentor. YMoH is determined by the following equation: YM.OH = x/SO -S, where x is cell concentration (grams of dry cell weight per liter), SO is methanol concentration in the feed (grams per liter), and S is methanol concentration in the fermentation broth (grams per liter). Methanol vapor in the exit air stream was monitored by gas chromatography and found to be negligible. The maximal specific growth rate of isolate DL-1 was determined by the washout technique at temperatures of 32, 37, 42, 45, and 50 C. In these experiments, once the chemostat came to steady state at some known dilution rate, the dilution rate was increased to an arbitrary value greater than the maximal growth rate of the organism. A non-steady state balance on the cells in the chemostat yields the following equation: Alnx = (A -D)At, where x is cell concentration (grams of dry cell weight per liter), A is the specific growth rate of the organism (generation per hour), D is dilution rate (per hour), and t is time (hours). For a culture growing at its maximal specific growth rate (Amax), Alnx is a linear function of time, with a slope equal to Ama. -D. Therefore, maximal specific growth rate can be determined by following cell concentration as a function of time at a known dilution rate such that D > Ama.. The slope of the relationship is determined graphically. Studies with 14-liter fermentor. A number of 14-liter, semi-batch fermentations were run for the purpose of determining (i) the cellular yield on oxygen (Yo2, grams of dry cell weight per gram of 02 consumed), and (ii) the highest attainable cell density. A 14-liter New Brunswick fermentor was charged with 10 liters of mineral salts-methanol medium, at a methanol concentration of 5 ml/liter. The fermentation was run aseptically at 37 C and pH 4.5. Both temperature and pH were controlled. During the course of the fermentation, additional nutrients were added discontinuously. Dissolved oxygen in the broth was monitored by means of a galvanic probe (4), and aeration was adjusted to maintain >50% saturation dissolved oxygen. To avoid a foam problem, aeration rate was kept low (<5 liters/min) and the inlet air stream was supplemented with pure oxygen as needed to meet the oxygen demand of the culture. Cellular growth was followed by measuring optical density with a Klett-Summerson calorimeter, and periodic sampling for dry weight analysis. The oxygen yield (Yo,) was determined by Yo0 = jux/Na, where A is specific growth rate (per hour); x is cell concentration (grams of dry cell weight per liter), and Na is oxygen uptake rate (grams of 02 per liter per hour). The oxygen uptake rates were determined by making a mass balance for oxygen on the fermentor. These balances were performed only during those parts of the fermentation when inlet air was not supplemented by pure oxygen. Oxygen input was therefore taken to be 20.9% of total air fed. Oxygen content of the exit gas was measured directly with a Leeds and Northrup magnetic oxygen analyzer. Other relevant parameters, such as volumetric gas flow and fermentor volume, were determined by direct measurement. Feeding study. The feeding study comprised two sections, the single-dose acute toxicity test and the sub-acute toxicity test. Both tests were carried out under the direction of Ronald Shank (Department of Nutrition and Food Science, Massachusetts Institute of Technology). For the single-dose acute toxicity test, five Sprague Dawley Charles River male rats (average body weight 100 g) were given, by gastric intubation, 1 g (wet weight) of methanol yeast per kg of body weight. Matched control animals were given bakers' yeast at the same dose. Both yeast preparations were suspended in distilled water (1:1) to permit intubation. The animals were fed rat chow ad libitum for 12 days and were then weighed and decapitated; liver and kidneys were fixed in buffered, neutral 10% Formalin, sectioned, and stained with hematoxylin and eosin. For the sub-acute toxicity test, five Sprague Dawley Charles River male rats (average body weight 100 g) were fed semisynthetic agar gel diets in which 16% of the protein was supplied by yeast, that is, the diet contained 7% yeast on a dry weight basis. Feeding continued for a total of 34 days, except for days 26 to 29, when these animals were fed rat chow because of depletion of the yeast diet. Matched control animals were fed a similar diet made up with bakers' yeast. Growth was followed by body weight measurements. On day 34, the animals were decapitated and examined histologically as above. In both tests, the yeast pastes were heat treated at 60 C for 1 min to stop metabolic activity before incorporation into the diets. RESULTS Isolation. A few weeks after initiation of the continuous enrichment culture, the apparatus contained a mixed population of microorganisms (bacteria, yeast, mycelial forms, and protozoa). From this mixed culture, a methanol-utilizing yeast was isolated and examined. Although isolated initially in simple mineral salts-methanol medium without growth factors, the pure culture of the yeast would not grow in this medium. An examination of the growth requirements showed that the isolate had an absolute requirement for biotin, whereas the addition of thiamine facilitated growth significantly. Since a significant increase in growth rate was noted in the presence of thiamine, all further experiments were carried out in a mineral salts-methanol medium with biotin and thiamine added. Identification of isolate. A slant of our isolate was sent to the CBS typing service for identification. They identified the culture as a strain of Hansenula polymorpha (Table 1). Identification tests performed in our own laboratory agree with these observations except that we have not observed sporulation of this yeast. On the basis of our initial observations and the keying guides provided in Lodder (10), we tentatively classified our isolate as a strain of Candida silvicola (6), which is the haploid form of H. holstii. However, comparison of our isolate to culture collection strains of H. polymorpha NRRL-Y-1798 and H. holstii NRRL-Y-2154 shows it to resemble H. polymorpha in assimilative pattern and colony morphology more closely than it resembles H. holstii. The isolate DL-1 has been assigned culture collection numbers NRRL-Y-7560 and ATCC 26012 and is available from these agencies. Growth characteristics. At 37 C the isolate has a pH optimum from 4.5 to 5.5 as determined in shake flask experiments (Fig. 1). At pH 4.5 the isolate showed optimal growth from 37 to 42 C as determined by continuous culture washout experiments (Fig. 2). The maximal specific growth rate of the organism under these conditions was 0.22 per h. At methanol levels less than 0.5% (vol/vol), the isolate follows the Monod growth model. Analysis of chemostat data indicates a K8 constant of 120 mg of methanol per liter and a maximal specific growth rate of 0.23 per h. In shake flask experiments, greater than 1% methanol (vol/vol) inhibited growth, and no growth was observed with 10% methanol; however, subsequent transfer to medium with less methanol showed that the cells were not killed by these concentrations. The growth yield on methanol as a function of dilution rate is shown in Fig. 3. Maximal yield of 0.36 g of dry cell weight per g of methanol occurred at a dilution rate of 0.13 per h. The growth yield on oxygen, as determined in a batch culture using a New Brunswick 14-liter fermentor, was 0.37 g of dry cell weight -per g of 02 (Table 2). Our isolate has a protein content of 46%, which is independent of the dilution rate, a total nucleic acid content varying from 5.0 to 7.0%, and a growth rate varying from 0.08 to 0.20 per h. Figure 4 summarizes cell composition data for the isolate growing at varying dilution rates and temperatures. These data Table 3. tivity as a function of dilution rate. The data show good internal consistency and expected behavior. Feeding study. The isolate was found to have no toxic effects on any of the experimental rats used in these studies. In the single-dose acute toxicity test, there were no significant differences in body weight between the test group and the control group. Examination of the animals' internal organs showed no evidence of toxicity of the yeast. During the sub-acute toxicity test, there were also neither external nor internal indications of yeast toxicity. After the first 12 days of this test, there were no significant differences in body weight between the test animals and the control animals. At 34 days, the test group weighed 14% less than the control group; however, the reasons for the weight difference are not clearly due to any inherent property of the methanol-grown yeast. It is believed that the difference is the result of an interruption of the diet and a 4-day feeding of rat chow. The results of the toxicity -tests as stated by the experimenter are: "There is no evidence to indicate that the methanol yeast is more toxic than Baker's yeast, and therefore would generally be regarded as non-toxic." DISCUSSION We used the technique of continuous culture as a tool both for the isolation of H. polymorpha DL-1 and for studies on the characteristics of this yeast. Continuous enrichment culture permitted us to impose multiple selective pressure during the isolation; by its very nature, continuous culture selects for the organism or organisms best able to compete for the utilization of methanol. Agreement of morphological, fermentative, and assimilative characteristics of isolate DL-1 with the type strain of H. polymorpha (10) is good. Isolate DL-1 differs from the type strain only in its latent fermentation of maltose and in its extremely poor sporulation. Lodder describes H. polymorpha as fermentative negative for maltose and as an "abundantly" sporulating species. The cellular yield of H. polymorpha DL-1 on methanol (Fig. 3) progressed through a definite maximum equal to 0.36 g of dry cell weight per g of methanol at a dilution rate of 0.13 per h. The rising part of the curve can be partially explained in terms of the decreasing effect of 10 maintenance energy with higher growth rates. The falling part of the yield curve is probably explained partially by increased secretion of 8 a metabolites into the medium. An examination (, of the fermentation broth for 260-nm absorbance on a per-gram-of-cell-dry-weight basis 6 shows an increasing absorbance with increasing i dilution rate; however, no other data are availa-0 ble to indicate the nature of the product or even Z if this excretion is able to account for the 4 observed decrease in cellular yield. ,, bIn calculating these averages we have only used experimental values that tend to support each other. The FAO reference (1973) for combined methionine and cystine equals 3.5 g of amino acid per 100 g of protein. "The FAO reference for combined tyrosine and phenylalanine equals 6.0 g of amino acid per 100 g of protein. The yield on methanol of 0.36 g of dry cell weight per g of methanol obtained with our isolate is similar to average yields (0.3 to 0.4) reported for other bacteria and yeast utilizing methanol. In comparison to other yeast isolates, our isolate does not have as high a yield as that of Asthana et al. (3) at 45%, while being somewhat higher than the 29% obtained by Sahm and Wagner (16). Using the methods described by Payne (15), one may calculate a theoretical yield on methanol of 0.6 g of dry cell weight per g of 02 based on the number of electrons available for transfer to oxygen. An explanation of this discrepancy between the theoretical yield and the actual yield reported for our organism and for others will be found probably upon the elucidation of the pathways of methanol oxidation and incorporation. It has been shown that microbial methanol dehydrogenation is carried out by a nicotinamide adenine dinocleotide-independent enzyme (2,7,17), and it is quite probable that different cofactor requirements will greatly affect energy yield. A double-reciprocal plot (not shown) of the specific growth rate versus the chemostat broth methanol concentration demonstrates a surprisingly good adherence of our isolate's growth to the Monod model y = [Mlma. (S/K8 + S)]. The isolate's fit to the model is good not only in terms of the near linearity of the data but also in the closeness of the graphically determined value of maximal specific growth rate, 0.23 per hour, to the experimentally determined value of 0.22 per h. The adherence is surprising in that methanol inhibition of growth is described in the literature for other organisms and has been noted in our own work. Apparently no significant inhibition effect occurs until higher concentrations (>0.5%) of methanol are present. A value for the Monod constant K8 equal to 120 mg of methanol per liter was calculated from the data. This value is useful for estimating residual substrate levels in a continuous culture. For continuous single-cell protein production (6) this value allows one to estimate the economic effect of substrate wastage and contamination of the recovered product. The value obtained is somewhat higher than those obtained for microbial growth on a wide variety of carbon sources, which generally range from 1 to 50 mg/liter. The variation of protein and nucleic acid content with growth rate follows the expected behavior. Figure 4 presents data for protein and total nucleic acid content of isolate DL-1 as a function of growth rate at 37 C. Data obtained from single growth rates at 32, 42, and 45 C are also shown. The plot is constructed using the format of Alroy (1), in that the data are plotted against a normalized dilution rate to allow for cross-comparison among data taken at different temperatures. Protein content shows little variation, being largely constant at 46%, whereas nucleic acid content shows a marked increase with increasing growth rate. Total nucleic acids varied from 5.0 to 7.0%. Data taken at 32 and 42 C show negligible variation from that taken at 37 C; at 45 C, however, both protein and nucleic acid contents of isolate DL-1 are significantly lower than the corresponding data at 37 C. Alroy presents a correlation of nucleic acid/protein ratio on the basis of dilution rates normalized against Dmax at 30 C for many different organisms grown under different conditions. Although Dmax at 30 C is not available for isolate DL-1 and so data presented here cannot be compared to Alroy's correlation, it is possible to compare composition data for isolate DL-1 at 32, 42, and 45 C to corresponding data at 37 C, on the basis of a dilution rate normalized against Dma. at 37 C. At 32 C the nucleic acid/protein ratio equals 0.13, and the corresponding value at 37 C equals 0.13. At 42 C the ratio equals 0.11, while the value at 37 C is 0.12. At 45 C the ratio equals 0.12, and at 37 C the ratio is 0.13. All these cases show good agreement and demonstrate an internal consistency among the data not readily apparent from Fig. 4. Actual protein contents of methanol-grown cells have been reported from 35% for yeasts (16) to 71% in bacteria (8). Working with yeast, Ogata et al. (12) found their Kloeckera sp. no. 2201 to have a protein content of 45.3%, and Asthana (Ph. D. thesis, University of Pennsylvania, Philadelphia, 1972) reports a similar figure of 50%, whereas Sahm and Wagner (16) report a lower protein content of 35.4%. Very little nucleic acid data are available; however, Asthana reports a ribonucleic acid content of 2.5%, and Ogata reports total nucleic acid content 5.4%. Our isolate's protein content of 46% and nucleic acid content of 5 to 7% were expected values for yeast and are in line with the data of other isolates. Figure 5 shows typical variation of cell density, substrate, and productivity with varying dilution rate. The drop-off of cell density at higher dilution rates is not a matter of oxygen limitation but of adherence to the Monod growth model. Although dissolved oxygen (DO) was not monitored during the fermentations from which these data were obtained, previous chemostats were set up with a galvanic 02 probe (4), and DO was found to be greater than 50% saturation over a wide range of conditions. Furthermore, if one calculates a theoretical oxygen demand for isolate DL-1 growing at Amax, at a cell concentration of 1 g/liter, it will equal 20 mM 02 per liter per h. This demand is well within the mass transfer capabilities of the fermentor. Amino acid analyses (Table 3) were performed on two pairs of duplicate samples of isolate DL-1. Agreement is quite good among the four samples except in the cases of methionine and cystine. For all the essential amino acids except methionine and cystine, the data compare well with the Food and Agricultural Organization of the United Nations (FAO) reference levels. Isolate DL-1 is particularly rich in lysine. Although methionine and cystine are below the FAO reference level, they are not so low as to make isolate DL-1 an unattractive protein source. Furthermore, values reported here should be considered as only minimal values since these amino acids are unstable under acid hydrolysis. In general, isolate DL-1 compares favorably to other methanol-grown protein sources (5,8,12,16; U.S. patent 3,546,071, 1970) in lysine, methionine, and cystine content. No data were obtained for tryptophane content. Calculation of cellular protein content, on the basis of total protein measured as individual amino acids, gives a value of 42% for all samples. This value compares quite well with the direct measurement of protein content equal to 46%. Thus, amino acid recovery is quite high. To determine growth yield on oxygen and the highest possible cell density obtainable, a number of fermentations were carried out in a 14-liter New Brunswick fermentor. The highest cell density obtained was 24.4 g of dry cell weight per liter. At this point in the fermentation cell growth stopped. The tapering off of growth could be explained by the production of an auto-inhibitor or by the build up of toxic metabolites. However, these explanations do not appear to be adequate, since broth collected at the end of the fermentation, filter sterilized, and inoculated with isolate DL-1 showed exceptionally strong growth. Additional experiments are required to determine the reason for the observed stoppage of growth at 24.4 g/liter. A series of oxygen balances was made during the fermentation to be used in determining the cellular yield of isolate DL-1 on oxygen. All of these balances were performed during the early parts of the fermentations before the first addition of methanol. The oxygen requirement of the culture was low enough at this stage of the fermentation to allow low aeration with room air to meet the demand. Therefore, the oxygen content of the inlet air was accurately known. Also, during this phase of the fermentation, cell growth was consistent and strong, and so major changes in growth rate during the course of measurement were not a problem. Data used in determining the oxygen yield (Y(2) are presented in Table 2. As can be seen, agreement among the various data points is quite good. No consideration was given to the effect on Yo2 that the maintenance coefficient of oxygen would have. At the time of the experiments, the effect of maintenance was assumed to be small, but no data are available to allow this maintenance coefficient to be estimated. A theoretical yield on oxygen can be calculated by assuming complete reaction of methanol according to the equation: CH3OH + NH3 + 02 , cells + CO2 + H20. When YN1,0j, equals 0.35 g cells per g of methanol and a cellular composition of 50% carbon, 8% nitrogen, 7% hydrogen, and 20% oxygen, a material balance on the above equation results in a theoretical Yo2 of 0.37. This value is gratifyingly close to the measured Yo2.

v3-fos

2020-12-10T09:04:12.684Z

1973-04-01T00:00:00.000Z

237230343

s2

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with 33P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of 33P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with 33P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N × 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs. Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with 33P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of 33P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with 33P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs. Hungate (19) calculated adenosine triphosphate (ATP) yields from known pathways of ruminal fermentation of carbohydrates. By using a Y ATP = 10 (3,34), he concluded that about 10 g of microbial protein (about 15 g of total cell mass) can be synthesized for each 100 g of carbohydrate fermented. This value was thought to represent an upper limit of the synthetic capacity of anaerobic ruminal fermentation (19). This level of protein synthesis in the rumen is equivalent to 18.3 g of digestible protein per Mcal of digestible energy (36) and is clearly below the requirement of growing ruminants (36). From a husbandry viewpoint, ' this result implied that, for optimal growth or production of ruminants, procedures to allow dietary protein to escape ruminal degradation must be developed. Extensive direct experimental work on quantitative aspects of ruminal microbial protein synthesis indicates, however, that the limits set by Hungate (19) were too low. Thus, growth yield studies with strains of rumen bacteria (11,12,18) and ingesta passage studies with sheep by using purified diets or microbial cell markers (14,15,17,27) indicated that ruminal microbial protein synthesis ranged from 15 to 22 g of microbial protein per 100 g of organic matter fermented. Other recent reports (13,21) have also suggested that the ATP yield per mole of hexose fermented in the rumen is higher than previously anticipated. The experiments described below were initiated to measure absolute bacterial and protozoal protein synthesis rates in whole rumen contents at various times after feeding with short-term, zero time in vitro (6) incubations. Since membrane (phospholipids) synthesis and growth are directly related (31,33) and since growth and protein synthesis are directly related (28) in microorganisms, protein synthesis rates of ruminal microorganisms were assessed by measuring the rate of microbial phospholipid synthesis. MATERIALS AND METHODS Radioactive phosphorus-labeling pattern of cellular phosphorus containing constituents during in vitro incubations of mixed ruminal bacteria: experiment 1. The labeling patterns of cellular phosphorus by 22P-inorganic phosphorus were studied in suspensions of mixed ruminal bacteria in a high and low phosphorus medium in vitro. Rumen contents were collected as outlined by Purser and Moir (37) from sheep fitted with Jarret cannulae (fed ration 1, Table 1) before the morning feeding, placed in a vacuum bottle warmed to 39 C, gassed with oxygen-free C02, and transported quickly to the laboratory. Rumen contents from sheep are quite homogenous, and samples of 100 g or more are representative of whole rumen contents (37). The rumen contents were subsequently squeezed through two layers of cheesecloth. Rumen liquor (500 ml for high PO4 and 800 ml for low PO4 media) was then centrifuged at 500 x g for 15 min at 2 C. The pellet was discarded, and the supernatant was centrifuged at 10,000 x g for 15 min at 2 C. The resulting pellet (bacterial fractions; 300 to 400 mg dry weight for the high PO4 and 150 to 200 mg dry weight for the low PO4 media) was resuspended to the original rumen liquor volume in reduced anaerobic buffer (AB; Table 2) containing either 180 or 35 gg of phosphorus, pH 6.7, per ml. During the centrifugation steps, all tubes and beakers were gassed with 0,-free CO2 to insure anaerobiosis. The ruminal bacteria fractions were then transferred into prewarmed fermentation flasks (1 liter) and incubated at 39 C under 0,-free CO2. Radioactive phosphorus (9P-HP04; ca. 60 x 106 DPM) dissolved in AB was then added to the fermentation flask, and initial subsamples (80 ml) were secured. For the experiments with the high P04 media, substrate (0.66% glucose, 0.5% soluble starch, and 0.15% urea; expressed as percent weight of the final volume of the fermentation system) was added and further subsamples were obtained at 30, 60, 120, 180, and 240 min. For the experiments with the low P04 media, inorganic phosphorus uptake by resting bacteria and actively growing bacteria was studied in the manner outlined by Mitchell and Moyle (31). Thus, substrate (as above) was added after 90 min of incubation. Subsamples (as above) were obtained at 30, 60, 90, 120, 150, 210, 270, and 330 min for the low P04 media fermentations. To stop bacterial activity, subsamples were placed in prechilled beakers which were then placed in a solution of solid CO2 in ethanol. 25,1973 When the temperature of the subsample reached 2 to 5 C, the subsamples were centrifuged at 18,000 x g for 15 min at 3 C. The pellet was then resuspended and washed with a small quantity of deionized water and recovered by centrifugation. The pellets were then frozen, lyophilized, and stored for phospholipid, intracellular and total phosphorus, and asp activity analysis. Increases in cellular dry matter and protein (N x 6.25) content were determined on separate subsamples. A sample of cell-free media was secured and frozen for inorganic phosphorus and asP analysis. Radioactive phosphorus incorporation into microbial phospholipids of rumen contents in vitro from sheep: experiment 2. Sheep (fitted with Jarret cannulae) were placed on ration 2 (high protein) or 3 (low protein) ( Table 1, one sheep per ration) for a 3-week period. Rumen contents were collected, as described above, from the sheep before (0 h) and 2 and 4 h after the morning feeding. Upon arrival at the laboratory, 150 g of rumen contents was weighed into a wide-mouth fermentation flask (800 ml) (under an O2-free CO2 atmosphere), and 6 ml of a solution containing 3P-HPO4 (13.2 x 107 DPM) was added. The flask was sealed and then shaken for 30 s, and an initial subsample was removed. Preliminary work showed that 6 ml of 1% crystal violet could be well mixed with 150 g of rumen contents within 30 s under these conditions. Further subsamples were obtained at 30, 60, 120, 180, and 240 min of the in vitro incubation. After each sampling the flask was flushed with 02-free CO2 and resealed. The microorganisms were killed by the addition of 0.05 volume of saturated HgCl,. After washing to remove unincorporated 33P, the samples were frozen and lyophilized. Microbial phospholipid synthesis and microbial protein synthesis in whole rumen contents of sheep: experiment 3. Rumen contents were collected from sheep fed ration 1 (Table 1) before (0 h) and 2, 4, and 9 h after the morning feeding. One sheep served as the donor of rumen contents for two separate experiments. Short-term, zero time method (6,20) in vitro incubations were conducted with rumen contents as described above, except 750 g of rumen contents was placed in the fermentation flasks. After the additions of 33P-H.PO4 (about 44 x 107 DPM), an initial subsample (350 g) was obtained. The final sample was obtained after 60 min. The microorgansims were killed with saturated HgCl2. Each subsample was divided in the following manner. From the well-mixed subsample, a 20-g sample was used to determine 33P incorporation into total microbial phospholipids. The particulate matter was spun down and then resuspended and washed in saline and centrifuged at 18,000 x g three times to remove unincorporated, free, and nonspecifically bound 33P. The residue was then frozen and lyophilized. The remaining rumen contents from the subsamples (305 g) were squeezed through two layers of cheesecloth. The particulate matter was then resuspended in 0.85% NaCl, stirred, and squeezed again through two layers of cheesecloth. Microscopy examination revealed that this procedure removed all except a few small protozoa from the residual plant matter and rumen contents. It was therefore assumed in subsequent calculations that total protozoal protoplasmic mass had been recovered quantitatively from rumen contents by this procedure. A small sample was removed from the rumen liquor for phosphorus and volatile fatty acid (VFA) analysis. The rumen liquor and extract from the second squeeze were then combined. The protozoa and bacteria were separated from the rumen fluid by differential centrifugation as described by Bergen et al. (4). Microscopy examination of the high-speed (bacterial) and 150 x g (protozoal) fractions were done in a separate (i.e., no isotope) study. It was found that the 150 x g (protozoal) and the high-speed (bacterial) fractions were devoid of gross plant material contamination. Previous work had shown that this differential centrifugation system (4) resulted in 150 x g (protozoal) and high-speed (bacterial) fractions which contained only 1.5 and 0.7% (based on dry matter) crude fiber (W. G. Bergen, unpublished data). The 150 x g (protozoal) and high-speed (bacterial) fractions were then frozen and lyophilized. Analytical procedures. Preliminary experiments were conducted to assess the efficacy of various extraction procedures for phospholipids from ruminal microorganisms and the salt wash procedure of Folch et al. (10). During these studies, microbial preparations were extracted and reextracted in organic solvents, and the extracts were then monitored for phospholipids with thin-layer chromatography. Similarly, the salt wash procedure was checked to assure that only inorganic, but not phospholipid, phosphorus was lost during the wash. In a number of trials, upon addition of 33P-HPO4 to chloroformmethanol (2: 1), after the salt wash only 0.3% of the initial radioactivity remained in the solvent. For the experiments described above, microbial phospholipids were extracted from lyophilized rumen contents, high-speed (bacterial), or 150 x g (protozoal) preparations by a modification of the method described by Katz and Keeney (24). Approximately 30 mg of sample was extracted with 5 ml of chloroformmethanol (2:1; vol/vol) in 15 ml of Teflon-lined screw-capped culture tubes. The tubes were rotated for 16 to 20 h at room temperature. The extracts were filtered through a fritted glass Buchner funnel and the nonlipid impurities were removed by the salt wash procedure (10). Sonic treatment of microbial preparations did not improve the yield of total lipid extraction. Phospholipid phosphorus was determined in the washed extract by the ascorbic acid procedure of Chen et al. (7) as modified by Rhee and Dugan (39). Total cellular phosphorus and phosphorus concentration in incubation media were determined by the procedure of Chen et al. (7). Intracellular inorganic phosphorus (IC-P,) was extracted from 30 to (26). Preliminary studies showed that this procedure recovered between 97 and 98% of the phosphorus from standard phosphorus solutions in the presence of 5% PCA. Nitrogen was determined with a micro-Kjeldahl procedure. VFA were determined from media treated with metaphosphoric acid (9) on a Teflon column (198 by 0.05 cm) packed with Chromosorb 101 (60-80 mesh) at 188 C with nitrogen as carrier gas and a hydrogen flame detector. Dry matter of subsamples was determined by drying at 110 C to a constant weight (this took 20-24 h). Suitable samples of all phosphorus-containing extracts (i.e., phospholipids and intracellular phosphorus) were placed in vials containing 10 ml of scintillation fluid (5 g of 2,5diphenyloxazole; 0.05 g 1,4 bis-[2-(4 methyl-5phenyloxazole) ]-benzene, 500 ml of toluene, and 500 ml of Triton X-100) and counted in a Nuclear Chicago 6848 liquid scintillation spectrometer. "3Pphosphorus activity (T 1½2 = 25.3 days) in the counted samples was corrected for counting efficiency and decay, with decay factors derived by Robinson (40). The 33P-phosphorus was obtained from New England Nuclear Corp., Boston, Mass., as H3-'3PO4 in 0.02 N HCl. All glassware used in the experiments was washed with detergent, rinsed in deionized water-12 N HCl (2:1), followed by a final rinse in deionized water. Calculations. To calculate the rate of microbial phospholipid phosphorus synthesis in whole rumen contents (experiment 3), the IC-P1 specific activity (SA) was used as an indicator of precursor pool SA. Since the SA of IC-P, may differ between protozoa and bacteria, these were assessed separately. In this incubation system (experiment 3), the SA of the IC-Pi was measured at the start (SA = 0) and the end of the incubation time. The rate of "3P uptake into cellular phospholipids is dependent on the rate of phospholipid synthesis as well as the rate of change of the SA of the precursor pool (IC-Pi) during the incubation. To properly describe the SA of the IC-P1, more frequent sampling would have been necessary. Thus, an approximation was made to determine the effective SA of the IC-Pi pool during the incubation period. The following formulae describe the calculations: (i) Intracellular phosphorus SA = (final IC-P,-SA + initial IC-P,-SA)/2. (iii) Total phosphorus (micrograms) incorporation into microbial phospholipids = phosphorus incorporation into high-speed pellet (bacterial) phospholipids (net counts/min of 3SP in the PL-PJ/SA of the IC-P1) + phosphorus incorporation into 150 x g pellet (protozoal) phospholipids (net counts/min of 33P in the PL-PJSA of the IC-P,). RESULTS Physical separation of microbial fractions. Sampling of rumen contents and the subsequent fractionation of the rumen contents into microbial fractions is one of the most difficult aspects of microbial investigations in the rumen. Throughout this work rumen samples were withdrawn from the ventral and dorsal sac of the rumen. More importantly, at least 1 liter (from a 4-liter rumen volume of sheep) of contents was removed to insure that a representative sample had been secured. For experiment 1, mixed ruminal bacteria were prepared similar to the procedure outlined by Baldwin and Palmquist (2). It must be recognized that many large bacteria and clumps of bacteria may be lost during the initial low-speed centrifugation step. As Hungate (19) pointed out, suspensions of washed mixed ruminal bacteria contain all kinds of rumen bacteria, but they are not necessarily in the same proportions as found in the total contents. In experiment 3, the 150 x g pellet was designated the protozoal fraction. Although, the protozoa could be nearly quantitatively removed from the rumen fluid, this fraction may also contain sizeable amounts of bacteria (49). These bacteria are large bacteria as well as those harbored within the protozoa. The metabolic activity of these organisms will contribute to the total activity of the 150 x g (protozoal) fraction. In the remainder of the paper, the 150 x g pellet will be called the protozoal fraction, whereas the high-speed pellet will be called a bacterial fraction. Experiment 1. The metabolism of 33P-H3PO, in mixed ruminal bacteria incubated in high or low phosphate medium was studied. Figure 1 depicts the results for the high phosphate medium studies. The SA of inorganic phosphorus medium stayed constant throughout the incubation period. In the presence of substrate there was a linear rise in 33P incorporation into phospholipids and in 33P uptake into IC-Pi and total cellular phosphorus. The increase in dry cell mass and cellular protein (N x 6.25) were significantly correlated (r = 0.99; P < 0.01), and the rate of 33P incorporation into cellular phospholipids and the increase in dry cell mass or cellular protein were significantly correlated (r = 0.91, P < 0.01; r = 0.94, P < 0.01). This high correlation indicates that cellular phospholipid synthesis can be 507 VOL. 25,1973 BUCHOLTZ AND BERGEN used as a marker of total cellular growth. During the 240-min incubation period, the SA of the IC-P, did not equilibrate with the SA of the inorganic phosphorus medium, indicating that, if phospholipid synthesis is to be measured, the SA of the inorganic phosphorus medium will overestimate the SA of the phosphorus precursor pool. Incubation of mixed ruminal bacteria in the low phosphate medium (Fig. 2) in the absence of substrate (0-90 min) resulted in some uptake of 33P into the IC-P1 pool. 33P-phosphorus was not incorporated into the phospholipids during this period. The addition of substrate caused a more rapid uptake of 33P into the IC-P1 pool, the incorporation of 33P into PL-Pi, and an increase (growth) of the bacterial cell mass. The SA of inorganic phosphorus medium showed little change. Again, the SA of the IC-P1 pool did not reach the SA of the phosphorus medium after 240 min of incubation after substrate addition. The rate of 33P incorporation into cellular phospholipids and the increase in cell mass were significantly correlated (r = 0.98; P < 0.01), indicating that cellular phospholipid synthesis can be used as a marker of total cellular growth. Mitchell and Moyle (31) showed similar patterns of 32P uptake into phosphorus containing constituents of S. aureus. In this organism, some 32P uptake occurred into the IC-P, pool, but 32P incorporation into phospholipid did not occur in the absence of a carbon source. After the addition of substrate there was 32P uptake into both PL-P1 and IC-P. As was the case for mixed ruminal bacteria, in S. aureus the SA of IC-P1 did not equilibrate with the SA of the inorganic phosphorus in the incubation medium (31). Experiment 2. Since experiment 1 showed that phospholipid synthesis was highly correlated with increases in cellular mass or protein content in mixed ruminal bacteria, experiment 2 was conducted to investigate whether rates of phospholipid synthesis in whole rumen contents (determined in vitro) could be related to dietary protein intakes of sheep. Under these experimental circumstances a significant amount of 33P incorporation into protozoal as well as bacterial phospholipid would be expected. Although protozoal phosphorus metabolism was not studied in detail as for mixed ruminal bacteria, it was assumed that for these organisms phospholipid synthesis would also reflect cellular growth. When a diet containing low levels of protein (N-limiting) is fed to sheep, an increase and then a decline in microbial growth (activity in the rumen) would be expected, whereas for a diet with adequate (or excess) protein a more sustained rate of microbial growth would be expected. Previous work had shown that, when urea is added to a low protein ration, microbial protein synthesis (16) and animal performance (23) are increased. As the results show (Fig. 3), before feeding and at 2 h after feeding the rate of 33P incorporation into microbial phospholipids (determined in vitro) did not differ between the two rations; however, for the high protein ration at 4 h after feeding the rate of 33P incorporation into phospholipids was still high, whereas for the low protein diet the rate of 33P incorporation into phospholipids declined to the prefeeding level. Experiment 3. Experiment 1 showed that phospholipid synthesis and increases in cellular protein were highly correlated and experiment 2 showed that, as expected, the addition of a NH3 source to a low protein diet resulted in more sustained (increased) microbial activity (growth). Thus, these studies indicated that phospholipid synthesis can be used as a marker of microbial growth in whole rumen contents. In this experiment microbial protein synthesis, at various times after feeding, in the rumen of a sheep fed a standard ration was studied. The results were expressed as grams of protein (N x 6.25) synthesized per hour per hypothetical 4-liter rumen. Sheep used in this work showed a rumen volume of 3.5 to 4.0 liters from polyethylene glycol disappearance curves (21). To calculate microbial protein synthesis from phospholipid synthesis, the two parameters had to be related. Walker and Nader (45) used a nitrogen-sulfur ratio to relate 35S incorporation to protein synthesis. Similarly, a nitrogento-phospholipid phosphorus ratio (N/PL-PI) was used to relate inorganic phosphorus incorporation into phospholipids to protein synthesis. Extensive preliminary studies showed that the N/PL-Pi was not constant between various strains of pure culture rumen bacteria or microbial preparations isolated from sheep (Table 3). Thus, the N/PL-P1 ratio had to be determined separately for each in vitro incubation to insure meaningful calculations. 33P-phosphorus and total phosphorus incorporation rates into microbial phospholipids and protein (N x 6.25) synthesis by whole rumen contents during 60-min in vitro incubation periods are given in Table 3. The apparent protein synthesis rate of the 150 x g (protozoal) fraction was equal to the rate noted for bacteria. Although protozoa per se were isolated by the physical separation methods used in this study, the extent of bacterial contamination of the 150 x g fraction was not ascertained. However, it is plausible that one-fifth to one-fourth or more of the cellular mass in the 150 x g fraction was of bacterial origin. Thus, it can be concluded that bacteria contribute 60% or more to overall protein synthesis in the rumen. Recent results by Hungate et al. (21) suggested that the rate of protozoal protein synthesis was nearly equal to bacterial protein synthesis. Overall, the highest rate of protein synthesis was observed at 2 h after feeding, whereas at 9 h after feeding the rate was substantially lower than the rate noted before feeding. There are no apparent reasons for a higher microbial protein synthesis rate at 15 h (0 h in Table 3, Fig. 4) than at 9 h after feeding. Since the 9-h rate was from a single experiment, the rate of protein synthesis may have been underestimated. These results expressed in terms of a 4-liter rumen are depicted in Fig. 4. The relative VFA production rate is plotted on the same graph and is parallel to the changes found for microbial protein synthesis. It is evident that immediately after feeding there was an increase in microbial activity (VFA production) and cellular growth (phospholipid and protein synthesis). Despite differences in rations, these results are in disagreement with the contention of Walker and Nader (46) that microbial protein synthesis in the rumen declines initially after feeding and then increases again to prefeeding rates. DISCUSSION Rumen microbial protein represents a major source of amino acids to the ruminant animal. Quantitative aspects of ruminal microbial protein synthesis have thus been the subject of extensive research efforts. In the past, procedures based on fermentation balances (19), ingesta passage studies with or without microbial cell markers (14), zero time in vitro incubations of rumen contents with 35S or 15N (1,45), single dose or continuous infusion of 15N-urea into the rumen (32,35), and rumen fermentation-turnover models with C, N, and H balance studies (21) have been used to assess microbial protein synthesis. Hungate (19), from known pathways of ruminal VFA production and supposed ATP yields and using Y ATP = 10 (3), calculated that about 10 g of microbial protein can be synthesized for each 100 g of carbohydrate fermented. This value represented an upper limit of the synthetic capacity in the anaerobic ruminal fermentation. This level of protein synthesis in the rumen is equivalent to 18.3 g of digestible protein per Mcal of digestible energy (36). As Purser (36) pointed out, this protein-calorie ratio is below the required protein-calorie ratio to meet the protein requirements of growing ruminants and less than such ratios established from empirical balance studies with growing ruminants. Growth yield studies with pure cultures of rumen bacteria (11,12,18) and ingesta passage studies with sheep, by using nonprotein nitrogen diets or markers for microbial cells (15,17,27), indicated that the microbial protein yield in the rumen was higher than predicted by Hungate (19). Overall, the above studies indicate that 15 to 22 g of microbial protein is formed per 100 g of organic matter fermented. There are a number of alternatives to explain these results. First, the Y ATP value may not be universal among bacteria (however, this has been rejected by Payne [40]), or substrate phosphorylation (43) may result in an extra energy yield to the microorganisms. Although the latter process has been implicated in ruminal organisms (44), a more tenable alternative appears to be the recent suggestion that the Y ATP value of 10 is universal but that previously assumed ATP yields per mole of VFA are too low (13,21). To further evaluate the potential of microbial protein synthesis in the rumen, we decided to use the short-term, zero time method. Walker and Nader (45) had used this approach with 35S, but their actual results on protein synthesis rates were quite low. They were able to correct their data by determining a VFA-to-protein synthesis ratio from the in vitro incubation and then multiplying this ratio by an assumed VFA yield. The major problem in the approach of the above authors is that they assumed that the SA of the intracellular precursor pool (in this case 35S-cysteine or 35Smethionine) for protein synthesis equalled the SA of medium 35S-sulfide. Al-Rabbat et al. (1) made similar assumptions in their 15N incorporation studies with ruminal microorganisms. In the present work, it was decided to use phospholipid synthesis as a marker for cellular growth since membrane synthesis is closely related to total cell growth (33,38,50), and since IC-P1 equilibrates readily with phospholipid precursors (5,48) and can be used to determine the SA of the intracellular precursor pool for phospholipid synthesis. The results of experiment 1 showed clearly that, with ruminal bacteria, the SA of inorganic phosphorus medium did not equilibrate with the IC-Pi pool. A very high correlation between cell growth and phospholipid synthesis was noted for mixed ruminal bacteria similar to the observations of Ohki (33) for E. coli. Preliminary work had shown that the N/PL-Pi ratios of the ruminal 150 x g fractions were not similar to the N/PL-Pi of ruminal bacteria. Since the 150 x g fraction is largely composed of protozoa, it was decided that the SA of the IC-Pi must be established for the 150 x g fraction and bacterial fraction separately. For all the calculations below, the rate of protein synthesis by both fractions have been combined to negate the implication ( Table 3) that (apparent) protozoal protein synthesis was equal in magnitude to the bacterial protein synthesis. The results from Table 3 and Fig. 4 were recalculated to estimate daily protein synthesis. The data (Table 4) are expressed as grams of protein produced in a 4-liter rumen per day. To estimate daily protein synthesis, the various measured rates were arbitrarily designated to represent an average rate of various time intervals (summation interval). The estimated daily rate of microbial crude protein (N x 6.25) and true protein (N x 6.25 x 0.85) (42) synthesis in the rumen of a sheep with a 4-liter rumen consuming 158 kcal of digestible energy per 0.75 kg body weight per day was 218 and 185 g, respectively. These values represent a rate of microbial crude and true protein synthesis of 26 and 22 g per 100 g of organic matter digested (fermented; DOM) in the rumen, respectively. These values are higher than the theoretical upper limit proposed by Hungate (19) but similar to the upper values of ruminal microbial protein synthesis per 100 g of DOM in the range of values reported from direct measurements (15)(16)(17)27). The system used in the present work to assess microbial protein synthesis measured absolute but not net rates of microbial protein synthesis which are obtained from ingesta passage studies. The values given above can be recalculated by taking microbial protein turnover into account. Although the extent of turnover of microbial protein and bacterial-protozoal protein interconversions has not been accurately defined, it may be estimated from stud- Time after feeding (hr) FIG. 4. Microbial protein (N x 6.25) synthesis and relative rate of volatile fatty acid production in rumen contents at various times after feeding. (Protein synthesis results, mean of two separate experiments; VFA production represents a single experiment; bars represent standard error.) (42). Estimated rates of microbial crude protein and true protein synthesis per day equals 218.0 and 184.9 g, respectively. Estimated microbial crude protein and true protein synthesis per 100 g of organic matter digested in the rumen [75% digestion (1)1 equal 26.0 and 22.0 g, respectively. ies of bacterial lysis in the rumen (22), from protozoa-bacteria interrelationships in the rumen (8,47), from the recent report on N turnover in ruminants (32), and from microbial turnover data (29) that 25 or more of the total protein synthesized in the rumen may be involved in turnover. On a 25 turnover basis the estimated ruminal protein synthesis rate was 16.1 g of true protein synthesis/100 g of DOM. This value represents a 30-g digestible protein-to-Mcal of digestible energy ratio which is adequate for growing calves and lambs (36).

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2020-12-10T09:04:12.791Z

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Fungi That Infect Cottonseeds Before Harvest As a part of an investigation of aflatoxins and other mycotoxins in cottonseeds at harvest, samples of seeds collected from the 1971 crop at locations across the U.S. Cotton Belt were examined to determine the kinds of microorganisms causing internal or seed-coat infection in the field. Aspergillus flavus infection was absent from all seeds examined from most areas but was present in some samples from Arizona, California, and Texas. Fusarium spp., Alternaria sp., and A. niger caused internal infection at many locations; Colletotrichum gossypii and Rhizopus stolonifer were present in seeds from some areas but were generally much less common. Many of the infections with A. niger were in the seed coat. Bacterial infections were fairly frequent. In a series of commerical samples from Arizona. A. flavus infection was found in 61% of seeds, with fiber showing the bright, greenish-yellow (BGY) fluorescence that is diagnostic for A. flavus boll rot. Aflatoxin contamination was also concentration in the same seeds. The above findings agree with previous data showing that aflatoxin contamination of cottonseeds before harvest occurs rarely, if at all, in most parts of the U.S. Cotton Belt and that when such contamination does occur, it tends to be concentrated in seeds with the BGY fluorescence in their fiber and seed fuzz. fore harvest. A. flavus boll rot can be detected by a characteristic bright, greenish-yellow (BGY) fluorescence caused by A. flavus in spots in the fiber (18) and is relatively uncommon. It has been noted particularly in certain western parts of the Cotton Belt (30). Aflatoxin contamination in the seeds at harvest appears to be similarly uncommon but has been found in some samples in the same areas as the boll rot (19). Aflatoxins have been detected most frequently and at highest levels in seeds whose fiber and seed fuzz display the BGY fluorescence (19,20). Aflatoxin contamination of U.S.grown cottonseeds at harvest thus appears from previously available data to be significant only in localized areas. However, because of the present and anticipated future importance of cottonseed products in both animal and human nutrition and the unusual hazards associated with the aflatoxins (15), we considered it desirable to make further observations. Fungi that infect cottonseeds before harvest (3,19). Seed infection with A. flavus has been noted in association with the characteristic BGY fluorescence in the fiber (19). As a whole, however, investigations of preharvest infections of cottonseeds have involved examination of only a relatively small number of seeds from limited cotton-producing areas. Mayne (21) reported that 17 of 41 fungal isolates from 28 samples of cottonseeds from six southern states were A. flavus, but the history of most of her seed samples was not known, and infection may have occurred in damp storage after harvest. Further background information on the cottonseedaflatoxin problem has been detailed and documented with references elsewhere (19). This paper reports results of examinations for fungal infection of cottonseeds grown across the U.S. Cotton Belt under widely variable condi-608 tions of culture and harvested at known dates. Confirmatory aflatoxin analyses on certain samples are also reported. MATERIALS AND METHODS Bolls from commercial cotton varieties were collected directly from plants on four to eight picking dates at 17 locations across the U.S. Cotton Belt ( Table 1). The maximum weathering period before harvest was not accurately known, but probably it was not in excess of that common with commercial cotton. The bolls were dried at the point of origin and sent to Beltsville. At Beltsville, each boll was ginned individually by hand, and the seeds were delinted in concentrated sulfuric acid, washed first in 1% sodium carbonate solution and then in water, dried, and treated overnight in chlorine gas as described previously to surface disinfect (20). Seeds that were immature or obviously damaged were avoided for the examinations summarized in Table 1. For each picking date, fifty seeds were examined. Five seeds from each boll were planted on 2% water agar in a petri dish and were incubated for 4 days at 30 C in a room with a 12-h light cycle provided by daylight fluorescent lamps, after which they were examined by microscope to detect any fungi or bacteria growing out of them. No distinction was made between A. flavus and A. parasiticus Speare. The 161 commercial seedcotton samples from fields in unspecified parts of Arizona were kindly provided by B. B. Taylor. The samples, as received, weighed in the range of 400 to 2,600 g. Seedcotton consists of seeds with fiber still attached and occurs in locks, one lock for each of the four or five segments of the boll and each lock containing about eight seeds. Analyses for aflatoxin B1 were by a mini-plate screening procedure which detects aflatoxin B1 at levels above 100 parts per billion (ppb), followed by a quantitative assay on 20-by 20-cm Schleicher and Schuell silica gel plates. The mini-plate method, to be detailed elsewhere, consisted essentially of the following. A seed meat sample was shaken with three times its weight of chloroform-acetonitrile (1:2) and onehalf of its weight of 10% aqueous ferric chloride, after which the extract was chromatographed on a 2-by 3-inch (5.08-by 7.62-cm) thin-layer chromatography plate with a 0.75-inch (1.905-cm) band of A120s at the bottom and a 2.25-inch (5.7-cm) band of silica gel on the remainder of the plate. Development was for 8 min with diethyl ether-methanol-water (96:3: 1). For a more quantitative estimate of the aflatoxin level, a sample of the above extract was diluted with benzeneacetonitrile (98:2), and this diluted extract was then spotted on a Schleicher and Schuell plate and developed in an unlined tank with diethyl ether-methanolwater (96:3: 1). Table 1 records data on fungal and bacterial infection of seeds from 17 locations across the Cotton Belt. A. flavus was detected only in the collections from Tulare County, California, and Vernon, Texas, and at low levels at these locations, even though the overall level of microbial infection in most of the samples was First Last slmpick pickB lackville, S.C. 6 8/27 11/29 300 1 0 10 2 17 1 27 0 1 59 College Station, Tex. Stoneville, Miss. 8 In almost all cases, infections of the seeds of Table 1 appeared to involve only a single fungus or bacterium per seed. The infecting fungus generally grew luxuriantly out of the seed at either the chalazal or funicular end. An exception to this was A. niger, which caused both internal (as above) and seed-coat infections. In the latter case, a sparse sporulating outgrowth of the fungus appeared over much or all of the surface of the seed, and hyphae could be seen inside the seed coat but not in the seed meats when the infected seeds were cut open. More than half of the observed infections with A. niger were in the seed coat. RESULTS The general absence of detectable A. flavus infection in seeds from the samples of Table 1 was believed to be real and not a result of any inadequacy in the detection method. The same method was used successfully to detect A. flavus in cottonseeds in previous work (19). Furthermore, it was successfully applied to a series of 161 commercial samples of the 1971 crop in Arizona in the present investigation, with results as shown in Table 2. All locks with BGY-fluorescing fiber were picked out of 25 samples, and 5 seeds from each fluorescent subsample were delinted and examined for fungal infection; of the 125 such seeds, 76 (61%) were infected with A. flavus. For comparison, 25 seeds from locks with non-BGY-fluorescing flavus infections were highly concentrated in the seeds with the BGY-fluorescing fiber. The BGY-fluorescing locks were low in number, constituting only 0 to 1.2% of the weight of the total seed cotton samples. The high general level of fungal infection in the seeds from nonfluorescing locks may have been related to breaks in the seed coat, readily seen in many of them, possibly caused by mechanical harvesting. A. niger was the predominant fungus isolated from these seeds ( Table 2). The fact that A. flavus infections in the 161 Arizona samples were highly concentrated in seeds with BGY-fluorescing fiber suggested that aflatoxins might also be similarly concentrated in these seeds. Aflatoxin analyses were made to check this point. Of the 161 seed cotton samples, 65 had no BGY-fluorescing locks and no detectable aflatoxin B1 by the mini-plate procedure. Of the 96 samples with BGY-fluorescing locks, 51 contained detectable aflatoxin B1 in the seeds from the fluorescing locks and 45 did not. From the same 96 samples, a random selection of non-BGY-fluorescing locks was made for each sample, and only three showed detectable aflatoxins present in their seeds. In each of these three cases, aflatoxins had also been detected in the seeds from the BGY-fluorescing locks in the same samples. Extracts from seeds of the BGY-fluorescing locks were spotted on 20-by 20-cm Schleicher and Schuell thin-layer chromatography plates, and the levels of aflatoxin B1 were estimated ( DISCUSSION The results here reported on A. flavus infections of cottonseeds are in accord with and substantiate previous evidence indicating that A. flavus boll rot and aflatoxin contamination of cottonseeds at harvest involve only a small fraction of the total U.S. cotton crop (19). Data indicate that all of these phenomena are very uncommon in the Southeast and mid-South. All have been obviously present in the Imperial Valley of California and occur at detectable levels in some still-undefined areas of Arizona. A. flavus boll rot has also been detected repeatedly at low to moderate levels in parts of Texas, especially in the region near Brownsville, but also in an area around Dallas. Evidence indicates that A. flavus boll rot is typical of hot, dry climates and that its incidence may be materially increased by insect attack on the bolls (19). Aflatoxin contamination of the seeds at harvest appears to be very uncommon, if present at all, in any growing area where the BGY fluorescence, caused by A. flavus, is not also easily detectable. Such contamination occurs especially in seeds whose fiber or seed fuzz, or both, exhibit the BGY fluorescence. The above observations provide no evidence regarding A. flavus infection and aflatoxin contamination of the seeds during humid transit and storage, matters still very inadequately investigated. The following comments may be made about the several fungi other than A. flavus which were found in the present work to infect cottonseeds before harvest. Alternaria. Cotton may now be added to wheat (4,6,17,22), barley (7,12,17), oats (17,25), rye (17), soybeans (10,24), corn (17), and the very many other plants (23) whose seeds are known to be infected frequently with this fungus in the field. Some isolates under some circumstances produce mycotoxins (5), but no information on such mycotoxins in the cotton crop is known to have been reported. A. niger. This fungus seems to be present in cotton particularly in the western parts of the U.S. Cotton Belt. This conclusion is based more on previously published fiber-infection data (29) than on the seed-infection data here presented. The fungus appears to be more common in field infections of cottonseeds than in field infections of seeds of other plants. C. gossypii. Extensive data on infections of fiber and bolls show this fungus to be geographically localized in areas with moderate to high rainfall (29, M. E. Simpson, P. B. Marsh, and E. C. Filsinger, Plant Dis. Rep., in press). The seed data reported here are less extensive but seem in general accord with this concept. Nigrospora oryzae (Berkley and Broome) Petch. This fungus has been reported by others to be a common cause of cotton boll rot in California (13) but was found rarely in the present study. If we were to select any major infecting fungus as distinguishing the fungal flora of cottonseeds at harvest from that of most other plants, it would be A. niger. We cannot, however, ascribe infection with this fungus to the oil component of cottonseed nor to any other chemical component. The fact that A. niger is a vigorous wound parasite capable of penetrating from wounded into living tissue of the boll wall may be relevant. In the peanut, also an oil-bearing seed, the fungal population before harvest includes numerous species of Penicillium and Aspergillus (14), but the flora in this case are probably influenced strongly by soil contact. The tendency for A. flavus to occur on small grains, especially under conditions of heating in storage, and the semixerophytic nature of the organism have been noted by Semeniuk (28). Because species of Alternaria and Fusarium cause field infections in the seeds of so many crops that have important food and feed uses, further investigations of mycotoxins produced by these fungi are especially important to U.S. agriculture. Some readers might wonder whether our surface sterilization of cottonseeds in chlorine gas, after delinting in sulfuric acid (20), might kill some fungi found internally in the seeds or whether the chlorine might selectively kill A. flavus. Actually, bolls incubated for several days with A. flavus, as previously described (18), and then examined by this method have shown a high internal seed infection with the fungus; the 61% infection of the seeds from BGY-fluorescing locks recorded in Table 2 also suggests no major killing of the fungus internally in the seeds. Uninfected seeds germinate in high percentage after the chlorine treatment and produce seedlings of normal appearance. Some insight may also be gained by comparing the microbial population of the seeds with that of field-exposed cotton fiber (29) because, in the latter case, no sterilization was used. With the fiber, the microbial population consisted mainly of actinomycetes, Altemaria sp., A. niger, bacteria, C. herbarum, Fusarium spp., and R. stolonifer. In the seeds, all of these were detected except the actinomycetes and C. herbarum. We believe, however, that the absence of these two forms from the detected seed population did not result from their elimination during the sulfuric acid-chlorine treatment. Rather, we think that internal infection of the seeds occurs primarily through the chalazal opening, that the rapidly growing fungi tend to preempt the infection court, and that the slower growing actinomycetes and C. herbarum are eliminated by selective competition. We would agree that a real question about the adequacy of our methods for detection of microorganisms in both cotton fiber and seed may exist in respect to D. gossypina, which probably does not produce recognizable fruiting bodies during the relatively short incubation periods used.

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2018-04-03T03:10:21.988Z

1973-12-01T00:00:00.000Z

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Degradation of Parathion by Bacteria Isolated from Flooded Soil Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathion- amended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite. release nitrite from p-nitrophenol as described for Pseudomonas sp. The same bacterium was also tested for its ability to liberate nitrite from intact parathion. Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite. Folidol, a commercial formulation of parathion (O, O-diethyl-0-p-nitrophenyl phosphorothioate), is extensively used in India for control of common insect pests of rice. Although parathion is known to be relatively less persistent than chlorinated hydrocarbon insecticides, recently this compound was reported to persist for more than 16 years in a sandy loam soil (13). Parathion undergoes rapid degradation in flooded rice soils either via reduction of the nitro group (12) or via hydrolysis at the P-O-C linkage after repeated applications (10). Degradation of parathion by Flavobacterium sp. isolated from diazinon-amended flooded rice soil ceased at the p-nitrophenol stage (11). This paper reports more extensive degradation of parathion past the p-nitrophenol stage by a single bacterium isolated from flooded alluvial soil and identified as Pseudomonas sp. Formation of nitrite as an end product in the degradation of p-nitrophenol by a species of Bacillus was also investigated. MATERIALS AND METHODS Preparation of parathion-hydrolyzing enrichment culture. One milliliter of aqueous 1,000-ppm solution of parathion was added to 20 g of an alluvial soil from the Institute farm at 2-week intervals. The soils contained in test tubes (25 by 220 mm) were flooded with 24 ml of distilled water. Within 24 to 48 h after the third addition, the standing water over the soils in certain tubes turned yellow, indicating the hydrolysis of parathion to p-nitrophenol (10). This standing water, together with soil suspension exhibiting parathion-hydrolyzing ability, was pooled from several tubes and employed as an enrichment culture. Isolation of bacteria. A dilution of the enrichment culture was mixed with molten modified Wakimoto nutrient agar medium (8) and incubated. Isolates from the agar medium were transferred to a sterile mineral solution [(NH4)2HP04, 0.5 g; MgSO4 7H20, 0.2 g; FeSO4 7H20, 0.001 g; K2HPO4, 0.1 g; Ca(NO3)2, 0.01 g; distilled water, 1,000 ml] containing parathion or p-nitrophenol as the sole carbon source. None of the isolates decomposed parathion, but a species of Bacillus capable of decomposing p-nitrophenol was isolated (10). In another experiment, the enrichment culture was serially diluted, and 1 ml of each dilution was incubated with parathion in a sterile mineral solution following the methods described for diazinon-hydrolyzing Flavobacterium sp. (8). The lowest dilution (10-6) which exhibited parathion-hydrolyzing ability was chosen for further studies. The active parathionhydrolyzing agents in the 106 dilution were multiplied by incubating 1 ml of this dilution with 4 ml of sterile mineral solution containing parathion as the sole carbon source. After 48 h, this solution was streaked on a modified Wakimoto agar medium (8). Individual bacterial isolates developing on the agar medium were transferred to a sterile mineral solution containing parathion. The medium which was incubated with isolate P-6 turned yellow within 24 h, and the yellow color faded within the next 24 h. This isolate was further purified and identified as Pseudomonas sp. Degradation studies. The ability of Pseudomonas sp. to decompose parathion was tested as follows. The mineral solution containing aqueous parathion was passed through a membrane filter (Millipore Corp.; 0.45 Am pore size), and 25-ml samples of this sterile solution (pH 7.1) were distributed in 250-ml Erlenmeyer flasks. The medium was inoculated with 0.1 ml of bacterial suspension in sterile distilled water prepared from 3to 7-day-old cultures. The incubation mixture was incubated at 27 C in a biological oxygen demand (BOD) incubator. Uninoculated media served as control. Methods for extraction (11) and analysis (10) of parathion residues in the incubation mixture have been described earlier. Residues in the medium were extracted three times with 20 ml of chloroform-diethyl ether (1:1), and the solvent fraction was pooled. The residues were evaporated to dryness at room temperature and then dissolved in 2 ml of methanol. The PARATHION DEGRADATION residues spotted on 300-Mm-thick Silica Gel G plates were developed for a distance of 15 cm by employing hexane-chloroform-methanol (7:2:1) as a developing agent. After drying, the authentic compounds of parathion and p-nitrophenol were located by spraying the chromatoplate with 0.5% palladium chloride in 2% HCl followed by 2.5 N NaOH. The silica gel areas of the samples corresponding to parathion were scraped carefully and transferred to a test tube. One milliliter of 2.5 N NaOH was added to each tube, and parathion was converted to p-nitrophenol by alkaline hydrolysis in a water bath for 1 h. After cooling, the volume was made up to 25 ml, silica gel was removed by centrifugation, and the supernatant was read in a Klett-Summerson calorimeter employing a 420-nm blue filter. The amount of parathion in the samples was obtained by multiplying the values for p-nitrophenol by 2.094. p-Nitrophenol in the silica gel areas of the samples opposite to the authentic compound was directly eluted in 0.1 N NaOH. After centrifugation of the silica gel suspension, p-nitrophenol in the supernatant was determined colorimetrically against an appropriate blank as described earlier. In a test to determine whether nitrite was formed during the bacterial decomposition of p-nitrophenol, 20-ml samples of sterile mineral solution without (NH4)2HPO4 and Ca(NO3)2 supplemented with pnitrophenol were inoculated with 0.1 ml of a suspension of Pseudomonas sp. in sterile water. The samples were drawn periodically, and nitrite in the samples was analyzed calorimetrically by using sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (2). Bacillus sp. which decomposed p-nitrophenol as the sole carbon source (10) was tested for its ability to release nitrite from p-nitrophenol as described for Pseudomonas sp. The same bacterium was also tested for its ability to liberate nitrite from intact parathion. RESULTS The bacterial isolate P-6 was gram negative, rod, and aerobic, and was identified as a nonfluorescent species of Pseudomonas. The isolate showed many characteristics of P. multivorans Stanier et al. and appeared to belong or to be closely related to this species. Large inclusions, which were presumably polyhydroxy butyrate, were very clear with the Gram stains. All tests for spores were negative. When Pseudomonas sp. was incubated with parathion, the insecticide was rapidly degraded. The color of the incubation medium turned yellow within 4 h of incubation, indicating the formation of p-nitrophenol. At 20 h, however, the yellow color disappeared, evidently because of the further metabolism of p-nitrophenol. Quantitative analysis of parathion and p-nitrophenol confirmed these findings. At 4 h, about 50 g of added parathion was hydrolyzed and 19 gg of p-nitrophenol was recovered as the major metabolite (Table 1). No other metabolite could be detected in the thin-layer chromatogram of the solvent extract. At 20 h, however, parathion was completely destroyed and no p-nitrophenol could be detected. No appreciable degradation of parathion occurred in the uninoculated control during 20 h of incubation. When the bacterium was grown in a nitrogen-free medium with p-nitrophenol as the sole carbon source at 27 C ± 2 C in a BOD incubator, nitrite nitrogen was released from the organic nitro molecule ( Table 2). The amount of nitrite formed was proportional to the amount of p-nitrophenol degraded. Within 16 h of incubation, 157 ug of p-nitrophenol was decomposed, liberating 51 tig of nitrite. In the uninoculated control, nitrite was not detected. In a preliminary resting-cell experiment, nitrite was formed when living resting cells of Pseudomonas sp. were exposed to parathion in phosphate buffer (pH 7.1). These studies indicated that nitrite was formed from p-nitrophenol. Bacillus sp., isolated from flooded alluvial soil, was reported earlier to utilize p-nitrophenol as a sole carbon source (10). In a test to determine the end product formed during the breakdown of p-nitrophenol by Bacillus sp., the bacterium was incubated with p-nitrophenol in a mineral solution as described for Pseudomonas sp. Within 24 h of incubation, 166 tg of p-nitrophenol was metabolized, releasing 43 Mg of nitrite (Table 3). To test whether Bacillus sp. could release nitrite from intact parathion, the bacterium was incubated with the insecticide for 120 h. p-Nitrophenol and nitrite were not formed. Pseudomonas sp. was subcultured repeatedly on parathionor p-nitrophenol-free modified Wakimoto agar. After the fifth subculture, the bacterium was tested for its ability to degrade parathion or p-nitrophenol as the sole carbon source in a mineral solution as described earlier. The bacterium retained its ability to hydrolyze parathion within 3 h of incubation and then release nitrite from p-nitrophenol within 24 h despite five transfers on parathionand p-nitrophenol-free media. DISCUSSION Parathion metabolism in plant and insect systems via oxidation, reduction of nitro group, or hydrolysis is well established (5), but the stepwise degradation of this insecticide in microorganisms has not been investigated (1). Until recently, the major pathway of parathion metabolism in soils and microorganisms appeared to be the reduction of the nitro group. Now, we have clear evidence that parathion can be hydrolyzed biologically at the nitrophenyl C-O-P bond, both in flooded soils after its repeated additions (10) and in pure culture by Flavobacterium sp. (11,12). Degradation of parathion by Flavobacterium sp., however, ceased Iat the p-nitrophenol stage (11). The results presented in this study clearly established the degradation of parathion past the nitrophenol stage by Pseudomonas sp., leading to the formation of nitrite as an end product. Nitrite formed appeared to persist in pure culture studies with Bacillus sp. and Pseudomonas sp. during the 24-h incubation period. However, in flooded soil nitrite does not accumulate (6), because of its rapid denitrification to molecular nitrogen in -an anaerobic environment. Various nitrophenols are known to be metabolized, liberating nitrite in the process. Corynebacterium simplex formed ni-trite from phenolic substrates containing the nitro group in the para position (3). A species of Arthrobacter released nitrite from a herbicide 3,5-dinitro-o-cresol (4). Similarly, both Bacillus sp. and Pseudomonas sp. used in this study readily released nitrite from p-nitrophenol, utilizing the latter as a sole carbon source ( Table 2, 3). This finding is in agreement with that of Raymond and Alexander (7). Pseudomonas sp., in addition, possessed an enzyme system(s) capable of hydrolyzing parathion. This result is apparently the first report on the degradation of parathion to p-nitrophenol and then to nitrite by the same bacterium. Metabolism of parathion in flooded soil or in pure cultures of microorganisms isolated from flooded soil involves (i) nitro group reduction, leading to the formation of aminoparathion as a major metabolite (9); (ii) hydrolysis to p-nitrophenol as a major metabolite which resists further degradation (11); and/or (iii) hydrolysis to p-nitrophenol followed by the formation of nitrite (this report). The findings reported in this paper show that microorganisms in flooded soils contribute to the rapid breakdown of parathion via the hydrolytic pathway to the end product, nitrite.

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2020-12-10T09:04:16.695Z

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Trends in Wort Carbohydrate Utilization A gas chromatographic method suitable for any type of low-molecular-weight carbohydrate analysis has been utilized to determine the individual wort sugars in corn adjunct wort from a Western Canadian brewery. The fluctuations in each sugar during primary lager fermentation have been graphed. “End fermented” wort has been shown to contain some maltotriose, a small amount of maltose, and the nonfermentable carbohydrates, including maltotetraose, maltopentose, and dextrins. Qualitative and quantitative carbohydrate analyses of wort have become increasingly more important to the brewer to detect differences in the composition of wort from brew to brew, mainly to predict or ensure the degree of attenuation (10). For a number of years, however, reliable analytical work on wort or beer carbohydrate composition depended on the timeconsuming separation of individual carbohydrates by paper chromatography followed by elution, and the application of a colorimetric and quantitative chemical assay (15,16,25). Most often, therefore, only fermentable carbohydrate (3, 10), or total carbohydrate by the anthrone method (9,17,21) or by specific gravity determination (5), is followed. The desire to eliminate tedious paper chromatographic methods has led to interesting developments in gas-liquid chromatographic (GLC) analysis (22). The application of this newer method to carbohydrates, however, awaited the contribution of Sweeley et al. (24), who prepared and chromatographed a number of carbohydrates in the form of volatile trimethylsilyl derivatives. Since that time, a number of other papers have appeared (for review, see references 20 and 22). For the brewing industry, the reports by Marinelli and Whitney (12,13), Otter and co-workers (18,19), and Clapperton and Holliday (4) have been most significant. The application of this method for individual sugar analysis, except in quality control of initial wort, or final beer, has been more limited. For example, the work of Harris and co-workers (7, 8) with paper chromatography has not been extended to show the disappearance of individual sugars in wort during commercial fermentation by using the more sensitive and more rapid GLC method, even though Griffin (5, 6) has used the method with the top fermenting ale yeast, Saccharomyces cerevisiae, growing in a stirred laboratory fermenter. A gas-liquid chromatography system in the present study has been used to qualitatively and quantitatively determine the fermentable carbohydrate levels in beer wort throughout the course of a Western Canadian commercial lager fermentation. We have made use of this method to apply the results toward a study on the effect of yeast environment on flocculation of S. carlsbergensis (manuscript in preparation). MATERIALS AND ME'rHODS Fermentation. A typical closed 8,600-gallon (39,000-liter) brewery fermentor was used, maintained at 57 F (14 C) throughout primary fermentation until the cooling stage was reached. Samples were taken from a lower port and a higher port (upper) near the center of the tank. Only the lower port samples have been discussed and plotted. Gas chromatography (12,13). All GLC analyses were performed with a Hewlett Packard model 5750 B chromatograph equipped with a thermal conductivity detector at 355 C with a 120-mA bridge current. The column used was copper (2 ft by 1/4 inch [60.96 by 0.635 cm] outside diameter) packed with 3% SE-52 Silicone gum rubber on 60/80 mesh Chromosorb W AW-DMCS (Chromatographic Specialties, Brockville, Ontario). The carrier gas was 80 ml of helium per min. The oven program from 150 to 350 C was carried out at a rate of 6 to 10 degrees per min with the injection port at 375 C. An attenuation of 1 was usually used. The strip chart recorder was a Hewlett Packard model 7127A run at 0.5 inch (1.27 cm)/min. The method described by Marinelli and Whitney (12,13) was used to prepare trimethylsilyl derivatives but with the incorporation of phenyl-ft-D-glucopyranoside as an internal standard for quantitation. To each vial containing 0.5 ml (approximately 60 mg solids) of wort was added 1.0 ml of pyridine (silylation grade, Pierce Chemical Co., Rockford, Ill.) containing 5 mg of phenyl--D-glucopyranoside per ml. Then 0.9 ml of 1,1,1,3,3,3-hexamethyldisilazane and 0.1 ml of trifluoracetic acid (Aldrich Chemical Co., Milwaukee, Wis.) were added. The vials were shaken for 30 s and then allowed to stand at 20 to 22 C for 15 min with occasional shaking. Reactions are quantitative using this method (20). When insoluble components were seen, the reaction mixtures were warmed prior to injection. Injections of 10 to 50 liters were done in duplicate by using a Hamilton syringe (Hamilton Co., Whittier, Calif.). Peak areas were calculated by cutting and weighing xeroxed peaks. The retention times were obtained by injection of standard solutions of sugars found in wort. Relative response values were also calculated for all sugars of lower molecular weight than maltotriose. Yeast dry weight determinations. Wort samples were centrifuged by using a Sorvall SS-1 super-speed angle centrifuge (Ivan Sorvall Inc., Norwalk, Conn.) at a relative centrifugal force greater than 5,400 x g. Supernatant fluid was discarded, and cells were washed twice and resuspended in 0.05 M phosphate buffer (pH 6.4) in h6s the volume of the original sample. Triplicate 2-ml samples of the resuspensions and of the resuspending buffer were transferred to preweighed aluminum foil pans. Dishes were dried to constant weight at 105 C, and cell mass/ml of wort was calculated. Chemical determinations. Total N in wort was determined by the Kjeldahl method as adapted by Bremner (2). Prior heating of samples in 0.5 ml of concentrated H2SO4 was used to prevent excessive foaming during digestion (1). The distillate was titrated with standard H2S04 to an end point of pH 4.9 by using a Radiometer automatic titrator type TTT1 (Radiometer, Copenhagen, Denmark). Protein nitrogen in wort was estimated by using the method of Lowry (11) with crystalline (X3) egg albumin (Nutritional Biochemical Corp., Cleveland, Ohio) as standard. Results were read at 750 nm (9) on a Spectronic 20 colorimeter (red phototube and filter). Triplicate anthrone tests for total carbohydrate in wort and cell samples were carried out by the method outlined by Herbert et al. (9), adapted for more reproducibility from Morris (17). Standard curves were prepared for each essay. A Beckman B spectrophotometer at 625 nm rather than a colorimeter was used as advised by Herbert et al. (9). (14). Primary fermented wort in Table 1 is actually beer after the initial 5 to 6 day fermentation and cooling cycle. At this time, all fructose, sucrose, and glucose, most of the maltose, and 80% of maltotriose have disappeared from the wort (94% of all fermentable sugar). Maltotetraose, maltopentose (detected but not quantified by this GLC method), and the larger-molecularweight, nonfermentable dextrins remaining after primary fermentation are not attacked by S. carlsbergensis, but were measured by anthrone. RESULTS AND DISCUSSION In Fig. 1, the trends of total fermentable carbohydrate, wort nitrogen levels, and yeast mass in suspension have been recorded. The decrease in total nitrogen is almost entirely a decrease in low-molecular-weight nitrogen compounds (amino acids, small peptides, and inorganic nitrogen). Protein nitrogen, for example, as measured by the Lowry method (11), shows little decrease during the fermentation, with some of this decrease due to utilization of Lowry-positive amino acids. Yeast mass throughout this time increases dramatically (450%) in the wort. In the later stages the decrease in mass is due to the phenomenon of flocculation, as yeast sink by the lower sampling port to sediment at the bottom. Figure 2 is a representation of the fermentable carbohydrates in a typical chromatogram of corn adjunct brewers wort. By using peak weights (areas) and the calculated relative response values in Table 2, Fig. 3 thoughout primary fermentation. Figure 3 reflects the ease of following environmental changes in a medium by this method compared to previous studies (7, 8). Sucrose decreased rapidly to an undetectable level during the first 5 h of the fermentation. The sucrose probably is hydrolyzed by invertase (sucrase), a yeast enzyme situated between the cell wall and cell membrane (23). However, Harris et al. (7) state that glucose is used first from wort, followed by fructose and then sucrose. In this experiment, the sucrose was hydrolyzed first. Fructose levels increase to the 5-h sampling period, reflecting hydrolysis of su- Phenyl-ft-D-gluco-4.38 Internal standard pyranoside a RRV/mg of sugar = (area sugar peak x attenuation/area internal standard x attenuation) mg of sugar in 0.5 ml of freeze-dried simulated wort. Amount of sugar (mg/100 ml) in unknown wort = (200 x area sugar peak (wort) x attenuation/area internal standard x attenuation) RRV/mg of sugar. b The maltose response factor may be used for maltotriose and maltotetraose, since it is difficult to purchase these compounds in pure state (Marinelli, personal communication, 1972). 24 Glucose, 16%, is unchanged for the first 4 to 5 h, probably because its rate of utilization is comparable to its rate of formation from sucrose. However, by 24 to 48 h, measurable levels of glucose disappear. Maltose and maltotriose hydrolysis to glucose must therefore be slower than glucose utilization. Maltose (66% of fermentable sugar) is not significantly attacked during the first few hours, presumably because maltase permease or the hydrolysis reaction are glucose repressed. A very rapid utilization is seen between 10 and 50 h, eventually resulting in 93 to 96% utilization. Maltotriose utilization is similar to maltose, eventually leading to 75 to 80% metabolism by the yeast. Residual maltose and maltotriose are attacked in subsequent fermentation and aging steps in the brewing process, since bottled beer from this process contains only nonfermentable carbohydrate and small amounts of maltotriose. The ability to monitor wort carbohydrates or any other carbohydrates by gas chromatography is certainly an advantage to a quality control or research laboratory. The method is rapid, quantitative, and can be applied to a large variety of studies, including lactose in milk, maltose and glucose ratios in syrups, and enzymatic attack on carbohydrates. It has already been used to detect glucose, fructose, and sucrose levels in tomato, cabbage, apple, carrot, and potato tissues (22

v3-fos

2020-12-10T09:04:17.326Z

1973-08-01T00:00:00.000Z

237231060

s2

Induction of Red Color Formation in Cabbage Juice by Lactobacillus brevis and Its Relationship to Pink Sauerkraut Membrane-filtered cabbage juice, when fermented by Lactobacillus brevis under conditions of controlled pH, frequently produced a water-soluble red pigment. The pigment, presumably responsible for imparting a highly objectionable discoloration to sauerkraut, was formed during the post logarithmic phase of growth. Color development is pH dependent (5.2 to 6.3) and can be suppressed by chemical reductants or anaerobic conditions of growth. The compound responsible for discoloration was purified and partially characterized. The grade of commercial canned sauerkraut is established by a composite value derived from the singular evaluations of character, cut, defect, flavor, and color. Of these factors, color and flavor are given the highest, but equal, considerations (1). The maintenance of a bright, cream to light straw color is necessary for producing a high-quality fermented product. The inability to conform to the highest color standard results in the downgrading of quality; if discoloration is serious, the product may be rejected. One such serious color defect is the formation of red or pink kraut. One type of discoloration is a light pink to deep burgundy coloration, which causes considerable economic losses to processors in Holland (10) and the United States. Since climatic conditions and the varieties of cabbage grown for kraut production vary widely within each locale, it would appear that factors related to the fermentation processes are primarily responsible for inducing the objectionable color formation. Furthermore, the red color develops during the course of the fermentation or immediately prior to processing. The initiation of red color formation in cabbage and sauerkraut apparently proceeds via a series of complex chemical reactions. Recent studies pertaining to the effects of dehydration upon cabbage indicate that ascorbic acid -amino acid interactions are responsible for ' Approved by the Director of the New York State Agricultural Experiment Station as Journal Paper No. 2033, April 24, 1973. producing non-enzymatic discoloration in the dried product (6), whereas in pink kraut the color has been attributed to the formation of a leucoanthocyanidin (4). Since the kraut fermentation arises as a result of a heterogeneous microbial population, it is difficult to assess the role each species contributes to color formation. Although yeasts are known to impart color to kraut (2, 3, 5, 10; E. Steinbuch, Antonie van Leeuwenhoek J. Microbiol. Serol., Yeast Symposium Suppl. no. 35, F39, 1969), the potential induction of red color by pure cultures of lactic acid bacteria associated with the "normal" fermentation of kraut has, to our knowledge, never been reported. Therefore, this paper describes those conditions which contribute to the onset of discoloration, with particular emphasis on the role of Lactobacillus brevis, in inducing a cerise color in filter-sterilized cabbage juice. Growth measurements. Fresh cabbage juice (variety Glory) was prepared as previously described (9). The cabbage sera, sterilized by membrane filtration in a glass assembly, were dispensed aseptically in 10-ml volumes (16-by 150-mm test tubes) for growth and color studies. Each sample was inoculated with one loopful (about 1.8 x 104 cells/ml) of a 24-h culture grown previously in cabbage juice. The viable cell counts were taken at 24-h intervals and were esti-161 mated by plating on tryptone-glucose-yeast extractsalts agar medium (8). Sodium hydroxide (0.5 N) or solid calcium carbonate were used for maintaining preselected pH values. Sterile CaCO., when added to cabbage juice at concentrations of 0.25, 0.50, 0.75, 1.0, and 1.5% (wt/vol), provided final pH values of 4.2, 4.4, 5.2, 5.8, and 6.0, respectively, whereas the maintenance of pH by NaOH was controlled by pH-stat. In addition to using smaller volumes (test tubes) for the studies of color and growth development, larger quantities of juice (40 to 80 ml) were fermented in a sterile fermentation assembly equipped with sampling ports, a magnetic stirring bar, and pH electrodes and maintained at 32 or 22 C by a constant temperature water bath. Measurement and purification of colored materials. Since it was desirable to establish the chemical composition of the unknown product(s), larger volumes of juice (5 to 10 liters) were fermented in the hope of obtaining increased quantities of red pigment. It was found, however, that in addition to being difficult to filter-sterilize, the fermented juice was extremely vulnerable to extensive browning, a condition which resulted in excessive losses in the yield of red color. It was observed that juice, when prepared in 150-ml quantities and contained in 250-ml Erlenmeyer flasks was more stable, and therefore was used for subsequent fermentation studies. After fermentation and maximum color production (about 8 days), the juices were centrifuged (20,000 x g) for 20 min. The clear, red juice was extracted four times with 25-ml volumes of diethyl ether. The aqueous phase, containing the red color, was concentrated sixfold under vacuum at 40 C. The red concentrate was treated with acetone (85% saturation) and filtered through paper. The resulting supernatant fraction was evaporated to dryness, dissolved in 10 ml of water, and applied to a polyvinylpyrrolidone (Polyclar AT) column (2.5-by 10-cm) which had been previously equilibrated with water. The column, retaining the adsorbed pigment, was washed with five bed-volumes of water and was subsequently eluted with 900 ml of methanol containing 0.01% hydrochloric acid. After elution, fractions containing the red pigment were concentrated to 0.2 ml, applied as a band on preparative cellulose thin-layer chromatography (TLC) plates, and further purified in the following solvent systems: (i) water-acetic acid-hydrochloric acid (80:25:5); (ii) 2% aqueous acetic acid; and (iii) upper phase of butanol-acetic acid-water (4:1:5). After irrigation, the red band was scraped from the plate, eluted with methanol (50 ml), and concentrated to 10 ml. A sample of the concentrate (4 ml) was subjected to ultraviolet light (UV) and visible spectroscopic analyses. The remainder of the concentrate (5 ml) was evaporated to dryness and the residue was mixed with KBr (0.10 g) and pressed into micropellets for infrared analysis. The sprays used for the qualitative analyses of functional groups included: 2,6-dichloroquinone chlorimide, ferric chloride, and phosphomolybdic-phosphotungstic acid, for phenolic compounds; silver nitrate for reducing materials; and ninhydrin for amino acids. RESULTS AND DISCUSSION While studying the effects of pH upon the growth rates of lactic acid bacteria (9), it was observed that L. brevis, when grown in cabbage juice containing calcium carbonate, imparted a brilliant red color to the fermented extract. Of the five microorganisms commonly associated with the kraut fermentation, L. brevis was the only species which induced color formation in the buffered juice (Table 1). Under these conditions, L. brevis produced a fivefold increase in color (A,,6) within 7 days of incubation at 32 C. In unbuffered juice, the culture produced no apparent changes in color, and the final absorbance values were similar to those displayed by the non-color-forming species. The inability of the cultures, other than L. brevis, to produce a red color in buffered juices cannot be attributed to differences in growth-sustaining properties of the buffered and regular extracts. This was confirmed by plate counts; each juice, when inoculated with about 1.8 x 10' cells per ml, provided final populations of 9 x 108 to 1.2 x 10' cells per ml after 3 days of incubation at 32 C. The rates of color formation as a function of the growth of L. brevis at two temperatures (32 and 22 C) in cabbage are shown in Fig. 1. It may be observed that the growth rates at each respective temperature in both buffered and unbuffered extracts were quite similar. After 5 days of incubation at 32 C, the juice containing no CaCO. yielded a total viable population of 5 x 108 cells per ml. During this time period the initial pH of the medium was abruptly lowered from 6.2 to 3.8, a value which remained constant throughout the 21-day incubation. No red color was formed under these conditions of growth. Although CaCO, had no apparent effect upon growth, it induced most markedly the development of red color. Color production was initiated at about 5 days of incubation at 32 C and reached its maximum intensity 5 days later (Fig. 1). It appeared that color production occurred during the latter stages of postlogarithmic growth and attained its maximum intensity during the stationary growth phase. Incubation beyond 10 days invariably resulted in a marked decrease in absorbance at 558 nm, and after 21 days of incubation the maximum color intensity was reduced more than 60%. The reduction in absorbance at 558 nm was accompanied by a concomitant increase in the 500-nm region suggesting the onset of browning. A comparison of color formation as a function of temperature is also shown in Fig. 1. As might be expected, a 10 degree down-shift in temperature, i.e., from 32 to 22 C, reduced both growth and color development rates by nearly 50%. At 32 C, 6 days were required to achieve maximum cell yields and 8 to 10 days were required for the formation of maximum color intensity, whereas at 22 C, 13 and 19 days were required, respectively, to achieve similar results. Again, as in the case of incubation at 32 C, color production was initiated during the latter phases of growth and occurred only in the juice containing CaCO,. To determine if CaCO, served as a buffering or chelating agent, the effects of various monoand divalent ions upon color production were examined. The addition of the chloride or sulfate salts of calcium, magnesium, manganese, iron, sodium, and potassium, when supplied at a concentration of 0.5%, produced B, viable cell count in juice-containing 1.0% CaCO.; C, red color formation injuice containing 1.0% CaCO,; final pH 5.2; D, color production in regularjuice, final pH 3.8. no enhancement in color response. Therefore, it was concluded that CaCO, played the role of a buffering agent in invoking the color devlopment. Additional evidence that pH was involved, in part, in color formation, was established by growing the culture in cabbage juice with varying concentrations of hydrogen ions. The culture, when maintained at constant pH by sterile NaOH (dispensed by pH-stat) provided color intensities similar to those obtained by the CaCO,-containing systems. As reflected in the increased absorbance values at 558 nm, pH markedly influenced color formation (Fig. 2). Increased quantities of red color were formed as the pH was raised from 4.5 to 6.0. Approximately equal levels of red color were attained in cultures constantly adjusted to pH values of 6.0 and 6.3; higher pH values were not studied. Since the growth rate of L. brevis is suppressed by lowering the pH of cabbage juice (9), the absorbances reported in Fig. 2 represent the maximum values attained as a result of growth at each respective pH. The absorbances observed at pH 5.0 or greater were obtained after 9 days of incubation, whereas those intensities produced at pH 4.5 or less were recorded after 21 days of incubation. These latter prolonged periods of incubation were used to permit the development of maximum color intensities under more acidic conditions. Direct microscopic count showed that each sample reached a minimum population of 9 x 108 cells per ml during the course of the fermentation. The failure of these extended incubations to provide color intensities equal to those observed under the more alkaline conditions show that pH plays a vital role in inducing color formation. This dependence of maximum color formation upon pH, i.e., >5.2, appears to be similar to that reported for the induction of color in dried cabbage by chemical mechanisms (6). However, the route of color generation in the cabbage extracts differs from that of the dehydrated product, in that color development in fermented juices is not only pH dependent, but also requires the presence of L. brevis. Further studies concerning the significance of the bacterium in initiating color response and the inability of sterile sera to undergo spontaneous color changes as a result of chemical or inherent enzymatic reactions likewise were investigated. A 48-h, unbuffered cabbage juice culture, containing 5.5 x 10' cells per ml, showed no evidence of color. However, when 25-ml samples of this culture (pH 3.9) were adjusted to pH 5.5 with NaOH (40%) and 1, color production was initiated absorbances arising as a result of bacterial L (Fig. 3). Maximum color intensity growth under atmospheres comprised of varying Ed after 4 days of incubation; viable air and nitrogen compositions show that aeros remained at about 1.1 x 10' per ml bic conditions were most conducive to color this period. No color development formation (Fig. 4). Although each juice, buf-'ig. 3) when the pH of the juice (3.9) fered at pH 5.7, showed evidence of color, those ared or when the pH of the juice was extracts incubated in the highest air-nitrogen 5.5 and the juice was sterilized by atmospheres (90 to 100% air, respectively), Dfore it was reincubated. provided intensities threefold greater than the ten fermented in 10-ml volumes for 8 corresponding extract grown under nitrogen t 32 C, often possessed a reddish hue only. liquid interphase. This suggested In addition to suppressing color formation, color formation. A comparison of the less aerobic conditions of growth produced lower cell yields in the unbuffered extracts than in the buffered series. As shown in Fig. 4 (8), pH also appears to produce vital interations in limiting growth in cabbage exfects of pH upon red color production by tracts. wn in buffered cabbage juice at 32 C. A, Since discoloration was initiated in part by fermented juices. pH maintained by aerobic conditions, the use of chemical reduc-3or NaOH (0.5 N) dispensed by pH-stat. Effects of pH adjustment upon induction of red color development by L. brevis grown in cabbage juice. Cabbage juice (75 ml) was fermented at 32 C for 48 h (to pH 3.9) and divided into three samples: A, no pH adjustment; B, pH adjusted to 5.5, and then the juice was filter-sterilized; and C, pH adjusted and maintained at 5.5 in a pH-stat. All samples were reincubated at 32 C. 164 reincubated within 16 h was achieve cell number throughout occurred (F was not alte adjusted to filtration be Juice, wh to 10 days a at the airair-induced Fig. 5. (Although S-methyl-cysteine is a nonreducing compound, it is a major sulfur amino acid found in cabbage [11] and its role as potential reductant remains to be clarified.) Of the above compounds examined, ascorbic acid was the more effective color suppressant at lower concentrations (less than 1 mg/ml) than was either cysteine or glutathione, whereas the reducing sulfur materials (cysteine and glutathione) were more effective retardants of color at higher concentrations (2.5 mg/ml). It was also observed that, once the pigment had formed, the color could not be reversed by the addition of the above compounds. Since Smethyl-cysteine was without effect, it appears that this compound provides no color-reducing properties in the kraut fermentation. Attempts to increase the final yield of red pigment by fermenting cabbage juice concentrates consistently resulted in producing a dark brown solution containing little red color. Lyophilized juice, reconstituted to provide 2-to 10-fold increases in soluble solids, or when added to regular juice to provide 2-fold concentrates, resulted in similar failures to produce a bright, red color. This loss in red color also occurred when fermented juices were stored at -50 C for 3 days. Not only is the red color unstable under conditions of rapid freezing and thawing, but it is labile to heat treatment. For example, the color intensities of two fermented juices, pH 3.5 and 5.5, decreased 35% when immersed in boiling water for 30 min. The effects of heat shifted the 558/500 nm ratio from 1.78 to 1.13 and produced browning. This suggests that temperatures used for processing E 0. commercial kraut (75 C) cannot be used advantageously for eliminating red color formation without imparting deleterious discolorations. At room temperature, the pigment is more stable under acidic than alkaline conditions. The adjustment of pH from 4.5 to 1.0 produced no significant change in absorbance at 558 to 568 nm (Fig. 6). At pH 8.5 the extract showed no visible absorption. Upon acidification with hydrochloride (concentrated) the alkaline solution regained its original red color. Although this pH-dependent color response is reversible, the yield, as measured by peak areas, decreased nearly 50%. The inability to extract the red color with ethers (petroleum or diethyl), amyl acetate, FIG. 6. Effect of pH upon the stability of red pigment produced in buffered cabbage juice by L. brevis. Juice (pH 5.2) fermented 8 days at 32 C, centrifuged, and pH adjusted to: A, 4.5 (concentrated hydrochloric acid); B, 1.0 (concentrated hydrochloric acid); C, 8.5 (40% NaOH); D, (C) acidified to pH 1.0 (concentrated hydrochloride) and absorbances were read immediately. chloroform, or n-butanol, but its complete solubility in water-miscible reagents, such as methanol, ethanol, and acetone, show that the pigment is an extremely hydrophilic compound. This pigment, purified by column chromatography and then applied to TLC cellulose plates and irrigated in three solvent systems, provided a visible, singular, red band with the following R, values: water: acetic acid: concentrated hydrochloric acid (80:20:5), 0.09; water: acetic acid (98:2), 0.11; butanol: acetic acid: water (4:1:5), 0.38. The above pigment, when subjected to various spray reagents, failed to show the presence of amino acids, reducing, aromatic, and indole constituents. Spectral analyses of the acidified methanolic elute (Fig. 7) show that the purified pigment vossessed three absorbance peaks, at 558, 272, and 226 nm, respectively. Although these UV absorbances show similarities to the spectral properties assigned to the red constituents of kraut by Gorin and Jans (4), the visible absorbance of the pigment produced by pure culture fermentation occurred at a wavelength considerably higher than they had reported (558 versus 540 nm). Furthermore, the infrared spectrum of the pigment (in KBr) showed absorption bands at the following frequencies: 675 (w); 746 (m); 1,045 (w); 1,075 (m); 1,125 (s); 1,283 (s); 1,387 (s); 1,470 (m); 1,610 (s); 1,737 (s); 2,870 (s); 2,940 (s); 2,960 (s); and 3,500 (m) cm-1. (Abbreviations [absorption intensities]: w, weak; m, medium; s, strong.) A correlation between the above band positions and types of grouping present indicates that the pigment (i) is aliphatic in character (strong signals in the 2,960-2,870 cm-' range), (ii) contains a carbonyl group(s) (1,737 cm-1), (iii) contains a methyl group(s) (1,387 cm-1), (iv) contains a number of hydroxvl groups (3,500 cm 1). Therefore, these data suggest that the red discoloration produced by L. brevis is not a flavonoid, anthocyanin, or anthocyanidin, but rather that the pigment is a saturated aliphatic ester, aldehyde, or diketone, or contains a five-membered ring ketone within its structure. Further determinations of the exact structure of the cabbage pigment were hampered by the difficulties encountered in producing large quantities of the red cabbage juice and by the general lability of the compound responsible for this discoloration.

v3-fos

2020-12-10T09:04:16.588Z

1973-12-01T00:00:00.000Z

237234766

s2

Virus Particles from Conidia of Penicillium Species Virus particles and their component double-stranded ribonucleic acid (dsRNA) have been isolated from conidia and mycelia of certain Penicillium species. The conidia and mycelia of P. stoloniferum NRRL 5267 contained 75 and 85 μg of dsRNA/g (dry weight), respectively. Of the total dsRNA released from NRRL 5267 conidia, 10% was nonencapsulated. Conidia of P. brevi-compactum NRRL 5260 and P. chrysogenum Q-176 contained 2 and 120 μg of dsRNA/g (dry weight), respectively, whereas mycelium from the two species contained 3 and 95 μg of dsRNA/g (dry weight), respectively. No viruses were isolated from conidia or mycelia of P. stoloniferum NRRL 859. A method is described for disruption of both conidia and mycelia. The technique facilitates the isolation and characterization of fungal viruses and their component dsRNA and also potentiates surveying of fungal isolates for the presence of virus. Polyhedral virus particles have been reported in a number of fungal species (5,12). Ellis and Kleinschmidt (11) were the first to present evidence that viruses occur in Penicillium stoloniferum. Banks et al. (3) isolated and characterized viruses and their component double-stranded ribonucleic acid (dsRNA) from P. stoloniferum. Bozarth et al. (6) described two serologically distinct viruses having different electrophoretic mobilities from P. stoloniferum NRRL 5267. Wood et al. (20) isolated a virus from P. brevi-compactum and demonstrated the double-stranded nature of its component RNA. The component dsRNA of a virus from a penicillin-producing strain of P. chrysogenum was characterized by Lemke and Ness (14). Viruses have been observed throughout the cytoplasm in thin sections of conidiospores of P. stoloniferum and P. brevi-compactum (13), of mushroom basidiospores (10), and of the zoospores of Plasmodiophora (1). However, the isolation and biochemical characterization of virus from fungal conidia have not been reported. We have isolated and characterized viruses and their component dsRNA from conidia of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, and P. chrysogenum Q-176. Although Banks et al. (4) have described an efficient pilot plant-scale isolation of viral dsRNA from several species of Penicillium and Aspergillus and Moffitt and Lister (17) have recently reported a rapid method for surveying fungal isolates for dsRNA-containing viruses, we developed a method for both the detection and isolation of viruses and dsRNA from subgram quantities of conidia and mycelia. The release of macromolecules, with time of disruption of conidia, provides the information necessary for quantitatively comparing concentrations of viral dsRNA in conidia and mycelia. The comparison provides an insight into the possible mode of transmission and rate of replication of viruses from the conidial to the hyphal stages of growth. MATERIALS AND METHODS Production and harvest of conidia. Cultures of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, P. chrysogenum Q-176, and P. stoloniferum NRRL 859 were furnished by the Agriculture Research Service Culture Collection, Northern Regional Research Laboratory. These cultures were maintained on potato-dextrose agar (PDA). Conidia of the organisms were produced by the method of Sansing and Ciegler (unpublished), as follows. White bread, containing no preservatives, was cut into 1.5-cm cubes; 200 g of the cubed bread was placed in a 2.8-liter Fernbach flask and autoclaved for 15 min. The bread was inoculated with 20 ml of a conidial suspension from PDA slant cultures and incubated for 10 days at 28 C. Conidia were harvested from the bread by adding 800 ml of a 10-'% solution of Triton X-100 and shaking the flask, after which the conidial suspension was filtered through cheesecloth to remove the bread cubes and the suspension was filtered through glass wool to remove bread fines. Conidia were pelleted by centrifugation at 2,000 x g for 15 min. The conidial pellet was suspended in 0.1 M potassium phosphate buffer, pH 7.2, and recentrifuged. This step was repeated twice to remove any starch particles remaining from the bread. Disruption of conidia. Known concentrations of conidia (as given under individual sections) were suspended in 0.1 M phosphate buffer (pH 7.2) and added to 75-ml glass Bronwill cell homogenizer flasks, each containing 45 g of 0.5-mm glass beads. Each sample was homogenized for a specific time (see assay sections) in a Bronwill mechanical cell homogenizer (Braun model MSK) at 4000 rpm under a cold CO2 stream. All homogenizing flask temperatures were maintained at 0 to 1 C. Samples of homogenates were examined microscopically to determine the percentage disruption of conidia. Disruption of mycelia. A 10% sucrose-2% yeast extract medium was inoculated with a conidial suspension and incubated at 28 C on a Brunswick shaker at 250 rpm in 2.8-liter Fernbach flasks (500 ml of medium) for 72 h. Mycelia were harvested by vacuum filtration and then suspended in 0.1 M phosphate buffer, pH 7.2 (5 ml of buffer/g [wet weight ] of mycelium). The suspension was added to 75-ml Bronwill flasks containing 45 g of 1.0-mm glass beads and homogenized for 3 min at 4000 rpm under a CO2 stream. Detection of virus. The homogenates resulting from the disruption of 1.5 g (dry weight) of conidia of each of the four Penicillium strains were centrifuged at 2,000 x g (10 min) to remove cell debris as described in Fig. 1. The supernatant fluid (SNF) was centrifuged at 105,000 x g for 2.5 h. The resulting pellet was suspended in 2 ml of 0.1 M phosphate buffer, pH 7.2, and centrifuged at 4,000 x g for 10 min. The supernatant was filtered through a 0.45-gm membrane filter (Millipore Corp.). The virus preparation was applied to carbon-coated Formvar grids, stained with 0.5% uranyl acetate, and rinsed twice with distilled water. The grids were examined by electron microscopy (RCA EMU-3 electron microscope) at an instrument magnification of x 32,000. The viruses were analyzed by polyacrylamide gel electrophoresis on 2.4% gels for 5 h (6 mA per tube) at 25 C as described by Loening and Ingle (15). Gels were scanned at 260 nm with a Gilford linear transport system. Isolation and quantitation of viral dsRNA. As shown in Fig. 1, two volumes of cold methanol were added to the SNF from each conidial homogenate, and the precipitates were sedimented by centrifugation at 2,000 x g for 10 min. Each precipitate was dissolved in 0.2 M sodium acetate and treated with an equal volume of aqueous 90% phenol containing 0.1% (wt/vol) 8-hydroxy quinoline (9). The mixture was shaken for 20 min at 25 C and centrifuged at 4,000 x g for 20 min. Nucleic acid was freed from phenol by repeated precipitation from 0.2 M sodium acetate with equal volumes of cold methanol. The nucleic acid precipitate was dissolved in a minimal volume of 0.15 M NaCl-0.15 M sodium citrate (SSC) solution, pH 7.4. The RNA samples were incubated with 1.0 gg of ribonuclease B (Sigma Chemical Co.) per ml of 0.3 M STE buffer (0.3 M NaCl, 0.01 M tris(hydroxymethyl)aminomethane, and 0.001 M ethylenediaminetetraacetic acid) at 37 C for 30 min. The remaining RNA was precipitated with cold methanol, the mixture was centrifuged at 8,000 x g for 10 min, and the pellet was redissolved in SSC buffer and subjected to electrophoresis. Mobilities of RNA components were compared to those of a purified dsRNA preparation (P. stoloniferum NRRL 5267) of known concentration and standard yeast transfer RNA (Sigma Chemical Co.). The dsRNA was measured by integration of the areas under the electrophoretic peaks compared to standards. Time release of macromolecules. Three 1.5-g (dry weight) samples of P. stoloniferum NRRL 5267 conidia were homogenized for 1, 2, and 4 min, respectively, by the previously described method. Three 3.0-g and three 6.0-g samples (dry weight) of conidia were homogenized for the same time intervals. Cell debris was removed from homogenates by centrifugation. The SNF was quantitatively assayed for total RNA, deoxyribonucleic acid (DNA), protein, and trehalase by the following methods. Quantitation of total RNA, DNA, and protein in conidial homogenates. Supernatants from centrifuged conidial homogenates were assayed for RNA by the orcinol method of Brown (7) and for DNA by the method of Burton (8). The pellet resulting from the hot trichloroacetic acid treatment of homogenates was dissolved in 1 N NaOH by heating at 90 C for 30 min. This solution was quantitatively assayed for protein by the method of Lowry et al. (16). Determination of trehalase activity. Trehalase activity was assayed by adding 0.1 ml of SNF to test tubes containing 2.0 ml of trehalose (2.0% wt/vol) in 0.1 M potassium phosphate buffer (pH 5.6) and incubating them at 25 C for 1 h. Glucose was assayed by the method of Nelson (19). Ratio of encapsulated to nonencapsulated dsRNA. Five 3.0-g (dry weight) samples of P. stoloniferum NRRL 5267 conidia were homogenized for 2 min. Cell debris was removed, and the resulting SNFs were pooled and centrifuged at 105,000 x g for 2 h to pellet virus particles. The virus-free SNF was quantitatively assayed for dsRNA. The virus pellet was suspended in 10 ml of 0.1 M phosphate buffer (pH 7.2), and 5 ml was assayed for viral dsRNA. The remaining suspension was diluted to 45 ml with buffer and homogenized in a Bronwill flask containing 45 g of 0.5-mm glass beads, as described for disruption of conidia. This homogenate was centrifuged at 105,000 x g for 2 h, after which SNF was assayed for dsRNA. Detection of virus and dsRNA in conidia. Analyses of conidial homogenates of P. stoloniferum NRRL 5267, P. brevi-compactum NRRL 5260, and P. chrysogenum Q-176 by electron microscopy demonstrated the presence of isometric virus particles similar in size to those reported from mycelia (2,6,11,13,14,18,20). Their electrophoretic mobilities were identical to those determined from mycelial sources in this investigation. No particles were detected in conidial or mycelial homogenates of strain NRRL 859 of P. stoloniferum. Electrophoretograms of viral dsRNA isolated from conidia of the three Penicillium species, after treatment with ribonuclease, are depicted in Fig. 2. The five dsRNA bands observed for the fast-and slow-moving virus species of P. stoloniferum are identical to bands observed from mycelial extracts (6). No attempt was made to determine concentrations of the fastand slow-moving dsRNAs. The virus particles from P. brevi-compactum conidia contained the same three dsRNA species reported for mycelial extracts of the organism (20). Similarly, the three distinct bands observed for the virus from conidia of P. chrysogenum correspond to those reported for mycelial extracts (18). The three bands of P. brevi-compactum have electrophoretic mobilities similar to those for P. chrysogenum. The concentration of viral dsRNA in conidia and mycelia of the three species is compared in Table 1 Electrophoretic profiles (polyacrylamide gel) of dsRNA extracted from conidia of Penicillium species. The isolated RNA from the three fungal species were separated electrophoretically on polyacrylamide gels (2.4%) for 3 h at 6 mA/tube. 5 6 almost twice that from P. stoloniferum (75 ig/g dry weight) and 60 times greater than in P. brevi-compactum (2 Mg/g dry weight). The concentration of viral dsRNA in the conidia of these species is quite comparable to that found in their mycelial forms. Time release of macromolecules. The relationship between concentrations of macromolecules (RNA, DNA, and protein), concentrations of conidia, and disruption time is shown in Table 2. Optimal breakage of conidia (88 to 94%) occurred at 4 min. At all conidial concentrations the percent of breakage increased with disruption time. The release of macromolecules also increased with increased disruption time at all three conidial concentrations; maximal release came at the 4-min time intervals. Trehalase activity increased as a 95 a These values were obtained by analyses of RNA extracted from 1.5 g (dry wt) of conidia of NRRL 5267 and Q-176, and 15 g (dry wt) of conidia of NRRL 5260. b These values were obtained by analyses of RNA extracted from mycelia harvested after 72 h of growth at 28 C as a shake culture. function of disruption time, with maximal activity occurring at 4 min. The amount of dsRNA released from disrupted conidia represents 0.8 to 1.0% of the total RNA released and is 0.003 to 0.004% of the total spore mass on a dry weight basis. Nonencapsulated dsRNA in conidia. Quantitative analyses of 15 g of conidia (dry weight) from P. stoloniferum NRRL 5267 indicated that 10% of the dsRNA exists free in the conidia. Homogenization of virus with subsequent repelleting resulted in no detectable dsRNA in the SNF. This failure to detect nonencapsulated dsRNA indicated that viruses are not being disrupted as a result of mechanical homogenization. DISCUSSION This report describes the first isolation and characterization of virus particles from fungal conidia. Electron microscope comparisons of particles isolated from conidia or mycelia of a particular isolate show that particles from either source are structurally identical. Biochemical and physical comparisons of mycoviral dsRNA isolated from conidia or from mycelia of a particular isolate show that the dsRNA is qualitatively the same. Quantitatively, the ratio of viral dsRNA per gram (dry weight) of conidia is equal to the ratio of viral dsRNA per gram (dry weight) of mycelia. The presence of virus in conidia explains a mechanism for viral sustainment during nonvegetative stages of the life cycle of fungal isolates.

v3-fos

2018-04-03T04:20:55.636Z

1973-05-01T00:00:00.000Z

38551136

s2

Isolation of Salmonellae from Pork Carcasses Four hundred and twenty pork carcasses from four abattoirs were examined for the presence of salmonellae by use of swabbing-enrichment techniques and contact plate imethods. Carcasses from only one abattoir were found to be contaminated by swabbing-enrichment (23.3%) and contact plate (17.9%) methods. The area of the skin side of the ham, near the anal opening, was determined to be the area to examine for isolating salmonellae from pork carcasses with the greatest frequency. The most frequently isolated species of salmonellae in this study were Salmonella derby, S. anatum, S. typhimurium, and S. indiana. The isolation of salmonellae from meat animals intended for food has been of interest since the first report on swine plague (2). Several authors have reported on the occurrence of salmonellae in meat products (4, 7-9, 11, 15, 26) and the use of various techniques to recover microorganisms, including salmonellae, from the carcasses of meat animals (11, 16, 18-20, 21, 26). Methods for assessing the bacterial content on the surface of raw foods are varied. Swabbir is one of the earliest and continues to be one u. the most widely used methods because of its versatility. Limitations of swabbing techniques were evaluated by Douglas (5) and Green and Herman (12). Variations of the swabbing techniques are discussed by Green et al. (13) and Walter (25). Dyett (6) proposed using a sharp, sterile knife to scrape the whole, exposed carcass surface and making an estimate of the number of microorganisms in the scrapings by a direct microscopy count or by . total viable count. Direct-surface agar plating was described by Angelotti and Foter (1), and application for sampling meat surfaces was described by L. ten Cate (3). Hall and Harnett (14) described a disposable plastic dish so constructed as to permit direct-contact plating of surfaces. The purpose of this study was to determine the incidence of salmonellae on pork carcasses in local abattoirs, to find the location on the carcasses most likely to be contaminated with salmonellae, and to report the species of salmonellae found. MATERIALS AND METHODS Four hundred and twenty pork carcasses from four different abattoirs were examined, after chilling, for the presence of salmonellae by swabbing an area approximately 25 cm2 on the skin surface of the ham near the anus and by placing a contact plate on the analogous area on the other half of the same carcass. Swabbing was accomplished by wetting a cotton swab in sterile, physiological saline, swabbing the designated area thoroughly, and then replacing the swab in the tube containing 10 ml of 0.85 M sterile saline for transportation to the laboratory. Samples (1 ml) from the tubes containing swab" were transferred into tetrathionate brilliant green (TET) broth (10) and incubated at 37 C for 24 h. After incubation, samples of the TET broth were streaked upon brilliant green agar (BGA, Difco) plates which were then incubated for 24 to 36 h at 37 C. Ctlonies showing typical growth of salmonellae on BGA plates (11) were inoculated into triple sugar-iron agar (Difco) and incubated at 37 C for 24 h, and those tubes showing positive reactions (10) were sent to the Southeaste.rn Salmonella Serotyping Laboratory, Atlanta, Georgia, for positive species identification. Contact plates, as described by Hall and Harnett (14), filled with BGA and bismuth sulfite agar (BSA; Difco) were applied to the surface of the carcasses in such a manner as to insure full and complete contact between the medium and the surface being examined. These plates were then transported to the laboratory and incubated at 37 C for 24 to 36 h, and suspected colonies from plates showing positive growth of salmonellae were treated as described above for the swabs in saline tubes. In a subsequent experiment, 91 pork carcasses from abattoir A were examined at three locations: (i) on the skin surface of the ham near the anus; (ii) on the skin of the jowl; and (iii) on the flesh si-le of the jowl, for a total of 273 samples. In this experiment, adjacent areas on the same carcass were selected for examination by swabbing and contact plates, thus eliminating variability between halves of carcasses. The isolates obtained were analyzed for salmonellae as described above. RESULTS AND DISCUSSION In a survey of pork carcasses from four different abattoirs, salmonellae were recovered from only one establishment (Table 1). None of the abattoirs surveyed obtained hogs from the same farms. Although they did receive hogs from the same general areas or locales on a regular basis, none of these areas overlapped. Galton et al. (9) concluded that there may be true regional variations and attributed these to (i) differences in the incidence of salmonellae infection in hogs, (ii) variation in the nature or control of processing procedures employed, or (iii) climatic factors affecting the viability or multiplication of salmonellae in the environment of abattoirs. Cross-infection of healthy pigs by infected pigs from individual farms may occur in the holding pens (11,17), leading to a continuing incidence at a particular abattoir. The most probable source of salmonellae is the animal from which the meats were obtained (4). Carcasses from abattoir A were examined on nine different occasions, or days, throughout a 12-month period, and salmonellae were found on all occasions. The greatest incidence of salmonellae was recovered from carcasses examined on sampling day 4. (Table 1). There was no apparent reason for the large number recovered at this particular sampling time. Total numbers of bacteria on all carcasses examined from abattoir A were in the order of 103 to 104 organisms per 25 cm2 of surface. No differences in total numbers were noted from those carcasses from which salmonellae were isolated. No salmonellae were recovered from abattoirs B, C, and D where 100, 65, and 34 carcasses, respectively, were examined. This indicates that the isolation of salmonellae from pork carcasses is not a simple, routine matter. Although no actual data were collected, it was noted that abattoirs B, C, and D were receiving hogs from geographic areas different from those of abattoir A. To determine their relative effectiveness in recovering salmonellae from pork carcass, the techniques of swabbing and the use of contact plates filled with BSA and BGA were compared. Results (Table 1) show that contact plates recovered salmonellae from 40 (17.9%) carcasses examined compared with 52 (23.3%) by swabbing and enrichment techniques. On only 3 sampling days were more salmonellae recovered by contact plates than by swabbing-enrichment techniques. On 5 other sampling days, more salmonellae were recovered by swabbingenrichment than by contact plates, and on three occasions salmonellae were not recovered at all by contact plates. Salmonellae were recovered by contact plates and swabbing from the same carcass in 12 instances. One important disadvantage of contact plates is that they are not representative of the entire carcass and only reflect the area that they touch. Either method of recovery may be satisfactory when the problem is of a gross nature. Recovery of salmonellae by contact plates seemed to depend upon the medium for recovery. Of 40 contact plates from which salmonellae were isolated, 26 of the isolations came from plates containing BSA and 14 came from plates containing BGA. Many of the plates containing BGA were overgrown. The increased efficiency of BSA or BGA for recovering salmonellae could be due to the greater inhibition of microflora by BSA. Taylor (23) found that, when the ratio of coliforms to salmonellae approached 50: 1, the appearance of a typical, well-isolated salmonella colony with its identifying characteristics for a given medium becomes the exception. In these overcrowded areas, the salmonellae are rarely able to disclose their distinguishing characteristics. However, Banwart and Ayres (2), by using pure cultures of salmonellae, found that BGA supported more luxuriant growth of the six species tested, whereas BSA was inhibitory to four. If a processor of pork carcasses desired to monitor the incidence of salmonellae on carcasses by the contact plate method, BSA would appear to be the medium of choice. BSA may require 24 to 48 h to develop characteristic growth of salmonellae. The incidence of salmonellae from pork carcasses processed by abattoir A, 17.9% by contact plates and 23.3% by swabbing-enrichment technique, is much lower than the figure of 56% reported by Weissman and Carpenter (26) for APPL. MICROBIOL. ISOLATION OF SALMONELLAE this same establishment. This decrease probably is due to extensive changes in slaughtering and processing techniques instituted in this particular plant in the interim between the periods of time when the two sets of data were collected. Recovery of salmonellae from three sampling locations on pork carcasses ( Table 2) was greater from the area examined on the skin side of the hams, near the anal opening, than from the skin or flesh side of the jowl for both methods of recovery, contact plate and swabbing-enrichment technique. No salmonellae were recovered by contact plates on either the skin or flesh side of the jowl, probably because of the difficulty of obtaining a satisfactory area for application of the plate in the case of the flesh or inside of the jowl. Salmonellae were recovered by swabbing-enrichment technique on both sides of the jowl, but with higher incidence of recovery from the skin side or outside. It might be expected that the jowl or neck area would be subject to greater contamination because of washings from the entire carcass fouling that area during processing. Koelensmid and van Rhee (16) found that water that drips from the skin of pork carcasses after singeing is not sterile. They isolated salmonellae from five samples of scrapings taken from 50 carcasses after inspection by veterinarians. In a study on beef, Mulcock (18) found the greatest number of bacteria on neck tissues (106) compared with sides (103) after incubation at 22 C for 5 days. Patterson (20) reported that, in sheep and cattle, contamination acquired during the butchering process is not spread evenly over the carcass. He found greater numbers on the brisket than on the foreleg or rump; yet he concluded that sites of heaviest contamination will vary from one abattoir to the next depending upon methods employed, washing, and other treatments used. Galton et al. (9) examined cultures from anal swabs from living and slaughtered hogs and swabs from sides of (26) concluded that no single area of a pork carcass was likely to be more contaminated than another, but suggested examining the area of the ham near the anal opening as the area of choice because of the possibility of fecal contamination. Results of this study ( Table 2) also indicate that this would be the most useful area to examine when monitoring pork carcasses for salmonellae. Table 3 lists the species of salmonellae isolated from pork carcasses in this study. The three most frequently isolated species, S. derby, S. anatum, and S. typhimurium, are common among isolates of salmonellae found in red meats. The presence of these organisms might be expected because the Center for Disease Control regularly lists these organisms among the 10 most commonly reported from nonhuman sources. However, isolation of S. indiana from red meats has been reported only once since 1967. Six isolations of S. indiana from swine have been reported during the same period. S. indiana is more commonly isolated from poultry and egg products (24). The serotypes isolated were not uniformly spread over the 9 sampling days. S. typhimurium and S. anatum were more frequently isolated on days 1 to 5. All of the S. indiana serotypes were isolated on day 4, whereas S. derby was isolated on days 5 to 9. Other serotypes listed were isolated on various days throughout the sampling period.

v3-fos

2020-12-10T09:04:12.942Z

1973-02-01T00:00:00.000Z

237232656

s2

Lysogeny in Lactic Streptococci Producing and Not Producing Nisin Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species. Lysogeny has hitherto not been observed in group N streptococci even though bacteriophages have been isolated from these bacteria by many workers (4)(5)(6)(7)(8)(9)(10). The purpose of this work was to determine whether lysogeny occurs in lactic streptococci, including strains producing the antibiotic nisin, and to examine some properties of host-phage systems in these bacteria. MATERIALS AND METHODS Strains. Eighty-seven strains were investigated, including 46 strains of Streptococcus lactis, 24 strains of S. diacetilactis, and 17 of S. cremoris; 12 of the S. lactis strains produced nisin. The strains were kept at 4 C and transferred once a week to a 10% water suspension of defatted powdered milk. The strains came from the Institute of Dairy Industry, Warsaw; Pure Dairy Cultures, Laboratory of Olsztyn; Department of Industrial Microbiology, Technical University of L6dZ. Medium. The medium consisted of the following: beef heart infusion, 1,000 ml; peptone (Gurr), 10 g; yeast extract (Difco), 10 g; NaCl, 5 g; glucose, 10 g; 1 M CaCl2, 1 ml; final pH of the medium, 7.2. The medium was autoclaved at 117 C for 15 min. This medium will be referred to as X. Equipment and chemicals. The equipment and chemicals used were an ultraviolet (UV, bactericidal lamp (Westinghouse G 30 T8), Berkefeld N2 and Seitz EKX filters; and mitomycin C (Nutritional Biochemicals Corp.). Determination of the plaque-forming units. Determination of the plaque-forming units was performed by the method of Horvath and Alffildi (4) or Gratia (see reference 1). Number of viable bacteria. The number of viable bacteria (colony-forming units) was determined by the plate method. The strains were incubated in a water bath or incubator at 30 C. RESULTS AND DISCUSSION Search for lysogens. Search for lysogens was performed by Fisk's method (2) looking for phages in bacterial culture filtrates. In the case of filtrates which hindered the growth of indicator strains, 10to 10-3 dilutions of the filtrates were applied, as well as the undiluted filtrates. This was necessary to distinguish between inhibition of the growth of indicator strains caused by phages and that due to other factors, e.g., nisin, produced by lactic streptococci. This distinction is possible if the filtrates are diluted to 10-3 since at this concentration only the presence of phages, but not the activity of other factors, is detected. The culture filtrates were screened for phages after 3 and 18 h of incubation of the strains and after 3 h of incubation of strains which were treated with UV (usually 260 ergs per mm2) immediately prior to incubation. Each strain was treated as a potential lysogen and as a potential indicator strain. Five lysogenic strains were found among 46 S. lactis strains examined. Four of them (37, 40, 41, 45) liberated phages which could only multiply in one indicator strain, S. lactis 28. A fifth strain, 31, liberated phages which could multiply in several strains belonging to the three examined species. Strains 40, 45, and 28 produced nisin. Phages were obtained sporadically from five other S. lactis strains, but reproducible results were not obtained. Two lysogenic S. diacetilactis strains (84 and 87) were found among the 24 investigated strains. Phages liberated by these strains were able to multiply in a number of strains from the three examined species. No lysogens were found among the 17 tested strains of S. cremoris, in spite of the use of various experimental conditions. Spontaneous liberation of phages. Eighteen-hour cultures were centrifuged and the cells were washed twice with Ringer's solution. The bacteria were then resuspended in a volume of Ringer's solution equal to the volume of the initial culture. The suspensions were diluted 1: 100 with X medium. After taking samples to determine the number of infective centers (CI), the strains were incubated to examine the kinetics of the increase of the number of phages and living bacteria. The controls performed indicated that a maximum of 1 out of 500 observed plaques does not represent CI. Table 1 contains data on the frequency of spontaneous induction which was 1 to 2% and 0.04 to 0.07% for S. lactis and S. diacetilactis strains, respectively. Effectiveness of induction depending on the dose of UV. Bacteria from 18-h cultures were washed twice with Ringer's solution and then resuspended in the same solution; 2.5 ml of this suspension was poured onto each petri dish (5.5 cm in diameter) and irradiated with UV. The number of induced cells and of surviving bacteria were determined immediately. A correlation between the number of induced At the optimal UV dose (260 ergs/mm2), 20 to 37% of the cells were induced in S. lactis and 7 to 10% were induced in S. diacetilactis strains. Only in the case of S. lactis strain 31 was no increase in the number of induced cells after UV treatment found, as compared with spontaneous induction (data not presented in the table). Sometimes, a 90% decrease of the number of phages was observed in this strain after irradiation. In this strain, neither mitomycin C at various concentrations nor a raised temperature was effective in the induction of phages. Investigations now being performed suggest that in this strain the prophage is not integrated into the chromosome. One-step growth of phages. Lysogen suspensions after UV irradiation (260 ergs/mm2-) were diluted 1:100 with X medium and incubated. Samples were taken for determination of the plaque-forming units and the number of living bacteria. One-step growth of the same phages was determined in parallel after infecting sensitive bacteria. The results are presented in Table 2. The course of one-step growth of all S. lactis phages was similar after induction. The latent periods were 80 to 100 min long, and the periods of growth 30 to 60 min; 40 to 46 phages were liberated from each cell. For both lysogens of S. diacetilactis, the latent period was 40 to 60 min, the period of growth 60 to 80 min, and 10 to 25 phages were liberated from each cell. When sensitive bacteria were infected with these phages, the periods of one-step growth of the phages were shorter, but the yields of phages were similar to those obtained after UV induction of lysogens. Lysogenization of sensitive bacteria. To check whether the isolated phages lysogenize indicator strains, suspensions of S. lactis )genic lactic streptococci by using various UV phages (with the exception of phages from strain 31) were applied to S. lactis strain 28, and those from S. diacetilactis) strains of S. diacetilactis 86. All phages were able to lysogenize indicator strains, as most bacterial colonies which grew in the presence of the phages contained phage-resistant cells which liberate phages spontaneously and after UV induction. Delysogenization and relysogenization of nisin-producing strain S. lactis 45. This strain was irradiated on solid medium with a UV dose of 650 ergs/mm2 which inactivated 99.9% of the cells. Among the colonies grown after UV irradiation, five nonlysogenic ones were found which were susceptible to phage 45 and produced nisin. The subclones thus obtained were susceptible to relysogenization with phage 45. Lytic spectrum of the phages. Phages at routine test dilution (RTD) and 1,000 x RTD concentrations were used for lytic spectrum determinations. As is shown in Table 3, phages from strains S. lactis 37, 40, 41, and 45 were able to reproduce in S. lactis strain 28 only. The phages from S. lactis strain 31 and S. diacetilactis strains 84 and 87 had a wide lytic spectrum and were able to multiply in a number of strains (including four nisin producers) belonging to the same and to the two remaining species. The lytic spectrum was slightly more narrow when the phages were used at RTD concentration. Repressor resistance of S. lactic strain 28 after lysogenization by a single phage to the remaining S. lactis phages. Investigations of the lytic spectrum and one-step growth of the isolated phages of S. lactis suggested a great similarity or even identity of these phages. To check whether these phages were identical, the repressor resistance of S. lactis strain 28 (lysogenized with one of the phages) to the remaining phages was determined; it was performed by lysogenization of strain 28 with one of the four phages and by subsequent treatment of this strain with the three remaining phages at RTD concentrations. The results presented in Table 4 suggest that phages 40, 41, and 45 produce a similar type of repressor, because cross-resistance to these phages is observed. It seems that this repressor differs from that produced by phage 37. This indicates that some subtle differences do exist between phage 37 and the remaining phages.

v3-fos

2020-12-10T09:04:12.631Z

1973-01-01T00:00:00.000Z

237235046

s2

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory. with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory. For some time the food industry has needed a rapid, simple, economic, presumptive Salmonella detection procedure that could be performed in reduced elapsed test time on large numbers of samples to eliminate negative samples early in the course of examination with minimal fear of false negatives. In addition, such a test should be incorporated easily into the standard Salmonella detection procedures (Association of Official Analytical Chemists [APHA]) currently used in food plants without the use of special equipment, antisera, or specially trained personnel. Culture methods currently used in qualitycontrol laboratories require a minimum of 3 to 4 days for presumptive results. Further biochemical testing requires another 2 to 3 days before the presumptive positive for Salmonella can be confirmed as positive. The enrichment serology (15,16) and fluorescent-antibody (FA) procedures, although more rapid than conventional techniques, require special equipment and antisera. A person having considerable experience with these techniques is usually needed to perform these tests. A simpler culture method for the rapid detection of Salmonella was described by Hargrove et al. (6). This procedure was developed primarily for detecting Salmonella in dairy products in a single enrichment step and was tested extensively with known Salmonella standards. The purpose of this study was to evaluate this procedure and to modify the technique for routine testing of a variety of foods, food ingredients, and feed materials for the presence of viable Salmonella. MATERIALS AND METHODS Cultures. Salmonella cultures (S. Montevideo, S. senftenberg, S. senftenberg 775W, and S. typhimurium) were used in this study. Representative strains of other Enterobacteriaceae (Enterobacter, Escherichia, Shigella, Citrobacter, and Proteus) were used also to determine the specificity of the method. HOBEN, ASHTOI Medium. The medium, lysine-iron-cystine-neutral red broth (LICNR), of Hargrove et al. (6) was modified as follows: (i) mannitol was substituted for lactose, (ii) a combination of L-lysine mono-and dihydrochlorides (8:2) was used instead of the monohydrochloride, and (iii) novobiocin was used at two concentrations, either zero added or at concentrations of 10 to 15 ug/ml of broth. The modified LICNR broth had the following composition: 10 g of L-lysine mono-and dihydrochlorides (8:2), 5 g of tryptone, 3 g of yeast extract, 5 g of mannitol, 1 g of glucose, 1 g of salicin, 0.5 g of ferric ammonium citrate, 0.1 g of sodium thiosulfate, 0.1 g of L-cystine, 0.025 g of neutral red, and 1 liter of distilled water. The medium was adjusted to pH 6.2 (1 N NaOH), dispensed in 10-ml quantities in metal-capped tubes, and autoclaved at 121 C for 15 min. The ability of this modified medium to differentiate Salmonella from non-Salmonella was determined with pure cultures, and its ability to detect Salmonella in various food materials was further confirmed. The basis of this test is a pH response to an indicator dye and formation of a black precipitate when hydrogen sulfide-producing salmonellae are present. All cultures which showed a color change from red to yellow in the LICNR broth after incubation at 37 C for 24 hr were further tested to eliminate the possibility that non-hydrogen sulfide-forming salmonellae were present. This test was accomplished by incubating for another 16 to 24 hr and then adding 0.1 ml of a 0.3% bromothymol blue solution to each tube and recording the color change. When salmonellae were present, the medium changed from yellow to dark green or blue, indicating an alkaline reaction. Color differences were obvious immediately. False results were observed to occur when the modified LICNR broth was incubated beyond 36 hr prior to testing with bromothymol blue. The bromothymol blue solution was prepared by mixing 0.3 g of bromothymol blue powder with 2 ml of 0.1 NaOH and diluting to 100 ml with 50% ethanol in distilled water. Salmonella antiserum. The antiserum used in this study for the direct FA technique was a commercially prepared polyvalent antiserum (Difco "spoly") which had been prepared from motile organisms representative of somatic groups A through S including 0 factors 25, 27, 28, 30, 34 through 41, 45, and Vi. The flagellar spectrum included H antigens a through i, n, p, r through u, and w through z. The antiserum was rehydrated by the addition of 5 ml of distilled water and diluted 1: 2 to obtain the proper staining titer. Microscopic examination. A Reichert "Zetopan" immunofluorescence research microscope, equipped with a mercury vapor light source, BG12 ultraviolet filter, and a X97 oil-immersion objective with iris diaphragm, was used. Test samples. Where possible, samples naturally contaminated with Salmonella were used in this study. In the case of cheddar cheese and powdered milk, Salmonella at 2 to 5 g was added because no naturally contaminated samples were available. Known culture assays. Viable cultures were inoculated directly into the presumptive broth and AND PETERSON APPL. MICROBIOL. incubated up to a maximum of 48 hr. Broths were observed for characteristic color changes and blackening of the medium during incubation. Foods and food ingredient assays. In the first study samples were enriched using 50 g of food material and 450 ml of tetrathionate broth. Enrichments were incubated at 41 C (40.5 C) for 20 hr. Following primary enrichment, 1 ml of each enrichment culture was placed into tubes containing 10 ml of the modified presumptive LICNR broth. A similar amount of primary enrichment culture was placed into tubes containing an equal amount of tetrathionate broth (secondary enrichment). Both secondary enrichments (presumptive LICNR and tetrathionate broths) were incubated at 37 C for 24 hr. Cultural testing was performed by streaking these secondary enrichments on Hektoen agar (10) plates. The Hektoen agar plates were incubated at 37 C for 18 to 20 hr. Presumptive colonies (blue-green to blue with or without black centers) picked from Hektoen agar were tested biochemically by placing onto slants of dulcitol lysine iron agar (DLIA) described by Taylor (17), and onto lysine iron agar (LIA) as described by Edwards and Fife (3). Slant and butt reactions were observed after incubation at 37 C for 24 hr. Typical Salmonella reactions on these agars are for DLIA: alkaline (red) slant, hydrogen sulfide blackening, and fractured acid (yellow) butt; and for LIA: alkaline slant (red) and alkaline butt with hydrogen sulfide blackening. DLIA slants which gave reactions characteristic of Salmonella were serologically tested with Difco polyvalent 0 and H Salmonella antisera (2). A small amount of each slant growth was mixed with a drop of the rehydrated antisera on a glass slide. Samples were considered positive if a tight granular precipitate was observed in 30 sec. FA results were obtained from smears made from presumptive broths after characteristic blackening of the medium occurred. Agar-coated slides were used to help retain the bacterial cells during staining and washing. Smears were fixed in an ethanolchloroform-formaldehyde solution (6:3: 1) for 3 min. Staining was accomplished (direct technique) using Difco fluorescein conjugated "poly" Salmonella antisera. Smears were stained for 30 min followed by rinsing twice in a phosphate-buffered solution (Difco), pH 7.2. After the excess antisera had been washed free, the slides were rinsed in distilled water and allowed to air dry. Dried smears were mounted using buffered gylcerol, pH 8.6, and examined for fluorescence. A smear was considered positive for Salmonella if bright, typically rod-shaped bacteria exhibiting +3 to +4 fluorescence (5) with or without attached flagella were present (4,18). In the second study, samples were pre-enriched using 25 g of food material and 225 ml of lactose broth. Samples were blended for 2 min at high speed using an Oster blender. The pre-enrichment culture was incubated at 35 C for 20 to 24 hr. Following pre-enrichment, 1 ml of each enrichment culture was placed into tubes containing 10 ml of tetrathionate broth and incubated at 35 C for 20 to 24 hr. After secondary enrichment in tetrathionate broth, 1 ml of each enrichment was placed in tubes containing 10 ml of the LICNR broth. Novobiocin was used during N. enrichment as follows: (i) at zero and at 10 to 15 yg/ml in the LICNR broth and (ii) at zero and at 10 to 15 Ag/ml in the tetrathionate broth enrichment. Samples were tested culturally by streaking the tetrathionate broth enrichments without novobiocin added onto Hektoen agar plates and picking typical colonies. These were then identified biochemically and serologically as previously described. RESULTS AND DISCUSSION Cultures of various known Salmonella and known type species of non-Salmonella were used in this study to evaluate the modified LICNR broth and to verify the results obtained by Hargrove et al. (6). Freshly grown cultures were inoculated into the modified LICNR presumptive broth. Color reactions were observed at 2-hr intervals. Salmonella were readily differentiated from related enteric bacteria except for Arizona. Results obtained (Table 1) were similar to those observed by Hargrove et al. Most known Salmonella produced a massive black precipitate. Arizona was observed to produce identical reactions to Salmonella. A hydrogen sulfide-negative Salmonella strain (S. seftenberg 775W) turned the presumptive broth yellow without blackening of the medium but was detected by the use of the bromothymol blue indicator which formed a green ring at the top of the tube. Pure cultures of Shigella, Enterobacter, Proteus, and Citrobacter did not change the red color of the LICNR broth. Species of Escherichia changed the LICNR broth from red to yellow without blackening. Escherichia could be distinguished from hydrogen sulfide-negative Salmonella by the fact that no green or blue color was produced upon addition of the bromothymol blue indicator. Food samples which turned the presumptive broth yellow and subsequently were tested for the presence of hydrogen sulfidenegative Salmonella did not give a clear-cut color result. In cases during incubation, competing non-Salmonella were able to raise the pH of the LICNR broth enough to cause questionable color results. This problem became almost non-existent in the later study when novobiocin was used. It should be also pointed out that hydrogen sulfide-negative Salmonella constitute less than 0.5% of all known salmonellae (6,11). These types have not normally been encountered in foods and food ingredients in this laboratory. An attempt was made to utilize the procedure as described by Hargrove et al. (6) for a rapid Salmonella detection procedure for foods, food ingredients, and feed materials. However, some food materials were observed to mask and interfere with the color reactions of the presumptive broth. To overcome that problem, the presumptive LICNR broth was used as a secondary enrichment medium and presumptive indicator. All materials were enriched in tetrathionate broth at 41 C because with feed samples this temperature appeared to be optimal for Salmonella detection and isolation. Other workers (1,7,12) Presumptive results were obtained within 32 to 38 hr. Samples that were found to contain Salmonella by conventional cultural procedures produced a massive black precipitate in the presumptive LICNR broth. Even when high levels of coliforms and Proteus species were present, the presence of Salmonella was readily indicated by loss of color and blackening of the medium. In 20% of the samples tested, some difficulty was encountered in detecting Salmonella by conventional cultural procedures. Eighteen different types of samples were evaluated for the presence of viable Salmonella. These samples included chicken parts, feed materials, powdered milk, raw frozen meats, pure culture slants, and environmental swabs. A positive correlation between this presumptive test and cultural results was obtained. (Table 2). Ninety-four percent of the samples found to be presumptively positive by this method was confirmed by cultural methods (AOAC, USDA, BAM, APHA). Of 110 samples tested, 58% were found to be positive for Salmonella by cultural methods as opposed to 62% with LICNR broth. Of the 110 samples tested only 50 were evaluated by all three techniques in parallel: cultural, FA, and the presumptive LICNR method. Salmonellae were found in 76% of those 50 samples by the cultural method and in 82% by both the FA and presumptive LICNR broth methods. The high percentage of samples positive for Salmonella by all techniques was due to the fact that samples were obtained as presumptive positives from previous screening programs. We think the difference between cultural results and those obtained by the presumptive and FA methods may have been due to the inability of the cultural method to detect Salmonella in the presence of high numbers of non-Salmonella organisms. False-positive results have been encountered by the FA method due to the presence of non-Salmonella organisms which share common antigens with Salmonella and consequently fluoresce (8). The presumptive LICNR broth, after hydrogen sulfide development, yielded smears that exhibited extremely bright (+3 to +4 fluorescence) peripheral staining. Many smears also exhibited excellent flagellar staining. FA smears in a few instances contained low numbers of fluorescing cells, indicating that low levels of Salmonella were present in some of the final enrichments. Similar results were observed by Reamer and Hargrove (14). Tetrathionate broth was used in this study because it has been found to be the single most suitable enrichment medium for use in this laboratory. Tetrathionate broth allows the enrichment of most Salmonella while inhibiting many of the unwanted organisms present in foods and food material (13). The combination of mono-and dihydrochlo- rides of L-lysine lowered the pH of LICNR broth and resulted in the ingredient materials going into solution more easily. Therefore, these materials were substituted for the monohydrochloride originally used by Hargrove et al. (6). Further work (pure and mixed culture) showed false-positive reactions (LICNR broth) could occur. These false-positive results were caused by a combination of a coliform and Proteus species that were isolated from eviscerated chicken samples. When grown in combination these organisms produced a false color result in the LICNR broth. None of these organisms alone gave false-positive results. This problem was eliminated by the incorporation of novobiocin at 10 to 15 Ag/ml in the LICNR broth. Jeffries (9) used novobiocin for suppressing competing organisms such as Citrobacter and Proteus. This suppressive effect on competing non-Salmonella organisms was substantiated by testing naturally contaminated samples for the presence of Salmonella. Food materials and ingredients were enriched using procedures currently in use in the quality control laboratory to see if the LICNR broth technique could detect the presence of viable Salmonella. What appeared to be falsepositive results were observed when novobiocin was not used during enrichment. When novobiocin was used in the LICNR broth, identical presumptive and cultural results were obtained (Table 3). Presumptive results from samples enriched with novobiocin present in the tetrathionate (rather than in the LICNR broth) broth enrichment, however, did not correlate as closely with those obtained culturally (Table 4). These results suggest that novobiocin was more effective in eliminating false positives when it was used in the LICNR broth. No false negatives were encountered in this study. It should be noted that presumptive LICNR results were compared with cultural results and that further cultural isolation might have resulted in the detection of Salmonella in sam- ples reported as negative. The assumption inherent in this approach is that conventional cultural procedures yield correct and absolute results, and this is not necessarily true. Using this new medium (LICNR broth), it was possible to detect Salmonella in food samples within 3 days which is 1 day faster than standard Salmonella detection procedures (AOAC, USDA, BAM, APHA) permit. This procedure, using modified LICNR broth, requires no special equipment or antisera. Its use as a routine quality control screening procedure should be of value in shortening the holding time for foods and food ingredients while awaiting cultural results. Presumptive results can be further evaluated by any of several presently recognized confirmatory procedures with no delays or increase in time beyond that presently required. The number of such samples requiring the more extensive confirmatory procedures should be substantially reduced through the use of this screening procedure. The savings from this reduction in sample volume are obvious.

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2020-12-10T09:04:13.096Z

1973-02-01T00:00:00.000Z

237229347

s2

Survivor Curves for Leptospira autumnalis Akiyami A Based on Most-Probable-Number Values The validity of survivor curves for Leptospira autumnalis Akiyami A based on most-probable-number values is supported by the following observations: (i) linear regression lines fell within most of the 95% confidence intervals; (ii) linear correlation coefficients (r) were consistently high (i.e., near -1); and (iii) statistical tests for goodness of fit usually accepted the linear model. These tests are consistent with an exponential death rate for the test organism in defined solutions. The influence of temperature and pH on survival was demonstrated by showing a statistically significant difference in survivor curve slopes. It was previously demonstrated that a single cell of Leptospira autumnalis Akiyami A could be recovered with a supplemented Fletchers medium (Difco) and that the density of washed-cell suspensions could be estimated by the most-probable-number (MPN) procedure (4). This report concerns an investigation of the applicability of the MPN procedure for generation of survivor curves. MATERIALS AND METHODS Test organism. The test strain of L. autumnalis Akiyami A was obtained from A. D. Alexander, Water Reed Army Medical Center. The organism was maintained in Fletchers medium (Difco) containing 10% rabbit serum (Pel-Freez) with transfer every 5 weeks. Antigenic stability was verified periodically with antiserum provided by the Center for Disease Control. Preparation of cells. A 1-ml amount of stock culture was used to inoculate 10 ml of a medium containing (per liter): Na2HPO4, 1.0 g; KH2PO4, 0.3 g; NaCl, 1.0 g; NH4Cl, 0.25 g; thiamine, 0.005 g; and rabbit serum, 100 ml. After incubation at 30 C for 86 hr, the cells were removed by centrifugation at 3,000 rev/min for 30 min. The cells were washed twice with 5 ml of buffer (5.33 mm phosphate, pH 7.6) and resuspended in 10 ml of buffer. Cell concentration was standardized at 101 per ml by direct count with a Petroff-Hausser chamber and dark-field microscopy. A 1-ml amount of the standardized suspension was added to 100 ml of test solution to give an initial cell concentration of 990 per ml. ' time long enough to permit study of the influence of other variables. The survivor curves presented here were obtained with two test solutions, Ringer's solution and sodium thiosulfate (4.95 mM), each buffered at a final phosphate concentration of 5.33 mM. Ringer's solution contained (per liter): NaCl, 537.5 mg (9.19 mM); KCl, 18.75 mg (0.25 mM); CaCl2 .2H20, 39.7 mg (0.273 mM); and NaS20S. 5H20, 125 mg (0.504 mM). To study the influence of pH in solutions of sodium thiosulfate (4.95 mM), the concentration of buffer was increased to 10 mm total phosphate. Test solutions were prepared with water passed through an ion-exchange column and glass-distilled. Thiosulfate concentrations were determined by titration with standard iodine solution. Test solutions were sterilized by filtration through a 0.22-agm membrane filter (Millipore Corp.), and 100 ml of solution was transferred to a sterile cotton-stoppered 250-ml Erlenmeyer flask. After inoculation, the flasks were held static in the dark at 20 C unless temperature was a variable. MPN procedure. Dilutions for MPN determinations were made in 9.0-ml blanks of Leptospira medium EMJH (Difco) containing 1% rabbit serum. Amounts of 0.1 ml of three decimal dilutions were transferred to three series of 5 or 10 tubes each (plastic, 12 by 75 mm, Falcon Plastics) containing 4.0 ml of Fletchers medium (Difco) supplemented with (per liter): ZnSO4.7H20, 0.2 mg; CaCl2.2H20, 10 mg; MgCl2.6H20, 10 mg; asparagine, 150 mg; and rabbit serum (Pel-Freez), 100 ml. Filter-sterilized asparagine and rabbit serum were added aseptically to autoclaved medium before dispensing. Incubation was at 30 C for up to 17 days. Positive tubes were those showing visible evidence of growth, usually obvious by the characteristic Dinger's ring of Leptospira. MPN values were obtained from available tables. Statistical tests. The goodness-of-fit test for a linear model was completed by calculating the chisquare test statistic given by: freedom and a = 0.05, then the linear model was rejected. Slopes were tested for significant difference by calculating the chi-square test statistic given by: where b, = slope of curve 1; b2 = slope of curve 2; S,2 = variance of curve 1; and S22 variance of curve 2. If the calculated value of X2 was greater than that from the chi-square distribution for one degree of freedom and a = 0.05, then the hypothesis of equal slopes was rejected. Figure 1 presents a survivor curve for L. autumnalis in Ringer's solution at pH 7.10 and 20 C. MPN values are based on 10 tubes per dilution and are plotted with their 95% confidence intervals. The linear regression line is presented as the best straight-line fit of the data. Figure 2 Figure 4 shows the influence of temperature on survival of L. autumnalis through differences in the slopes of survivor curves. Temperatures above 20 C increased the death rate. The effect of pH shown in Fig. 5 indicates that pH values below and above 7.4 increased the death rate, with a greater sensitivity to acid pH values. RESULTS A statistical test for goodness of fit on the survivor curves presented in Fig. 1, 2, and 3 accepts a linear model at a = 0.05 ( Table 1). The same test rejects a linear model for one of the pH survivor curves ( Table 2) and one of the temperature survivor curves ( Table 3). The temperature curve would be acceptable at a = 0.01. Statistical analyses for significant difference in slopes between each pair of pH survivor curves and each pair of temperature survivor curves rejects a hypothesis of equal slopes at a = 0.05. In other words, each of the pH curves has a slope significantly different from the other two curves, and the same is true for the temperature curves. DISCUSSION The validity of the survivor curves for L. autumnalis based on MPN values is supported by the following observations. (i) The linear regression line for log MPN versus time fell within most of the 95% confidence intervals; the nine linear regression lines presented missed 9 of 114 (7.9%) confidence intervals. (ii) The linear correlation coefficient was consistently high, i.e., near -1 (range: -0.870 to -0.987); this coefficient is a measure of the strength of the linear relationship between the variables. (iii) A statistical test for goodness of fit usually accepted a linear model for log MPN versus time. These three tests are consistent with an exponential death rate in the test solutions and support the reliability of the MPN procedure for estimating the number of survivors. The influence of temperature and pH on survival was demonstrated through statistical analyses for difference in survivor curve slopes. The results generally agree with other reports on the effect of temperature (2, 3) and pH (1,5) on survival of Leptospira. The results substantiate the sensitivity of this method for studying the influence of environmental factors on survival. The entire procedure is offered as a more quantitative model for survival studies with Leptospira.

v3-fos

2020-12-10T09:04:12.923Z

1973-01-01T00:00:00.000Z

237234866

s2

Characterization of a Heat-Stable Protease of Pseudomonas fluorescens P261 A heat-stable, extracellular proteolytic enzyme was isolated from Pseudomonas fluorescens P26. Brain heart infusion broth (Fisher Scientific Co.), pH 7.5, and incubation at 21 C provided the optimal conditions for bacterial growth for enzyme production. The organism had a D value of 2.6 min at 62.8 C (145 F). The enzyme, however, was quite heat-stable, requiring 15 hr at 62.8 C, 8 hr at 71.4 C, and 9 min at 121 C for complete inactivation. Milk, whey, and casein each had a protective effect on the enzyme against heat inactivation. Purification was accomplished by growth of organisms in broth, centrifugation, sterilization by filtration, ammonium sulfate precipitation (55% satura-tion), dialysis (against six changes of water), and protein separation by passage through a Sephadex G-100 column. Ultracentrifugation revealed a single band with a sedimentation coefficient of 1.51 which suggested a molecular weight of approximately 23,000. As little as 0.2 unit of the purified enzyme caused detectable flavor defects during 30 days of storage at 4 C, and the Hull test for liberation of tyrosine compared favorably in sensitivity with the sensory method in milk. casein MATERIALS AND METHODS Selection of protease producers. Psychrophilic bacteria from the stock culture collection of the Department of Food Science and Nutrition were screened for production of protease by streaking on casein digest agar (15% skim milk in plate count agar). Cultures which were proteolytic were grown in nutrient broth at 21 C for 48 hr. Cells were removed by centrifugation, and the supernatant fluid was sterilized by filtration. Filtrates (0.1 ml) were absorbed in 12-mm filter paper discs which were placed on surfaces of casein digest agar plates. Zones of proteolysis were measured after incubation at 21 C for 48 hr. Quantitation of protease activity. Enzyme activity was determined by the method of Hull (3), in which the quantitites of free tyrosine and tryptophan are measured spectrophotometrically after incubation of the enzyme with the substrate. The standard curve was based on analyses of solutions of tyrosine. One unit of enzyme was defined as the milligram of purified protein that would produce one milligram of tyrosine (equivalent) per milliliter from a solution of 2.5% casein in 48 hr at 21 C. The quantity of protein per unit was determined by adding 2.5 ml of two fold serial dilutions (1: 1 through 1:256) of purified enzyme in sterile phosphate buffer (pH 7.45) to 7.5 ml of 2.5% casein solution. After 48 hr at 21 C, the enzyme was inactivated with trichloroacetic acid and the quantity of tyrosine and tryptophan was determined. HEAT-STABLE PROTEASE OF P. FLUORESCENS P26 Optimal medium, pH, and temperature. The following media were tested for ability to support protease production: brain heart infusion (Fisher Scientific Co.), nutrient (Difco), Trypticase soy (Difco), and tryptone glucose extract broth (Difco). Each was adjusted to pH levels of 5.5, 6.5, 7.5, and 8.5, with 1 N HCl or NaOH. Incubation temperatures of 10, 21, 32, and 37 C were used for each combination of medium and pH. Also, the organism was grown in brain heart infusion broth from three different manufacturers, and the enzyme concentration in each was determined. Enzyme preparation and examination. Brain heart infusion broth (Fisher Scientific Co.) was inoculated with 1% of an actively growing culture of P. flourescens P26 and incubated for 48 hr at 21 C. Cells were removed by centrifugation (4,500 x g for 30 min) and filtration (0.45-agm membrane filter). The filtrate (crude enzyme) was stored at -10 C in 5-ml portions. Purified enzyme was prepared by ammonium sulfate precipitation from the crude enzyme. Initial stepwise addition of ammonium sulfate indicated best yields of enzyme at 55% of saturation during storage at 4 C for 12 hr. The precipitate was removed by centrifugation and resuspended in phosphate buffer at pH 7.45. Salts were removed by dialysis against six changes of water over a 24-hr period and against phosphate buffer for 12 hr. Dialysate (320 mg of protein) was filtered through a 4-by 78-cm column of Sephadex G-100 which had previously been equilibrated against the eluent buffer (phosphate buffer, pH 7.45). Eluate was collected in 3-ml fractions, and protein content was determined on a Hitachi Perkin-Elmer model 124 double-beam spectrophotometer at 280 nm (bovine serum albumin standard curve). Fractions were screened for proteolytic activity by using saturated filter paper discs on casein digest agar plates. Proteolytic fractions were further evaluated by the Hull test, and strongly proteolytic fractions were pooled and stored at -20 C. Homogeneity of purified enzyme was confirmed by agar plates. Proteolytic fractions were further evaluated by the Hull test, and strongly proteolytic as follows: cyanogum 41 gel (7%), 0.4 M trisglycine buffer at pH 9.3, 200 v for 3.5 hr, amido black stain, methanol, and glacial acetic acid dye solvent. Ultracentrifugation was accomplished in a Beckman model L analytical ultracentrifuge by using a 4to 12-mm sector valve type synthetic boundary cell operated at 59,780 rev/min at 20 C. Purified enzyme was acid hydrolyzed under nitrogen at 110 C for 21 hr (4). The hydrolysate was taken to dryness, redissolved in 2 ml of 0.125 M norleucine in pH 2.2 citrate buffer, and then analyzed by automated cation exchange chromatography using a Bio Cal BC200 analyzer (1). D value for P. fluorescens P26. Sterile skim milk (10 ml) was inoculated with 5.0 x 10" organisms per ml and heated at 62.8 C. Tubes were removed to an ice bath at "come up time" and each minute thereafter for 30 min. Plate counts were made in duplicate. Inactivation of enzyme by heat. Samples (3 ml) of crude enzyme at 4 C were placed in screw-cap tubes (12 by 125 mm) which were immersed in a water bath at 62.8 ± 0.2 C. Time for temperature to reach 62.8 C ("come up time") was 3.8 min, as determined by repeated measurement with a thermister (model 42SF Telethermometer, Yellow Springs Instruments) immersed in the liquid. When the temperature reached 62.8 C, and each hour thereafter for 18 hr, one tube was removed. Upon removal, tubes were immediately immersed in an ice bath and held until heating was completed for the lot. The same technique was used for determining the rate of inactivation at 71.4 C (± 0.2 C) except that samples were removed at "come up time," at 15, 30, 60, 90, and 120 min and at 4, 6, 8, and 10 hr of heating. Tests for inactivation at 121 C were done in thermal death time tubes to which 6.0 ml of crude enzyme were added prior to sealing the tubes with an oxygen flame. The thermister was placed in one tube, and the opening was sealed with epoxy. All tubes were submerged in a thermostatically controlled mineral oil bath at 121 0.2 C. Tubes were removed to an ice bath at "come up time" and at 1-min intervals up to 25 min. Activity of each sample was determined by the Hull test. Three milliliters each of skim milk, whey (from acidified pasteurized skim milk), casein solution (2.5% acid casein), and water were placed in separate test tubes. Three milliliters of crude enzyme was added to each tube, and the contents were pasteurized at 71.4 + 0.2 C. One tube from each group was removed and placed immediately in an ice bath at "come up time," after 15, 30, 45, 60, 90, and 120 min of heating, and at hourly intervals to 10 hr. Off-flavor production. Skim milk was sterilized by heating in flowing steam for 30 min on 3 consecutive days. Purified enzyme was added to 50-ml samples to give final concentrations of 4, 2, 1, 0.5, 0.4, 0.2, 0.1, 0.05, and 0.04 units per ml. Controls were the sterilized skim milk and sterilized skim milk plus 4 ml of buffer, the amount added with the enzyme. Samples were randomly coded and stored at 4 C for 30 days, after which the amount of proteolysis was determined by the Hull test, and flavor and odor were determined by two experienced judges. RESULTS AND DISCUSSION Selection of protease producers. Tests were conducted on 47 different strains of psychrophilic bacteria: 3 Achromobacter, 3 Aerobacter, 3 Enterobacter, 1 Escherichia, 1 Flavobacterium, 1 Proteus, and 36 Pseudomonas. The Escherichia strain and 14 of the pseudomonads were proteolytic according to the disc assay method. Six of the pseudomonads produced proteases that remained active after heating at 62.8 C for 30 min. Each belonged to the fluorescent group of aerobic pseudomonads (8). The most heat-stable of these enzymes was produced by P. fluorescens P26, which was studied in detail. Optimal medium, pH, and temperature. Brain heart infusion broth was slightly better than Trypticase soy broth for enzyme production. The organism produced little enzyme in nutrient broth or tryptone glucose meat extract broth. In the latter, growth was excellent. Slightly more activity was found at pH 7.5 than at 6.5, but broths adjusted to pH 5.5 or 8.5 had less than half of this activity. Incubation at 21 C proved far superior to 37, 32, or 10 C. Enzyme activity in broths produced at 37 C was only 3% of that associated with incubation at 21 C. The generation times for P. fluorescens P26 at 4, 10, and 21 C were 10, 5.5, and 4.5 hr, respectively. The three brain heart infusion media, BBL, Difco, and Fisher, supported growth equally well. Average plate counts were within 10% of each other. But enzyme activity was nearly four times higher in the sterile filtrate from the Fisher broth than in filtrate from Difco broth, and activity of the latter was 10 times that of filtrate from the BBL broth. Characteristics of the enzyme. We recovered 5.7% of the protein applied to the Sephadex column (Table 1). Specific activity of the crude and purified preparations indicated concentration by some 392 times. The single band observed after polyacrylamide electrophoresis indicated the preparation was electrophoretically pure (Fig. 1). All protein migrated from the spot, as evidenced by the lack of stain at that location. A zone of proteolysis developed when an unstained slice of the polyacrylamide gel was placed on casein agar. The zone developed 12.5 cm from the slot, the same distance at which we observed the stained band. Ultracentrifugation also revealed a single band (Fig. 2) which had a sedimentation coefficient of 1.51, suggesting a molecular weight of 23,000. Amino acid analysis (Table 2) revealed the absence of cystine. Tryptophan is destroyed by acid hydrolysis; hence, it does not appear in the table. Proline (17.9%), glycine (17.9%) and glutamic acid (12.9%) were present in greatest quantities. D value for P. fluorescens P26. The test organism was rapidly killed in skim milk heated at 62.8 C. The D value was 2.6 min, which agrees closely with the D value of 2.5 min reported by Davis and Babel (2) for organisms producing slime on cottage cheese. Inactivation of enzyme by heat. "Come up times" for heating at 62.8, 71.4, and 120 C were 3.8, 4.6, and 8.5 min, respectively. The enzyme lost about 30% of its initial activity in this time during heating to 62.8 and 71.4 C. However, about 14 hr were required to inactivate 90% of the enzyme at 62.8 C (Fig. 3). More than 8 hr of exposure were required for complete inactivation at 71.4 C (Fig. 3). About 9 min were required when the temperature was 120 C (Fig. 4). The lengthy "come up time" at the highest temperature caused a loss of about 70% of the initial activity. Skim milk, whey, and 2.5% casein protected the enzyme from heat denaturation at 71.4 C (Fig. 5). Skim milk was significantly more protective than whey or casein, and they produced significant protection (P < 0.05) compared to water. The data suggested that the effect of skim milk was due to the additive effects of whey and casein. Off-flavors produced. The purified enzyme caused an unclean flavor to develop, followed by a bitter flavor ( Table 3). Evidence of proteolysis, the presence of free tyrosine and tryptophan in the milk, was directly related to the presence of bitterness. As little as 0.2 unit of enzyme was able to produce an off-flavor during storage of the milk for 30 days at 4 C.

v3-fos

2020-12-10T09:04:17.321Z

1973-10-01T00:00:00.000Z

237234090

s2

Sporicidal Properties of Hydrogen Peroxide Against Food Spoilage Organisms The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H2O2 were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with “D” values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H2O2 concentration and the temperature reduced the lag period. Packaging systems that utilize paper or plastic-based packaging materials are used increasingly by the food industry because of the simplicity of operation, the lower raw-material costs, and the easier disposal of the empty container. According to Hsu (4), the most widely accepted of these systems are suitable for aseptic operation, but the nature of the packaging material excludes the use of heat as a sterilizing agent. Among the chemical sterilants available, hydrogen peroxide (H.02) appears to be the most suitable because it does not impart an off-flavor to the product and small residues on the packaging material can be tolerated without adverse effects. The use of H202 for the sterilization of pipes, filters, etc., in the food industry has been reported as early as 1916, viz: Schumb et al., (5). Swartling and Lindgren (6) observed slow sporicidal activity of 11 to 22% H202 at room temperature on spores of Bacillus subtilis and Bacillus cereus var. mycoides which were inoculated on glass and polyethylene surfaces. However, exposure for 15 s to 22% H202 at room temperature, followed by hot-air heating at 125 C for 10 s, substantially reduced the numbers of survivors. Cerf and Hermier (2) reported that 3.5 min of exposure to 15% H202 at 80 C was necessary to achieve four-decimal reductions in populations of the most resistant of 21 Bacillus strains tested. von Bockelmann and von Bockelmann (8) 'Present address: Department of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland. reviewed the literature on the sporicidal properties of H20,. Many of the studies referred to the use of 15 to 20% H202. The absence of data on H202 inactivation of dry spores and the apparent differences in results by using different techniques were discussed. The present study was undertaken to establish the relative resistance of food spoilage organisms to inactivation with H202 and to identify the factors that affect spore inactivation by H202 during aseptic packaging. MATERIALS Preparation of test suspensions. B. subtilis var. globigii and B. coagulans were grown in tryptic soy broth (Difco) and incubated at 37 and 55 C for 4 to 6 days respectively. The spores were harvested by centrifugation, washed three times, suspended in physiological saline, and stored at 4 C until used. B. stearothermophilus and B. subtilis SA 22 were grown on nutrient agar slants (Difco) in 900-ml screw-capped prescription bottles incubated at 55 and 37 C respectively. After 5 days, the spores were recovered from the slants by washing the surface with saline and collecting the suspended cells. S. aureus ATCC 6538 was grown in micrococcus medium (peptone, 5.0 g; yeast extract, 3.0 g; beef extract, 1.5 g; glucose, 1 g; in 1 liter of distilled water, pH 7.4), and incubated at 37 C. Samples from the actively growing cell suspension were used in the tests. Clostridium sp. NCA 3679 was grown in thioglycolate broth (Difco), sealed with mineral oil, and incubated at 37 C. Spores were recovered by successive centrifugation and suspension in saline as was previously discussed. Cultures were tested for the extent of sporulation before harvesting by staining smears with malachite green. Counts taken of samples from spore suspensions plated directly from stock and those plated after heating at 80 C for 20 min showed no significant differences, indicating that the stock suspensions were essentially all spores. Enumeration of viable organisms. Standard plate count techniques were used. B. subtilis var. globigii, B. stearothermophilus, S. aureus, and B. coagulans were plated on plate count agar (Difco) and incubated at their optimal growth temperature. B. subtilis spores were plated on tryptone glucose extract agar (Difco). Clostridium sp. 3679 spores were plated on thioglycolate agar and incubated at 35 C in an anaerobic chamber. Initial spore counts were made by plating a sample of the test spore suspension that had been heated for 20 min at 80 C. Spores treated with H202 were not heat shocked so that they were able to retain maximum resistance to H202. H202 treatment: (i) Wet spores at room tempera- immersed in a water bath until the H,02 reached water bath temperature. One ml of spore suspension at room temperature was then introduced to the test tubes rapidly by using a 5-ml plastic disposable syringe fitted with a cannula. After the desired contact time, 1 ml of the mixture was removed by using a sterile syringe and cannula and immediately discharged into the catalase solution, and the numbers of survivors were determined. Average temperatures to which the spores were actually exposed after mixing the cold spore suspension with hot H,02 were determined by recording the temperature change in control test tubes by using H202 and water. (iii) Dry spores. A 1-ml amount of spore suspension was introduced into sterile 25-by 150-mm culture tubes and dried over CaCl2 in a desiccator at 25 C for 72 h. No evidence of spore injury due to drying was found as shown by similar counts obtained from the dried spores and that from the same volume of wet spores. H202 was diluted with distilled water to the same concentration used for the wet-spore treatments after the 4 to 1 ratio mixing and added to the dry spores in the culture tube. A small, sterile, magnetic stirring bar was then introduced, and the mixture was agitated continuously over a magnetic stir plate. At appropriate time intervals, 1-ml samples were removed, and the numbers of survivors were determined, as was previously described for the wet spores. RESULTS AND DISCUSSION Comparative resistance of different microorganisms to H202. The resistance of various microorganisms to 25.8% H202 at 24 C is shown in Fig. 1. As expected, S. aureus was less resistant than any of the spore forms requiring only 1 min of exposure to reduce populations by 593 VOL. 26,1973 six log cycles. Thus, the most rigorous treatment necessary to eliminate spore-forming spoilage organisms should eliminate S. aureus as well. Data reported by Dittmar (3) indicated a very low resistance of this microorganism to HO,, requiring only 10 min in 0.05% H,O, for inactivation. Amin and Olson (1) worked with more resistant strains of S. aureus and reported as much as 171 min of exposure to 0.05% HO, for 99.9% destruction of cells in 250 ml of a 10' suspension per ml. Both groups of investigators used 0.05% H02, a much lower concentration than the 25.8% H02 used in the present investigation. Survival curves for most of the spore-forming organisms showed a lag period after initial exposure where there was a slow reduction in count, followed by a rapid rate of inactivation representative of first-order reaction kinetics. The less the resistance of the organism, the shorter the lag period, and for the two least resistant organisms, the survival curve followed first-order kinetics from the initial exposure. If there was a lag period for these organisms, it was not observed after the initial 30-s interval. The shapes of the survival curves were the same as those reported by Swartling and Lindgren (8) for B. subtilis ATCC 95244 spores in 10% H202. No tailing was observed in the survival curves of the organisms tested. Cerf and Hermier (2) reported a tail in the survival curve of a Bacillus strain isolated from milk when it was exposed to 23% H202 at pH 7.7 and 26 C. According to their data, the tail appeared to be less pronounced at pH 2.9. The present study was conducted by using stabilized H202 (pH 3.8) without adjustment of pH. Schumb (5) points out that HO, has maximal stability at pH 3.5 to 4.0, but becomes increasingly unstable at very low and very high pH values. The presence of the tail in Cerf and Hermier's survival curve could be due to decomposition of H202, resulting in decreased activity at the later stages of exposure. Figure 1 shows that the order of resistance was B. subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus. The "D" values determined from the straight line portion of the inactivation curves were 7.3, 2, 1.8, and 1.5 min, respectively, in 25.8% H02 at 24 C. Clostridium sp. 3679 and S. aureus showed very low resistance to H202. By comparison, Swartling and Lindgren (7) reported a "D" value of 2.3 min for B. subtilis ATCC 95244 in 20% H202, and Cerf and Hermier (2) reported a "D" value of 3.5 min in 23% H,02 at pH 4.6 for the most resistant Bacillus strain isolated from milk. Thus, B. subtilis SA 22 is much more resistant than the organisms previously tested by other investigators. The resistance of B. subtilis var. globigii is comparable to organisms previously tested. Effect of combined heat and HO, treatment on spore survival. Heat shock of spores at 80 C for 20 min prior to H2O2 treatment enhanced their destruction. Because the heatshocked spores were quickly cooled in ice water and immediately treated with H202, the possibility of germinated spores being responsible for the decreased resistance to H202 was eliminated. The inactivation curve for heat-shocked spores of B. subtilis var. globigii (Fig. 2) showed a rapid rate of destruction after 4 min of exposure to H202 compared with untreated spores. This is evidenced by the "D" values of the straight line portion of the curves which were 0.5 and 2 min, respectively. The mild heat treatment alone did not significantly reduce the spore count, but it was sufficient to decrease their resistance to H202. When the unheated spores were exposed to 25.8% H202 for 4 min at 24 C and then, after destroying the H202 by catalase, heated at 80 C in a water bath for 20 min, a six-log cycle reduction in count was observed. The 4 min of treatment with H202 by itself did not reduce the spore count by more than 1 log cycle (Fig. 2), and the heat treatment by itself did not signifi- SPORICIDAL PROPERTIES OF H202 cantly alter the count. Yet, the H202 treatment considerably reduced the resistance of spores so that a mild heat treatment inactivated the injured spores. This result is in agreement with data given by Swartling and Lindgren (6) who showed that 11 s of exposure to 22% H202 at room temperature did not inactivate spores of B. subtilis, but that exposure of the spores to hot air at 125 C for 8 to 10 s following the H202 treatment resulted in a 99.2% destruction of spore population. Effect of H202 concentration. Increasing the concentration of H202 increased its sporicidal properties. The survival curves of B. subtilis var. globigii in varying concentrations of H202 at 24 C (Fig. 3) showed that increasing H202 concentrations lowered the time during which the curves showed the initial change in slope, reduced the exposure time, and also decreased the "D" value. Ten minutes of exposure at 10 and 20% was insufficient to start the curve on a exponential decline of surviving spore populations, whereas 6, 4, and 1 min were sufficient when 25.8, 35, and 41% H202, respectively, were applied. "D" values were 2, 1.5, and 0.75 in Fig. 1 through 4 reveals that simple multi-At of temperature. The temperature at plication of the "D" value by the number of log H202 was incorporated has a very marked cycles for the spore inactivation desired is n spore inactivation. Figure 4 shows that insufficient to obtain the required exposure e of inactivation in 25.8% H202 increased time for inactivation because of the initial creasing temperature. persistence of spores, particularly those of B. nination of the inactivation curves shown subtilis SA 22 and B. subtilis var. globigii. However, in most of the curves, the lag does not 10% persist for more than one log cycle of initial A~o * decrease in spore population. Thus, the "D" 0\ \A concept utilized in heat sterilization can also be A.\ \ \\0 utilized for H202 sterilization if, in addition to 20\ \ <% the "D" value, the time required for the first log cycle reduction is incorporated into the calcula-0 tion. In Fig. 5, the "D" value and time required to achieve the first log-cycle reduction in spore Based on the "D" value, the "z" value for B. subtilis var globigii in 25.8% H202 is 40 C. This compares with "z" values of 46 C, 52 C, and 47 C determined from the data reported by Swartling and Lindgren (7) for B. subtilis in 10%, 15%, and 20% H202, respectively. The "z" value is the temperature change required to bring about a 10-fold change in the "D" value. Comparative resistance of wet and dry spores. In a well-mixed system, dry spores are less resistant to H202 (viz. Fig. 6). Spores of B. subtilis SA 22 in 25.8% H202 at 24 C had a first log-cycle inactivation time and a "D" value of 8.5 and 7.3 min respectively when wet, compared to 4.8 and 4.7 min respectively when dry. Spore inactivation started immediately after contact of dry spores with H20., whereas a short induction period was required for wet spores. Because the wet-spore suspension was replaced by water in the H202 solution used for the dry-spore treatment, the actual H202 concen- In an unmixed system (treatment was similar to wet spores at room temperature except that spores were allowed to dry inside the syringe prior to H202 treatment), data scatter was very * pronounced, and in most instances more of the eventually appears in the packaged food and a reasonable margin of safety is allowed in the elimination of pathogenic and food-spoilage microorganisms. Acceptance of H202 has been slow because of uncertainties with regard to its sporicidal properties and the lack of quantitative information necessary to evaluate the sterilizing effectiveness of a specific processing condition within a system. The system described by Hsu (4) is now used primarily on refrigerated or on acid foods. Swartling and Lindgren (7) described microbiological studies on which treatments used in the above system were based. By using 10 to 20% H202, the conditions of exposure to H202 were 0 20 30 40 50 60 TO 80 90 100 sufficient to reduce the population of B. subtilis 95244 (the one species of spore-forming orga-TEMPERATURE 0C nisms studied) by four to six log cycles. Our 5. Effect of temperature of 25.8% H202 on results show that organisms exist that are more :due and time for first log cycle inactivation of resistant than the B. subtilis used by Swartling of B. subtilis var. globigii. and Lindgren (7) and may be used to evaluate 596 APPL. MICROBIOL. "I sterilizing treatments needed for paper-based packaging materials. The organisms in the present study were able to survive the treatments they recommended. A safer process would result if the most resistant organism in our study, Bacillus subtilis SA 22, is used as a basis for developing requirements for sterilization in H202. Although an anaerobe, Clostridium sp. PA3679 appears to have very low resistance to H202, and the resistance of other anaerobes such as Clostridium botulinum to H202 should be further investigated. The danger from anaerobic organisms in aseptically packaged foods using plastic or paper-based packaging materials is minimized because of the permeability of these materials to oxygen and the minimal vacuum in the container. Our results show that "D" and "z" value concepts used in determining inactivation times in heat sterilization could be applied to H202 if the time required for inactivating the first 90% of the population was used in addition to the "D" value. We have presented data on the "D" values of six microorganisms at 24 C and the "z" value of one organism in 25.8% H202. When more data are compiled on the "D" and "z" values of various microorganisms at different H202 concentrations, it should be possible to evaluate conditions in any aseptic packaging system in which H202 is used for sterilization.

v3-fos

2020-12-10T09:04:16.753Z

1973-01-01T00:00:00.000Z

237230630

s2

Effects of Homologous Bacteriophage on Growth of Pseudomonas fragi WY in Milk Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml. Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml. (2) isolated two strains of bacteriophages and their bacterial hosts from raw skim milk. They isolated 36 other strains from additional refrigerated foods. This created speculation that the growth of bacteria within refrigerated foods might be slowed by homologous bacteriophage, thus extending shelf life of the food. The present report describes conditions which had to exist if a homologous bacteriophage of Pseudomonas fragi WY was to significantly affect growth of the host bacterium in refrigerated milk. MATERIALS AND METHODS Bacteriophage and host. P. fragi WY and its homologous bacteriophage, wy, were isolated from ground beef, and their characteristics have been reported (3). A suspension (160 ml) of bacteriophage particles containing 2 x 1012 plaque-forming units (PFU) per ml was prepared by propagation on 25 large petri plates (150 mm) by the double-layer method (1). The semisolid layer of each confluently lysed plate was macerated in 6 ml of tryptic soy broth (TSB) and combined in a beaker. After 5 hr of incubation at 4 C to complete lysis, the slurry was centrifuged for 15 min at 10,000 x g, and the supernatant fluid was sterilized by consecutive filtration through membranes with pore sizes of 1. Anti-phage serum. Four adult rabbits were subcutaneously inoculated twice weekly for 3 weeks with 5 ml of the high-titer phage suspension. One week after the last injection, rabbits were bled by cardiac puncture. The blood was allowed to coagulate at 37 C in vaseline-lined centrifuge tubes before overnight storage at 4 C. After centrifuging for 10 min at 5,000 x g, the serum was drawn off, filtered through a membrane filter (0.45 Am diameter pore size), stored at 4 C, and assayed for anti-phage activity. After assay, the sera were pooled and stored frozen. Assay of anti-phage activity. Serum was diluted 1:100 and 1: 1,000 in TSB, and high-titer phage stock was diluted to 107 PFU/ml. Phage suspension (0.1 ml) was added to 0.9 ml of each dilution of antiserum at room temperature. At 5-min intervals, 0.1-ml samples of the phage-serum mixture were added to 9.9 ml of TSB to stop the antibody reaction, and 0.1-ml samples of this dilution were plated by the agar layer method (1). If phages were not inactivated, about 1,000 plaques appeared after incubation; 90% inactivation resulted in about 100 plaques, and 99% inactivation (the desired level) resulted in about 10 plaques. Effect of phage on its host in skim milk. Skim milk, obtained from the University of Missouri Dairy, was sterilized by heating at 121 C for 15 min. The milk was divided into 400-ml lots and then inoculated with P. fragi WY in two concentrations: 1,000 and 100,000 cells/ml. (Previous observation showed that a 24-hr TSB culture contained approximately 2 x 108 cells/ml; this figure was used as a basis for dilutions prior to inoculation.) The host cells were mixed thoroughly with the milk by stirring 5 min with a Teflon spinbar driven by a magnetic stirrer. Each 400-ml lot of milk containing either 10' or 10' host cells per ml was subdivided into four 100-ml samples; these samples were inoculated with 0, 0.1, 10, or 1,000 PFU/ml. These ratios of phage to host were consid-ered to be those which might occur in milk normally. Controls consisting of milk containing no phage particles or bacteria and one containing 100 phages per ml but no bacteria were also incubated. All samples were incubated at 7 C for 72 hr (a time and temperature at which grade A milk might be held in practice). Samples were then plated in triplicate to determine bacteria and phage counts. Plates were incubated at 21 C; phage counts were made after 15 hr, and bacteria were counted after 48 hr. Antiserum was added at a 1: 1,000 concentration to the first dilution bottle used in making bacterial counts to prevent additional infections of bacteria. These experiments were replicated three times. RESULTS AND DISCUSSION Anti-phage serum was used to preclude lysis of host cells during plating of samples to determine bacterial counts. Our preliminary experiments had suggested this as a possibility. Our serum inactivated 99% of the bacteriophages at room temperature in 5 min when diluted 1: 100 and in 20 min when diluted 1: 1,000. No bacteria were found in controls with only added phage, and no phage were found in controls with only added bacteria. When the ititial concentration of P. fragi WY was 100,000/ml, addition of 10 or 1,000 PFU/ml of homologous phages caused significantly lower bacterial counts after 72 hr at 7 C (Fig. 1). The count appeared to be lower when only 0.1 PFU/ml was added, but the difference was not statistically significant. Bacteriophage titers were significantly different at the end of incubation, with the lowest and highest counts corresponding to the lowest and highest inputs of phage, respectively. Effects of added phages were much less pronounced when the initial bacterial concentration was 1,000/ml (Fig. 2). In fact, the bacterial count of the control was lower (insignificantly) than the average counts of each sample to which phage was added. Numbers of phages increased markedly in samples to which 10 and 1,000 PFU/ml were added. There was no detectable multiplication of phage in the samples to which only 0.1 PFU/ml was added. These results indicate that bacteriophage can have an influence on shelf life of refrigerated milk, but the conditions necessary for significant effect are improbable. These conditions are (i) a relatively high population of the host bacterium, (ii) presence of its homologous

v3-fos

2018-04-03T01:53:42.447Z

1973-11-01T00:00:00.000Z

31429940

s2

Preparation of Antimicrobial Compounds by Hydrolysis of Oleuropein from Green Olives' Oleuropein, an intensely bitter glucoside, was isolated from green olives. Hydrolysis products obtained from oleuropein in sufficient quantity for further tests were: (i) l-3, 4-dihydroxyphenylethyl alcohol prepared by acid hydrolysis of oleuropein; (ii) elenolic acid obtained by methanolysis of oleuropein, isolation of the intermediate acetal, and subsequent acid hydrolysis; and (iii) oleuropein aglycone formed by the action of f3-glucosidase on the parent glucoside. Mass spectral verification of the isolated compounds and ultraviolet absorption data are given. Oleuropein and its aglycone had similar threshold levels for detection of bitterness, whereas elenolic acid and f-3, 4-dihydroxyphenylethyl alcohol were not judged to be bitter. The bitter principle of olives, oleuropein, was named and studied by Bourquelot and Vintilesco (1). Later Panizzi et al. (8) showed that the oleuropein molecule contained glucose, fl-3, 4-dihydroxyphenylethyl alcohol, and an acid (Fig. 1). These workers suggested that this acid was identical to a hypotensive agent designated as elenolic acid (W. L. C. Veer, U.S. Patent 3,033,877, 1962; reference 10) which was prepared by hydrolysis of olive extracts with phosphoric acid. Fleming et al. (5) isolated a compound from green olives that appeared to have the antimicrobial properties noted earlier during the fermentation of brined olives (3,4). The compound, a bitter phenolic material, was considered to be an enzymatic degradation product of oleuropein (5). Others (8) proposed that oleuropein is hydrolyzed in vitro by fl-glucosidase into glucose and a bitter tasting aglycone. Because of the importance of a proper fermentation on the preservation of brined green olives, a complete understanding of oleuropein's role was needed. The present study was undertaken to develop a procedure to produce sufficient amounts of oleuropein and its hydrolysis products so that the chemical and antimicrobial properties could be determined. Effects of these compounds on selected species of bacteria and yeasts are reported in a separate paper (6). ' Paper no. 4108 of the Journal Series of the North Carolina State University Experiment Station, Raleigh, N.C. MATERIALS AND METHODS Extraction and purification of oleuropein. Oleuropein was extracted from steam-heated (100 C, 20 min), green, Manzanillo variety olives (500 g) as described earlier (5) except that the residue from the methanol extraction was shaken with hexane to remove lipids before extraction with ethyl acetate. The lipid-free ethyl acetate solution was evaporated to 20 ml, and the components were separated by counter-current distribution (CCD) with the solvent system of ethyl acetate: 0.1 M potassium phosphate, pH 4.5. By using absorption at 280 nm, the major band was located and collected, and the solvent was removed by evaporation. The isolate was a crusty, light-yellow material (7.2 g), consisting mainly of oleuropein. This dried extract was dissolved in methanol, and the oleuropein fraction was purified by preparative thin-layer chromatography (PLC) as described earlier (5) except that the solvent system was benzenemethanol-acetic acid (45:16:1). The oleuropein was purified further by CCD by using ethyl acetate as the mobile phase and distilled deionized water as the stationary phase. Oleuropein (2.01 g) purified in this manner was a light-yellow amorphous material. The final yield of purified oleuropein was about 0.4% of the weight of the pitted olives. Acid hydrolysis of oleuropein and isolation of the products. Purified oleuropein (500 mg) was hydrolyzed in 100 ml of 1 N H2S04 for 1 h at 100 C. The hydrolysate was cooled, adjusted to pH 2, and extracted with ethyl acetate. After drying, the solvent was removed in vacuo, yielding 304 mg of an oily product. The oil was dissolved in methanol and applied to thin-layer chromatography (TLC) plates coated with Silica Gel HF2,4. After development in WALTER, FLEMING, AND ETCHELLS benzene-methanol-acetic acid (45:8: 1), the plate was observed under shortwave ultraviolet (UV) light, and three compounds were noted. Compound 1 (R, 0.26) gave a positive reaction when sprayed with a phenolsensitive reagent (5). The R, was identical to that of authentic #l-3,4-dihydroxyphenylethyl alcohol. Compound 2 (R. 0.35) gave a faint blue color with the phenolic spray and was assumed to be the aglycone of oleuropein. Compound 3 (R. 0.43) gave a negative phenol test and had an R. value similar to that of elenolic acid. With the compounds tentatively identified, components of the oleuropein hydrolysate were separated by PLC by using the solvent system described above for analytical TLC. Each of the three zones was collected and dissolved in the appropriate solvent for purification by CCD. The solvent system used for CCD purification of the aglycone and elenolic acid was ethyl ether-water (compounds located by absorption at 224 nm), whereas ethyl acetate-water was used for fl-3,4-dihydroxyphenylethyl alcohol (located by absorption at 280 nm). Yields of the lyophilized isolated products were: fl-3,4-dihydrolyphenylethyl alcohol, 65 mg; aglycone, 14 mg; and elenolic acid, 15 mg. High-resolution mass spectra were obtained on each of these compounds. The glucose content in the hydrolysate, assayed by paper chromatography (5) followed by enzymatic glucose analysis (11), was 27.4% of the weight of the oleuropein sample. This value is similar to that previously reported (5) but less than the theoretical value of 33%. Macropreparation of compounds. Because direct acid hydrolysis of oleuropein gave very small amounts of elenolic acid and oleuropein aglycone, alternate methods were used to prepare quantities sufficient for microbiological studies. Elenolic acid was prepared by methylating a crude oleuropein extract with anhydrous methanolic hydrogen chloride. The resulting methyl-o-methyl elenolate (W. L. C. Veer, U.S. Patent 3,033,877, 1962) was isolated and converted into the free acid by hydrolysis in dilute mineral acid. Crude oleuropein (3.4 g dry weight) from an ethyl acetate extract of olives was dissolved in 10 ml of anhydrous methanolic HCl (5%), sealed in an ampoule under nitrogen, and heated at 65 C for 3.5 h. The methyl-o-methyl elenolate (1.06 g) was obtained from the reaction mixture by extraction with ethyl acetate, followed by a CCD separation using hexanemethanol, 1: 1, as the solvent system. The oily material (1.01 g) was dissolved in 5 ml of ether and added dropwise to 300 ml of 1 N H2S04, and the mixture was stirred at 70 to 80 C for 1.5 h. The solution was cooled and extracted with ethyl ether. The extract was washed several times to remove H2SO4, dried, and the solvent was evaporated, leaving 0.65 g of a colorless, oily material which TLC analysis indicated was primarily elenolic acid. This material was purified further by PLC and finally by CCD to give 0.150 g of pure elenolic acid. Oleuropein aglycone was prepared by enzymatic hydrolysis of oleuropein by using fl-glucosidase. A filter-sterilized 1% solution of pure oleuropein (500 mg) in 0.1 M sodium acetate buffer, pH 4.1, was mixed with one-ninth volume of filter-sterilized 2% fl-glucosidase solution (Sigma Chemical Co.) and incubated for 16 h at 32 C. The solution then was extracted with chloroform. After removal of the solvent, the resulting purple oil was purified by PLC followed by CCD. The purified aglycone was a lightbrown oil (78 mg). Analyses. Mass spectra were obtained at the facilities of the Research Triangle Institute on an AEI-MS-902 instrument. Elemental analyses and molecular weights were determined by Galbraith Laboratories, Knoxville, Tenn. UV spectra were recorded with a Cary model 15 instrument, and optical rotations were recorded with a Perkins Elmer model 141 polarimeter. CCD. CCD was performed with a 50-tube Post-Craig apparatus (H.O. Post Co., Middle Village, N.Y.). Solvents were reagent grade and redistilled prior to use. Water was distilled and deionized. TLC. Preparation of TLC and PLC plates using Silica Gel HF2.4 and Silica Gel PF2,4, respectively, was described previously, as was the procedure for developing the plates and detection of phenolic compounds (5). Compounds also were located on TLC plates by spraying with 50% H2SO and heating at 170 C for 30 min. PLC plates, after developing, were dried under nitrogen, compounds were visualized under UV light at 254 nm, and zones were collected (5). Reference compounds. A sample of elenolic acid as the calcium salt was provided by the Upjohn Company. A sample of ,B-3,4-dihydroxyphenylethyl alcohol was prepared by the Research Triangle Institute, N.C. Bitterness test. The bitterness of oleuropein and its aglycone was evaluated by a taste panel consisting of eight individuals. Whatman no. 1 filter paper was washed in ethyl alcohol, dried, and cut into 1-cm squares. The compounds were applied to the paper as ethyl alcohol solutions, and then the alcohol was removed by evaporation. Panelists were instructed to hold the paper squares on their tongues until they could detect bitterness or decide that no bitterness was present. They were given a blank square of paper first and then a series of papers containing increasing levels of the compounds. They were asked to discontinue the test at the first level where bitterness was detected and to describe any unusual characteristics of the bitterness. RESULTS AND DISCUSSION Purified oleuropein was obtained from green olives, portions were hydrolyzed, and the major fragments were isolated therefrom. Physical properties of the compounds isolated are summarized in Table 1. Oleuropein isolated by our procedure was essentially identical to that previously described (8), except that we observed a specific optical rotation value of -1780, whereas they reported -158°. The differences might be explained by the higher purity of our preparation. For example, PREPARATION OF ANTIMICROBIAL COMPOUNDS Our study confirms those of Panizzi et al. (8) and Cruess and Alsberg (2), who reported that oleuropein is hydrolyzed by fl-glucosidase. Shasha and Leibowitz (9) reported that the olive bitter principle is not attacked by the enzyme. The ability of fl-glucosidase to produce the aglycone from oleuropein is of considerable importance in olive fermentation due to the inhibitory nature of this moiety (6). A molecular weight of 540 for oleuropein was obtained by vapor pressure osmometry. This value agrees with that reported by others (8). The mass spectrum did not exhibit any fragments above 360 mass units, probably because of the nonvolatility of the oleuropein molecule. Some of the higher-molecular-weight fragments are given in Table 1. The mass spectrum for elenolic acid, whether from direct hydrolysis of oleuropein or from hydrolysis of methyl-o-methyl elenolate, was identical to the authentic reference compound. In addition, the mass spectrum of ,B-3,4-dihydroxyphenylethyl alcohol, from hydrolysis of oleuropein, was identical to that of the reference material. Some of the physical data are supplied in Table 1. Panizzi et al. (8) reported the formation of oleuropein aglycone on the basis of paper chromatographic examination of the products re-sulting from treatment of oleuropein with flglucosidase. No attempt was made by these workers to isolate or study the aglycone further. The structure given in Fig. 1, therefore, is tentative. Our preparation of the aglycone was subjected to several purification steps and is chromatographically pure (TLC). By the use of high-resolution mass spectrometry, we obtained an elemental composition of C 1H225O (molecular weight 378.132) for this compound, which corresponds to the product expected when glucose is hydrolytically cleaved from oleuropein. The highest-molecular-weight fragments and UV absorbance maxima are provided in Table 1. The structures given in Fig. 1 are those of Panizzi et al. (8) and other workers (W. L. C. Veer, U.S. Patent 3,033,877, 1962; reference 10). A recent paper, however, proposed a slightly modified structure for elenolic acid (7). The tentative structures are provided as an aid in observing the origin of oleuropein hydrolysis products. Oleuropein and its aglycone were bitter, the threshold levels for detection being about 50 sg for most of the panelists. Two individuals did not detect bitterness at a level of 200 Mg per paper square, which was the highest level tested. Although the threshold levels for detection of bitterness of both compounds were similar, some panelists described the taste of the aglycone as having a stinging, biting, or sharp sensation associated with the bitterness. Neither elenolic acid nor ,B-3, 4-dihydroxyphenylethyl alcohol was bitter at levels up to 200 Mg.

v3-fos

2018-04-03T01:37:01.575Z

1973-07-01T00:00:00.000Z

30195493

s2

Production of Ochratoxins A and B on Country Cured Ham Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm. Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm. During a recent survey on the mycoflora on country cured hams in the Southeastern United States, Sutic et al. (17) isolated two aflatoxinproducing strains of Aspergillus flavus Link ex Fries as well as other potential mycotoxin-producing aspergilli. Among these strains, one was identified as A. ochraceus Wilhelm. In a similar study, Strzelecki et al. (16) recovered two strains ofA. ochraceus from country cured hams but were not able to show any production of ochratoxins. Ochratoxin A and its dechlorinated analogue, ochratoxin B, are metabolites of several members of the A. ochraceus group (6) and of Penicillium viridicatum Westling (13), a mold which was also frequently found on country cured hams by Leistner and Ayres (8). The toxicity of ochratoxin A is well established, whereas ochratoxin B was first reported to be nontoxic (15). Later Peckham et al. (11) indicated that the toxicity of ochratoxin B was one-tenth that of ochratoxin A to day-old chicks. No carcinogenic effect has been observed for either toxin. Scott et al. (13) and Shotwell et al. (14) described cases of natural occurrence of ochratoxin A in moldy corn, wheat, and other agricultural products. In the present investigation, country cured hams from curing plants in Georgia were surveyed for occurrence of A. ochraceus. Isolates of A. ochraceus and P. viridicatum from cured hams were then screened for production of ochratoxin A and B by using media known to be suitable for obtaining high-toxin yields. I Present Address: Department of Agricultural Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland. 27 Since it was not known if cured ham itself could sustain ochratoxin formation, or how deeply the toxin or mold mycelium would penetrate if the surface of a whole ham were contaminated with A. ochraceus or P. viridicatum, these factors were also checked. MATERLALS AND METHODS Organisms. From five Georgia processing plants, 166 swabs were taken from 153 hams aged for 1 to 12 months. Sampling was restricted to colonies that appeared to have morphological and cultural characteristics of aspergilli. Each swab was inoculated initially on the Czapek-Dox agar plus 10% NaCl, thus favoring the growth of osmophilic species of Aspergillus which includes A. ochraceus (7), and then onto malt agar for final identification. From the 166 swabs, one strain of A. ochraceus (H 33) was found. This strain, as well as A. ochraceus D-1, isolated by Sutic et al. (17) Culturing. (i) On rice, defatted peanut meal, and corn. Spores (3 x 106) of each strain of A. ochraceus and P. viridicatum and 75 ml of sterile water were added to 150 g of rice or defatted peanut meal in 500-ml Erlenmeyer flasks and incubated at 25 to 27 C for 14 days. In addition, P. viridicatum was cultured on popcorn under the same conditions. These conditions were reported by Schindler and Nesheim (12) to yield maximal amounts of toxin. After steaming the cultures to facilitate extraction of ochratoxins, they were transferred to Mason jars and extracted. (ii) On country cured ham to screen for ochratoxin production. Boneless slices of fully aged country cured ham (0.5-1.0 cm thick) were procured from various commercial curing plants. Excessive fatty parts were removed and the slices were cut to a weight of 100 to 150 g. They were surface sterilized by dipping them into 1% NaOCl solution for 1 min, rinsing with sterile water, and blotting dry with sterile cheese cloth. For inoculation, the slices were swabbed with 0.5 to 1.0 ml of a spore suspension containing 106 spores per ml, thus obtaining a spore load of 0.5 x 106 to 106 spores per slice. The ham slices were then suspended by a string in sterile 1-qt (0.946-liter) Mason jars which were covered with three layers of no. 1 Whatman filter paper instead of Mason lids. The jars were incubated for various lengths of time at 5, 15, 25, 30, and 37 C and at a relative humidity of 70 to 75%. If necessary, saturated aqueous NaCl solution (relative humidity 75% at 20 C [10]) was added to the jars. After incubation, the ham slices were cut into small pieces for extraction. (iii) On country cured ham to determine toxin and mold penetration. From the center section of fully cured and aged hams, slices (5 cm thick) each weighing about 1,000-g were cut, surface sterilized as described above, and placed into sterile culturing chambers. The top surface (crossing the bone, 150 to 180 cm2) was inoculated with approximately 103 spores per cm2, whereas the edges were kept sterile by repeatedly cleaning them with NaOCl solution. Cultivation was at 25 C and 70 to 75% relative humidity for 21 days. The mycelial mat was then scraped from the surface and the slices were cut into layers (0.5 cm thick) which were assayed individually for ochratoxins. Also, slabs were cut from the slices in different depth, again surface sterilized, and incubated on rose bengal-streptomycin agar RBM-2 (18) at 25 C. Assays. Official methods of the Association of Official Analytical Chemists (1) were used to determine moisture and salt content of cured hams. To quantitate ochratoxins, all cultures were extracted with chloroform by using a Sorvall high-speed blender. The crude extracts were filtered through diatomaceous earth and concentrated to 50 ml. A clean-up step with column chromatography by the procedures proposed by Eppley (4) followed. The ochratoxins in the purified extracts were separated by thin-layer chromatography on Adsorbosil-1 (Applied Science Laboratories, State College, Pa.) with toluene-ethyl acetate-formic acid 5: 4: 1 (vol/vol/vol) as developing solvent (4). To exclude possible interference of 4-hydroxymellein with ochratoxin A, chloroform-acetone 93:7 (vol/vol) was used as a second solvent system (9). A Photovolt fluorodensitometer was used to compare intensity of fluorescence of the samples with that of standards obtained from the Bureau of Food Sanitation, Food and Drug Administration, Washington, D.C. It was possible to improve the sensitivity of this method by exposing the thinlayer chromatography plates to ammonia fumes for 2 to 4 min, a treatment which changes the fluorescence of both ochratoxin A and B from blue-green to intense blue. The reaction with ammonia is specific for both ochratoxin A and B. Additional confirmation was obtained by extracting the purified extract with 0.1 M aqueous sodium bicarbonate solution, acidifying with 2 M hydrochloric acid, and re-extracting with chloroform (4). This extract was concentrated and chromatographed as before. RESULTS AND DISCUSSION As the results of the screening tests in Table 1 indicate, both isolates of A. ochraceus recovered from country cured ham were able to produce ochratoxins A and B. Toxin yields were lower than those observed on shredded wheat by Schindler and Nesheim (12). Whereas P. viridicatum (ATCC 18411) produced ochratoxin A as expected, none of the isolates of P. viridicatum from cured hams formed detectable amounts of either ochratoxin A or B. This contrasts somewhat with the results of Scott et al. (13), who found only 5 nonproducers among 27 strains of P. viridicatum isolated from various grains, mixed feed, beans, and peanuts. Since the isolates of P. viridicatum from cured ham did not yield any toxin on rice, peanut meal, or corn, only A. ochraceus was grown on aged hams. Table 2 shows that again all three strains of A. ochraceus produced ochratoxins A and B. The temperature optimum for toxin production by strain H 33 was 25 to 30 C; very little toxin was recovered at 15 C and none was recovered at 5 or 37 C, at which temperatures only poor mycelial growth and almost no sporulation occurred. At 25 C, strain H 33 produced more toxin on hams containing 45% moisture than on those having 55%, the latter being above the equilibrium moisture at 75% relative humidity. These hams therefore lost 5% moisture during incubation. Aspergilli tend to show optimal growth at comparatively low water activities (8). Culturing at 90 and 100% relative humidity in ambient air resulted in poor growth and successful competition from other molds and bacteria present in hams. In aging rooms of commercial plants, 75% relative humidity prevails. In the study on mold and toxin penetration into ham (55% moisture, 5.1% NaCl), mycelial growth was observed as deep as 1 cm along the binding tissue where the meat often split open during the incubation period (21 days at 25 C and 75% relative humidity). In the lean muscle, growth occurred to a depth of approximately 0.5 cm. This figure was not very consistent, since contamination from other locations on the hams was difficult to prevent when the ham slabs were prepared for incubation. One-third (7 Mg) OCHRATOXIN IN COUNTRY CURED HAMS of the total ochratoxin A produced was found in the mycelial mat on the surface of the ham, two-thirds (14 Mg) was found in the upper 0.5-cm meat layer (260 Mg of ochratoxin A per kg of meat), only traces were found in the second 0.5 cm, and none was found in the deeper layers. Ochratoxin was detected only in the layer in which mycelial growth was also observed. Ochratoxin apparently penetrates into the meat along with the mycelial growth, whereas physical diffusion of the toxin is limited, at least during the 3-week period of this experiment. This is different from cultures of A. ochraceus in liquid media, where a large proportion of toxin is always found in the culture filtrate. In studies of aflatoxin in bread, Frank (5) recovered the toxin only in zones where mycelial growth occurred. There is evidence that A. ochraceus is able to produce more than negligible amnunts of ochratoxins A and B on country cured hams under conditions which are often encountered in commercial curing plants. Mycelial growth is not restricted to the ham surface, and toxin can penetrate as far as 0.5 cm into the muscle of the meat. The three strains of A. ochraceus used in this study also produced penicillic acid on cereals. Penicillic acid does not seem to be a problem on meat because it reacts easily with amino acids to form much less toxic or nontoxic compounds (2). None of the strains of P. viridicatum isolated from hams produced measurable amounts of ochratoxin A or B. However, besides also producing penicillic acid (2), P. viridicatum is a source of the mycotoxin citrinin (13). Its occurrence on aged hams and its importance are still to be determined.

v3-fos

2020-12-10T09:04:12.397Z

1973-06-01T00:00:00.000Z

237229462

s2

Identification of the Volatile Compounds Produced in Sterile Fish Muscle (Sebastes melanops) by Pseudomonas fragi Volatile compounds produced by Pseudomonas fragi strain 18 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. The fruity odor that developed in fish muscle during incipient spoilage was attributed to a synergistic flavor interaction involving the ethyl esters of acetate, butyrate, and hexanoate. Castell and Greenough (3) described a fruity or ester-like odor that commonly developed in chilled fish muscle during the early stages of spoilage. This distinctive odor, encountered more often on commercially prepared fillets rather than round or eviscerated fish, was reproduced in sterile fish tissue and fish media by bacterial cultures isolated from fish. The causative bacterial species was identified as Pseudomonas fragi, a psychrophilic organism which utilizes a variety of amino acids for odor production (4,5). This study was initiated to identify the volatile compounds produced in sterile fish muscle (Sebastes melanops) by P. fragi. Particular emphasis was placed on the identification of the compounds responsible for the characteristic fruity odor. MATERIALS AND METHODS Sterile muscle tissue. Sterile fish muscle was obtained from black rockfish (S. melanops) by a modified method of Lobben and Lee (10) plates for 48 h at 25 C, were collected and suspended in sterile, distilled water. Sterile muscle tissue (pH 6.4-6.7) was homogenized, inoculated, and dispensed in screw-capped vials as reported previously (13). An additional muscle homogenate, adjusted to pH 7.3 with sterile NaOH, was treated as described above. Duplicate samples (pH 6.4-6.7 and pH 7.3), supplemented with 0.2% ethyl alcohol, were also prepared. Sterile homogenized milk and milk fortified with 0.2% ethyl alcohol were prepared by autoclaving at 121 C for 10 min. All sample vials and appropriate controls were incubated at 5, 15, and 25 C and checked periodically for odor production and microbial counts. When the fruity odor was pronounced, the contents of selected vials were analyzed by combined gas-liquid chromatography and mass spectrometry. Gas-liquid chromatography and mass spectral analyses. The gas chromatographs, mass spectrometer, chromatographic columns, and methods of sample preparation and analysis used for the separation and identification of aliphatic methylamines and other low-boiling compounds were reported previously (11)(12)(13). RESULTS AND DISCUSSION An ester-like or fruity odor was produced by P. fragi strain 18 in sterile fish muscle (S. melanops) incubated at 5, 15, and 25 C. The characteristic odor developed more rapidly at the higher temperatures and was gradually superceded by a distinct sulfide odor. Since strain 18 did not reduce trimethylamine oxide to trimethylamine, no typical amine odor was apparent. 952 Preliminary gas chromatographic analyses of the volatiles produced by strain 18 in sterile muscle tissue at pH 6.4 to 6.7 and pH 7.3 indicated limited concentrations of several components that were suggestive of ethyl esters on the basis of past experience. In an attempt to enhance ester production, the sterile fish homogenates were supplemented with 0.2% ethyl alcohol, and the volatile compounds produced by strain 18 were identified by gas-liquid chromatography and mass spectrometry after 4, 8, and 12 days incubation at 15 C. Compounds positively identified and listed in Table 1 included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. Limited amounts of acetone were detected in the distilled water used for all analyses, and methylene chloride was considered a persistent contaminant in the atmosphere in which the samples were prepared and analyzed. The microbial count in fish homogenates (pH 6.4-6.7), supplemented with 0.2% ethyl alcohol, increased from 4.0 x 106 cells/g at 0 days to 1.1 x 1010 cells/g at 8 days. The olfactory evaluation of each component, eluting from the column, was facilitated by a splitter which was attached to the effluent end of the column (13). The compound that eluted with a retention time almost identical to that of ethyl butyrate had a strong, fruity odor but, because of the limited concentration, the mass spectrum was weak. Although the parent ion for ethyl butyrate, m/e 116, was not discernible, the relative intensities observed for mle 29, 60, 71, and 88 were strongly suggestive of an ethyl ester. Ethyl hexanoate was tentatively identified by relative retention time. A retention time of 99.7 cm for the authentic compound compared reasonably well with the retention time of 98.6 cm recorded for the peak in question. The fruity aroma produced by strain 18 in fish during the early stages of spoilage or incubation was attributed to a synergistic flavor interaction involving ethyl acetate, ethyl butyrate, and ethyl hexanoate (17). The strong sulfide odor that persisted during continued incubation was the result of marked increases in methyl mercaptan (tentative identification), dimethyl sulfide, and dimethyl disulfide. Castell et al. (4) reported that lipolytic and nonlipolytic strains of P. fragi isolated from fish produced fruity odors primarily from monoamino monocarboxylic acids. It was suggested that the fatty acids were produced by a number of different reactions involving deamination, methyl mercaptan and dimethyl disulfide could be formed as reported previously (13). Repeated attempts to produce fruity odors in sterile fish muscle inoculated with the strain of P. fragi obtained from the Department of Microbiology at this institution were unsuccessful. Apparently, this strain lost its ability to produce esters. Strong, fruity odors are characteristic of new isolates of P. fragi, but the ability to form esters is easily lost by continued subculturing of the organism under laboratory conditions. However, in some cases, ester production can be restored by growing the organism on a medium containing the necessary or suitable substrates. Since authentic strains of P. fragi characteristically produce a fruity aroma in many dairy products (7,14,17), P. fragi strain 18 was also cultured in sterile, homogenized milk supplemented with 0.2% ethyl alcohol. A typical flame-ionization detector chromatogram of the volatiles produced by strain 18 in milk incubated at 15 C for 4 days is illustrated in Fig. 1. Compounds identified are listed as follows with respective peak numbers: (1) methyl mercaptan (tentative identification), (2) dimethyl sulfide, (3) acetaldehyde, (4) ethyl acetate and acetone, (5) ethyl alcohol, (6) not identified, (7) ethyl butyrate, (8) dimethyl disulfide, (9) not identified, (10) ethyl hexanoate, and (11) heptanone. The small peak immediately after peak 6 had a retention time identical to that recorded for 2-butanone. Bills and Day (2) reported that acetaldehyde, dimethyl sulfide, butanone, and acetone are usually present in milk. Although the concentrations of the latter three compounds remained relatively constant throughout the 4-day incubation period, acetaldehyde increased between 12 to 24 h and then decreased substantially with continued incubation. An increase and subsequent decrease in acetaldehyde content during the early phases of incubation were also observed in fish homogenates inoculated with strain 18. Keenan et al. (9) previously observed a very active reduction of acetaldehyde to ethyl alcohol by several pseudomonads, including P. fragi. Marked increases in the concentrations of ethyl acetate, ethyl butyrate, and ethyl hexanoate were noted between 1 and 4 days of incubation at 15 C, and the ratio of peak areas of the esters at 4 days was approximately 4:2: 1, respectively (Fig. 1). The microbial count in homogenized milk increased from 4.9 x 105 cells/g at 0 days to 2.4 x 108 cells/g at 5 days, and a reasonable correlation with the production of esters was observed. In addition, there was no evidence of bacterial growth or ester formation in the uninoculated controls. The fruity aroma produced by P. fragi strain 18 in milk was due primarily to the production of ethyl butyrate and ethyl hexanoate. These results correlated well with data previously reported for recognized strains of P. fragi cultured in milk (16) and, therefore, further substantiated the reclassification of Pseudomonas type III no. 18 to a type II pseudomonad, P. fragi. Although P. fragi strain 18 produced a fruity aroma in homogenized milk and sterile fish muscle, quantitative differences in the resultant ethyl esters were quite apparent. Appreciable amounts of ethyl acetate, ethyl butyrate, and ethyl hexanoate were produced in homogenized milk (Fig. 1). In contrast, ethyl acetate was the major ester produced in sterile fish muscle, and only limited concentrations of ethyl butyrate and ethyl hexanoate were detected. Therefore, it is quite apparent that ester production can be influenced considerably by the medium or available substrates (15). The data presented above indicate that P. fragi, the cause of the fruity defect in dairy products, apparently plays a similar role in the spoilage of chilled fish muscle. Although sterile fish muscle homogenates were used in this investigation, the fruity and sulfide odors produced by P. fragi have been associated with naturally spoiling fish (6) and were also reproduced in sterile muscle blocks (6) as well as on heat-sterilized muscle and autoclaved fish media (3).

v3-fos

2020-12-10T09:02:26.751Z

1973-10-01T00:00:00.000Z

237234902

s2

Microbiology of Lebanon Bologna Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 104/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also. Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 104/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also. Lebanon bologna is a semidry, fermented, all-beef sausage which is smoked but not cooked. The sausage originated among the Pennsylvania Germans in the Lebanon, Pa., region. There is a paucity of literature concerning Lebanon bologna technology and microbiology; however, federal specifications for "Lebanon" style bologna have been published (3) and a nontechnical discussion of the processing of Seltzer brand Lebanon bologna has appeared (1). A few spice formulations for Lebanon bologna are known (8,9,13). Various aspects of the manufacture of Lebanon bologna have been presented elsewhere (Palumbo et al., manuscript in preparation). This paper is concerned with the microbiology of Lebanon bologna technology. MATERIALS AND METHODS Viable counts on meat or Lebanon bolognas were performed as follows: 50 g of beef cubes or 50 g of material from the center of a bologna were removed aseptically and ground at high speed in a Waring blendor with 200 ml of peptone water, and appropriate dilutions were then surface plated in triplicate. The types of media employed, temperatures, and times of incubation were: (i) total aerobic count on APT agar, 3 days at 25 C; (ii) micrococci on phenol red mannitol salt agar (MSA), 3 days at 32 C; (iii) lactic acid bacteria on acidified Rogosa SL agar (RSL), 3 days at 25 C; (iv) yeast on acidified potato dextrose agar (PDA), 3 days at 25 C; and (v) gramnegative bacteria on eosin methylene blue agar (EMB), 1 day at 37 C. All media were obtained from Difco Laboratories, Detroit, Michigan. Dilutions were made in 0.1% peptone (Difco) water. Gram stains of all colony types found on the various media were examined; in addition, the catalase test was performed on isolated colonies. RESULTS Viable counts were determined on 14 commercial brands of sausages, including 10 Lebanon bologna types. The viable counts are presented in Table 1 and types of microorganisms are presented in Table 2. The fermented sausages had catalase-negative, gram-positive rods (characteristic of lactobacilli) on APT and RSL except for the products produced by companies D and H, which contained catalase-negative, gram-positive cocci. Company D adds a pediococcus starter culture to their Lebanon bologna, and the predominant flora of thuringer has been shown to be pediococci (2). A number of the sausages contained lactic acid bacteria that produced gum on RSL plates. Lactics were not found in the sweet bologna produced by company C, and this is reflected in the high pH t89 a The pH was determined by inserting the electrode into the mass of meat or into the center of a bologna. b A "sweet" Lebanon is similar to the regular type except that more sugar is present. The pH is low but the sweetness masks the acid "tang." c The sweet bologna produced by company C was not a Lebanon variety nor was it a fermented product. of 5.6. Typical coliforms were not observed in any of the sausages. Micrococci were present in company A's sweet Lebanon and in both types of Lebanon bologna produced by company G as well as in the cervelat and dry Italian salami; micrococci were not found in the other sausages. The individual steps in Lebanon bologna manufacture were investigated to determine the microbial flora involved and the factors responsible for the development of the flora that brings about the desired acid formation and nitrate reduction. The data in Fig. 1 represent the course, with time, of the viable count in aging salted beef. The viable counts on all five types of media used show a gradual increase in numbers during the aging period. The colony types found on APT consisted of catalase-positive, gram-positive, and gram-negative rods; catalase-positive, gram-positive cocci were found on MSA. The organisms on EMB were catalase-positive, gram-negative rods, but the colonies were not typical coliforms in appearance. Yeasts were present on PDA and catalase-negative, grampositive rods characteristic of lactobacilli were found on RSL. The viable counts for the bolognas made from meat aged for varying periods of time are given in Table 3. The only bologna that had a low pH (4.5) was made from meat aged 14 days, at which time the lactic count on RSL was approximately 104/g ( Fig. 1). In general, if the lactic acid bacteria count of the salted beef was not in the 104/g range, then a low pH was not achieved in the bolognas. Both APT and RSL plates show catalase-negative, gram-positive rods, with a large number of gum producers on RSL; yeasts tend to disappear in the bolognas (Table 3). Typical coliforms were not observed; MSA contained catalase-positive, gram-positive cocci in all of the bolognas except the one in which the pH was 4.5; at the low pH, the cocci were replaced by catalase-positive, gram-negative rods. In aging beef for Lebanon bologna manufacture, the level of NaCl used is critical. As the salt concentration is increased, the total aerobic count (APT), lactic acid bacteria count (RSL), and the gram-negative bacterial count (EMB) decreased (Fig. 2). The counts on MSA and PDA were less affected by increasing salt concentrations. In the absence of NaCl, catalase-positive, gram-negative short rods were the predominant flora on APT, and at 10 days the meat had the strong fruity odor that is associ- Yeast was present on PDA only with company A regular Lebanon, G sweet Lebanon, and I dry Italian salami. ' There was no growth on RSL at 102 dilution. ated with pseudomonads. A few catalase-positive, gram-positive rods were found at 1% salt, and at 2% there were about equal numbers of catalase-positive, gram-negative and catalasepositive, gram-positive organisms (APT). At 3 and 4% NaCl, very few gram-negative orga- Influence of time on the viable count of salted beef aging at 5 C. Beef chuck was ground through a 3/4-inch plate, and NaCI was added to the ground meat to make the concentration of salt to 3%o. One kilogram of beef cubes was placed into individual plastic bags, and the meat was allowed to age at 5 C. At intervals, a bag was removed from the cooler; 50 g was used to determine the viable count, and the remainder was used to make bolognas. The starting pH of the meat was 5.8 and did not change during the aging period. a Spices, sugar, and KNO3 were added to the salted beef cubes and the mixture was ground through a %4-inch plate. The mixture was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The sausages were then mellowed at 5 C for 4 days. At the end of the mellowing period, a 50-g sample was removed aseptically from the bologna, and bacterial determinations were made. The viable counts for the meat are given in Fig. 1. b Number of days meat was aged at 5 C with 3% NaCl before making bolognas. Effect of varying salt concentrations on the viable count of beef aged for 10 days. Beef chuck was ground through a 3/4-inch plate, and 0, 1, 2, 3, or 4% NaCI was added. Each lot of beef was placed into individual plastic bags, and the meat was allowed to age at 5 C for 10 days. At the end of the aging period, 50 g was used to determine the viable count, and the remainder was used to make bolognas. The starting pH of the meat was 5.6 and did not change during the aging period. nisms were present on APT. The microorganisms found on RSL were catalase-negative, gram-positive rods; catalase-positive, grampositive cocci were present on MSA, and yeasts were found on PDA. In Table 4, the viable counts of bolognas prepared from meat aged with varying concentrations of NaCl are presented. The counts on APT and RSL were similar, and the organisms found on both media were catalase-negative, gram-positive rods. Micrococci were not found in the bolognas; the organisms found on MSA were catalase-positive, gram-negative and gram-positive rods. Bolognas prepared from meat aged with low salt had a suitably low pH but they were defective in odor and taste. The bolognas made from meat aged with 4% salt did not reach a low pH because the numbers of the lactic acid bacteria were low (Fig. 2). Under normal conditions of the manufacture of Lebanon bologna, the fermentation occurs PALUMBO APPL. MICROBIOL. during the smoking process; however, a satisfactory bologna with low pH and good color can be obtained by incubation of the stuffed sausages in a constant temperature-constant humidity cabinet (35 C and 85% relative humidity [RH]). Some studies were done by utilizing the incubator rather than the smokehouse because of the restrictions associated with the use of the smokehouse. Although bolognas of similar pH, color, and texture were obtained by incubating in the smokehouse or incubator, the sequence of the microbial flora observed differed between the smoked and incubated bolognas. The effect of the presence or absence of smoke on the viable count is illustrated in Fig. 3. The counts on APT, RSL, and MSA decreased markedly during smoking at 35 C and during the subsequent mellowing period at 5 C (Fig. 3A). Both APT and RSL agars contained catalase-negative, gram-positive rods. MSA had colonies that consisted of catalase-positive, gram-positive cocci at the beginning of the fermentation, but by the second day of fermentation the number of micrococci decreased. The cocci were replaced on MSA by catalase-positive, gram-positive and gram-negative rods; by 10 days of mellowing, no micrococci could be detected (< 1 x 102/g). With nonsmoked bolognas (Fig. 3B), the MSA count decreased rapidly during the fermentation period, but the APT and RSL counts were not as severely reduced as under smoked conditions. APT and RSL contained catalase- a Spices, sugar, KNO., and salt (to make the concentration to 3% except for the 4%) were added to the beef cubes, and the mixture was ground through a 5/4-inch plate. The material was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The sausages were mellowed at 5 C for 1 day and then a 50-g sample from each bologna was removed aseptically for bacterial determinations. The viable count for the meat is shown in Fig. 2 Beef chuck was ground through a 3/4-inch plate, and NaCI was added to make the concentration of salt to 3%. The salted meat was packed into a wooden barrel and allowed to age at 5 C for 10 days. Spices, sugar, and KNO, were added to the meat, and the mixture was negative, gram-positive rods; MSA showed a rapid die-off of micrococci which were replaced by catalase-positive, gram-negative and grampositive rods (the microbial pattern on MSA was quite similar in both the smoked and nonsmoked bolognas). Experiments were performed to determine if Lebanon bologna could be prepared from frozen beef. Viable counts on thawed meat and the bolognas prepared from thawed meat are presented in Table 5. Thawed meat, before the addition of salt, contained catalase-positive, gram-negative rods on APT, yeast on PDA, and catalase-positive, gram-positive cocci on MSA. After 10 days of aging in the presence of salt, the viable count and the types of organisms found in the thawed, salted, aged meat appeared to be similar to that of unfrozen, aged, salted meat: 10 20 30 40 DAYS ground through a 524-inch plate. The material was stuffed into fibrous casings, and the sausages were fermented at 35 C at approximately 90% RH for four days either in an incubator or smoke house. At the end of the fermentation period, the bolognas were mellowed at 5 C. At intervals during the processing, 50-g samples of meat or bolognas were removed for determination of bacterial numbers and pH. catalase-positive, gram-positive rods were found on APT, yeast was found on PDA, catalase-negative, gram-positive rods were found on RSL, and catalase-positive, gram-positive cocci were found on MSA. EMB plates had catalasepositive, gram-negative rods which were not typical coliforms. The bolognas made from the thawed beef had a low pH of 4.6 with good color and texture. The organisms found on APT and RSL were catalase-negative, gram-positive rods; catalase-positive, gram-positive and gram-negative rods were found on EMB; MSA had catalase-positive, gram-positive rods and cocci; and no yeast was present at 102 dilution on PDA. Unaged beef or beef aged at 5 C (in the presence of 3% NaCl) for short periods of time did not give Lebanon bologna with a normal low pH of 4.7 to 4.5 when the fermentation period was 3 to 4 days ( Table 3). The fermentation period was lengthened to determine whether bolognas made from fresh meat could attain the desired low pH. The data presented in Table 6 indicate that bolognas made from fresh meat did not reach a normal product pH after 12 days of incubation at 35 C and 80% RH. The microbiology also was different in some respects from the properly fermented product. At days 0, 1, and 2, catalase-positive, gram-positive cocci were the predominant organism on all plates that showed growth except for RSL, which had gum-producing catalase-negative, gram-positive rods. From day 3 on, APT and RSL had catalase-negative, gram-positive rods (gum producing on RSL); catalase-positive, gram-positive cocci were present on EMB and MSA. A portion of the same beef chuck that was used for the above experiment was ground through a 3/4-inch plate, salted (final concentration of NaCl was 3%), and then aged at 5 C for 12 days. The aged meat was made into bolognas in the usual way and incubated at 35 C and 80% RH. The pH of the meat going into the fermentation was 5.3; after 1 day, the pH was 5.3, at 2 days, the pH was 4.7, and at 3 days, the pH was 4.6. Thus, Lebanon bologna could be made from aged beef but not from fresh beef. However, if Lactacel MC (Merck & Co., Rahway, N.J.) starter culture were used with fresh beef and bolognas prepared in the usual manner, a fermented sausage with a pH of approximately 4.5 was produced within 24 h. DISCUSSION Semidry and dry sausages are meat products that have been fermented by lactic acid bacteria (2, 10). Our work (Table 1) and the work of others (2; L. B. Jensen and L. S. Paddock, U.S. Patent 2,225,783,1921) show that lactic acid bacteria are isolated in large numbers from Lebanon bologna. In the manufacture of Lebanon bologna, cubed beef plus salt is allowed to age at refrigerated temperatures for several days. Pseudomonads and lactic acid bacteria have been shown to be the predominant flora of ground beef during refrigerated storage in the absence of salt (4). At low concentrations, NaCl prevents the growth of pseudomonads (12) but permits the growth of the more salt-tolerant lactic acid bacteria (11). The role of salt as an inhibitor of bacterial growth has been reviewed by Ingram and Kitchell (6). Therefore, the primary reason for salting the cubed beef during the aging Fresh beef chuck was ground through a 3/4-inch plate, NaCl, spices, sugar, KNO, were added, and the mixture was ground through a %4-inch plate. The sausage mix was stuffed into casings 55 mm in diameter, and the bolognas were incubated at 35 C and 80% RH. At each time interval, a bologna was removed and bacterial numbers were determined by utilizing a 50-g sample. " No growth occurred at 10' dilution on PDA. c At 12 days, the pH of the bolognas was still 5.6. a Beef chuck was ground through a 3/4-inch plate, placed in plastic bags, and frozen for approximately 4 months. The meat was thawed for approximately 24 h at 5 C and a 50 g sample was removed to determine bacterial numbers. NaCl was added to make the concentration to 3% salt and a kilogram of salted meat was placed into individual plastic bags and was allowed to age at 5 C. b Viable counts were determined after 10 days of aging at 5 C. c Spices, sugar, and KNO, were added to the salted beef, and the mixture was ground through a W-inch plate. The mixture was stuffed into casings 55 mm in diameter, and the sausages were incubated at 35 C and 80% RH for 3 days. The bolognas were mellowed for 4 days at 5 C; bacterial numbers were determined on a 50-g sample. period in the manufacture of Lebanon bologna is to prevent the growth of the undesirable pseudomonads. During the aging or holding period used in these studies, microbial populations changed considerably. Deibel et al. (2), in the manufacture of summer sausage, found little or no change in the microbial flora during a short, 2to 4-day aging period. We found that such a short holding period was not sufficient to permit development of the lactic acid-producing flora. During the long aging period, at least 10 days in our studies, the lactic acid bacteria increased to about 104/g, and this concentration appears to be critical for adequate decrease in pH in the bolognas ( Fig. 1; Table 3). Therefore, a long aging period is necessary to allow the development of sufficient numbers of lactic acid bacteria. Another important function of aging is to allow the micrococci to develop. The micrococci reduce nitrate to nitrite and thus give bolognas a good cured meat color (F. W. Kurk, U.S. Patent 1,380,068, 1921; reference 10). The concentration of micrococci does not seem to be as critical as the concentration of lactic acid bacteria; good color was generally found in all bolognas regardless of the micrococcal count. Micrococci are more halotolerant than lactic acid bacteria and pseudomonads (12). With too much salt, the increase in the lactic acid bacterial population was very slow and the numbers at the end of the aging period were not high enough to produce bolognas of desirable low pH (data for 4% salt in Fig. 2 and Table 4). When meat was aged with low concentrations of salt (1-2%), pH decreased satisfactorily during processing but the excessive development of pseudomonads in the meat gave the bologna inferior flavor. Salt at the 3% level appears to be a good compromise between the concentration that inhibits the pseudomonads and one that is not too inhibitory to the lactic acid bacteria. Our results indicated that smoking is inhibitory to the lactic acid bacteria because the viable count of the lactic flora decreased drastically on both APT and RSL (compare A and B, Fig. 3). Apparently some component of smoke and not acid caused the killing because the pH decrease was similar under smoked and nonsmoked conditions. Handford and Gibbs (5), using liquid smoke, found that certain lactic acid bacteria and micrococci were inhibited by smoke constituents. The MSA count rapidly decreased during fermentation and mellowing under both smoke and nonsmoke conditions (compare A and B, Fig. 3). The loss of micrococci probably was due to the acid content of the sausage rather than to smoke. Also, the organisms might be sensitive to the nitrite formed by their reduction of nitrate. By day 20, few if any micrococci were found (Fig. 3); they apparently were superseded by the outgrowth of rod forms. The counts on PDA and EMB also decreased during fermentation and mellowing regardless of smoking or nonsmoking. The yeast and gram-negative bacteria are probably sensitive to the acid environment of the sausages. Sausage makers generally believe that the use of frozen meat does not produce a high-quality product (7). However, our experiments indicate that frozen beef chuck, when thawed, salted, and aged, yields a satisfactory Lebanon bologna. Prolonged incubation under fermentation conditions did not give a bologna of low pH when fresh rather than aged meat was used (Table 6). However, a portion of the same batch of meat that had been salted and aged at 5 C for 12 days did yield bolognas that reached a low pH in 2 to 3 days. One possible explanation for the failure of the fresh meat bolognas to ferment is that the lactic acid bacteria count never reached a critical level to initiate sufficient acid production. Aging at low temperatures may allow the selection and growth of salt-tolerant organisms that are active acid producers. In a typical Lebanon bologna process, the micrococci die off quickly once the fermentation leads to acid conditions, but in the bolognas made from fresh beef, the micrococcal count remained quite high (Table 6). When the number of lactic acid bacteria going into the fermentation is low, the continued high density of micrococci might interfere with lactobacilli proliferation by competing for nutrients. Apparently, whatever the reasons, Lebanon bologna of consistently good quality cannot be made from unaged meat. However, fresh meat can be used for Lebanon bologna manufacture if a suitable starter culture is used.

v3-fos

2018-04-03T04:14:46.136Z

1973-01-01T00:00:00.000Z

38242306

s2

A rapid, presumptive procedure for the detection of Salmonella in foods and food ingredients. A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory. with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory. For some time the food industry has needed a rapid, simple, economic, presumptive Salmonella detection procedure that could be performed in reduced elapsed test time on large numbers of samples to eliminate negative samples early in the course of examination with minimal fear of false negatives. In addition, such a test should be incorporated easily into the standard Salmonella detection procedures (Association of Official Analytical Chemists [APHA]) currently used in food plants without the use of special equipment, antisera, or specially trained personnel. Culture methods currently used in qualitycontrol laboratories require a minimum of 3 to 4 days for presumptive results. Further biochemical testing requires another 2 to 3 days before the presumptive positive for Salmonella can be confirmed as positive. The enrichment serology (15,16) and fluorescent-antibody (FA) procedures, although more rapid than conventional techniques, require special equipment and antisera. A person having considerable experience with these techniques is usually needed to perform these tests. A simpler culture method for the rapid detection of Salmonella was described by Hargrove et al. (6). This procedure was developed primarily for detecting Salmonella in dairy products in a single enrichment step and was tested extensively with known Salmonella standards. The purpose of this study was to evaluate this procedure and to modify the technique for routine testing of a variety of foods, food ingredients, and feed materials for the presence of viable Salmonella. MATERIALS AND METHODS Cultures. Salmonella cultures (S. Montevideo, S. senftenberg, S. senftenberg 775W, and S. typhimurium) were used in this study. Representative strains of other Enterobacteriaceae (Enterobacter, Escherichia, Shigella, Citrobacter, and Proteus) were used also to determine the specificity of the method. HOBEN, ASHTOI Medium. The medium, lysine-iron-cystine-neutral red broth (LICNR), of Hargrove et al. (6) was modified as follows: (i) mannitol was substituted for lactose, (ii) a combination of L-lysine mono-and dihydrochlorides (8:2) was used instead of the monohydrochloride, and (iii) novobiocin was used at two concentrations, either zero added or at concentrations of 10 to 15 ug/ml of broth. The modified LICNR broth had the following composition: 10 g of L-lysine mono-and dihydrochlorides (8:2), 5 g of tryptone, 3 g of yeast extract, 5 g of mannitol, 1 g of glucose, 1 g of salicin, 0.5 g of ferric ammonium citrate, 0.1 g of sodium thiosulfate, 0.1 g of L-cystine, 0.025 g of neutral red, and 1 liter of distilled water. The medium was adjusted to pH 6.2 (1 N NaOH), dispensed in 10-ml quantities in metal-capped tubes, and autoclaved at 121 C for 15 min. The ability of this modified medium to differentiate Salmonella from non-Salmonella was determined with pure cultures, and its ability to detect Salmonella in various food materials was further confirmed. The basis of this test is a pH response to an indicator dye and formation of a black precipitate when hydrogen sulfide-producing salmonellae are present. All cultures which showed a color change from red to yellow in the LICNR broth after incubation at 37 C for 24 hr were further tested to eliminate the possibility that non-hydrogen sulfide-forming salmonellae were present. This test was accomplished by incubating for another 16 to 24 hr and then adding 0.1 ml of a 0.3% bromothymol blue solution to each tube and recording the color change. When salmonellae were present, the medium changed from yellow to dark green or blue, indicating an alkaline reaction. Color differences were obvious immediately. False results were observed to occur when the modified LICNR broth was incubated beyond 36 hr prior to testing with bromothymol blue. The bromothymol blue solution was prepared by mixing 0.3 g of bromothymol blue powder with 2 ml of 0.1 NaOH and diluting to 100 ml with 50% ethanol in distilled water. Salmonella antiserum. The antiserum used in this study for the direct FA technique was a commercially prepared polyvalent antiserum (Difco "spoly") which had been prepared from motile organisms representative of somatic groups A through S including 0 factors 25, 27, 28, 30, 34 through 41, 45, and Vi. The flagellar spectrum included H antigens a through i, n, p, r through u, and w through z. The antiserum was rehydrated by the addition of 5 ml of distilled water and diluted 1: 2 to obtain the proper staining titer. Microscopic examination. A Reichert "Zetopan" immunofluorescence research microscope, equipped with a mercury vapor light source, BG12 ultraviolet filter, and a X97 oil-immersion objective with iris diaphragm, was used. Test samples. Where possible, samples naturally contaminated with Salmonella were used in this study. In the case of cheddar cheese and powdered milk, Salmonella at 2 to 5 g was added because no naturally contaminated samples were available. Known culture assays. Viable cultures were inoculated directly into the presumptive broth and AND PETERSON APPL. MICROBIOL. incubated up to a maximum of 48 hr. Broths were observed for characteristic color changes and blackening of the medium during incubation. Foods and food ingredient assays. In the first study samples were enriched using 50 g of food material and 450 ml of tetrathionate broth. Enrichments were incubated at 41 C (40.5 C) for 20 hr. Following primary enrichment, 1 ml of each enrichment culture was placed into tubes containing 10 ml of the modified presumptive LICNR broth. A similar amount of primary enrichment culture was placed into tubes containing an equal amount of tetrathionate broth (secondary enrichment). Both secondary enrichments (presumptive LICNR and tetrathionate broths) were incubated at 37 C for 24 hr. Cultural testing was performed by streaking these secondary enrichments on Hektoen agar (10) plates. The Hektoen agar plates were incubated at 37 C for 18 to 20 hr. Presumptive colonies (blue-green to blue with or without black centers) picked from Hektoen agar were tested biochemically by placing onto slants of dulcitol lysine iron agar (DLIA) described by Taylor (17), and onto lysine iron agar (LIA) as described by Edwards and Fife (3). Slant and butt reactions were observed after incubation at 37 C for 24 hr. Typical Salmonella reactions on these agars are for DLIA: alkaline (red) slant, hydrogen sulfide blackening, and fractured acid (yellow) butt; and for LIA: alkaline slant (red) and alkaline butt with hydrogen sulfide blackening. DLIA slants which gave reactions characteristic of Salmonella were serologically tested with Difco polyvalent 0 and H Salmonella antisera (2). A small amount of each slant growth was mixed with a drop of the rehydrated antisera on a glass slide. Samples were considered positive if a tight granular precipitate was observed in 30 sec. FA results were obtained from smears made from presumptive broths after characteristic blackening of the medium occurred. Agar-coated slides were used to help retain the bacterial cells during staining and washing. Smears were fixed in an ethanolchloroform-formaldehyde solution (6:3: 1) for 3 min. Staining was accomplished (direct technique) using Difco fluorescein conjugated "poly" Salmonella antisera. Smears were stained for 30 min followed by rinsing twice in a phosphate-buffered solution (Difco), pH 7.2. After the excess antisera had been washed free, the slides were rinsed in distilled water and allowed to air dry. Dried smears were mounted using buffered gylcerol, pH 8.6, and examined for fluorescence. A smear was considered positive for Salmonella if bright, typically rod-shaped bacteria exhibiting +3 to +4 fluorescence (5) with or without attached flagella were present (4,18). In the second study, samples were pre-enriched using 25 g of food material and 225 ml of lactose broth. Samples were blended for 2 min at high speed using an Oster blender. The pre-enrichment culture was incubated at 35 C for 20 to 24 hr. Following pre-enrichment, 1 ml of each enrichment culture was placed into tubes containing 10 ml of tetrathionate broth and incubated at 35 C for 20 to 24 hr. After secondary enrichment in tetrathionate broth, 1 ml of each enrichment was placed in tubes containing 10 ml of the LICNR broth. Novobiocin was used during 15 Ag/ml in the tetrathionate broth enrichment. Samples were tested culturally by streaking the tetrathionate broth enrichments without novobiocin added onto Hektoen agar plates and picking typical colonies. These were then identified biochemically and serologically as previously described. RESULTS AND DISCUSSION Cultures of various known Salmonella and known type species of non-Salmonella were used in this study to evaluate the modified LICNR broth and to verify the results obtained by Hargrove et al. (6). Freshly grown cultures were inoculated into the modified LICNR presumptive broth. Color reactions were observed at 2-hr intervals. Salmonella were readily differentiated from related enteric bacteria except for Arizona. Results obtained (Table 1) were similar to those observed by Hargrove et al. Most known Salmonella produced a massive black precipitate. Arizona was observed to produce identical reactions to Salmonella. A hydrogen sulfide-negative Salmonella strain (S. seftenberg 775W) turned the presumptive broth yellow without blackening of the medium but was detected by the use of the bromothymol blue indicator which formed a green ring at the top of the tube. Pure cultures of Shigella, Enterobacter, Proteus, and Citrobacter did not change the red color of the LICNR broth. Species of Escherichia changed the LICNR broth from red to yellow without blackening. Escherichia could be distinguished from hydrogen sulfide-negative Salmonella by the fact that no green or blue color was produced upon addition of the bromothymol blue indicator. Food samples which turned the presumptive broth yellow and subsequently were tested for the presence of hydrogen sulfidenegative Salmonella did not give a clear-cut color result. In cases during incubation, competing non-Salmonella were able to raise the pH of the LICNR broth enough to cause questionable color results. This problem became almost non-existent in the later study when novobiocin was used. It should be also pointed out that hydrogen sulfide-negative Salmonella constitute less than 0.5% of all known salmonellae (6,11). These types have not normally been encountered in foods and food ingredients in this laboratory. An attempt was made to utilize the procedure as described by Hargrove et al. (6) for a rapid Salmonella detection procedure for foods, food ingredients, and feed materials. However, some food materials were observed to mask and interfere with the color reactions of the presumptive broth. To overcome that problem, the presumptive LICNR broth was used as a secondary enrichment medium and presumptive indicator. All materials were enriched in tetrathionate broth at 41 C because with feed samples this temperature appeared to be optimal for Salmonella detection and isolation. Other workers (1,7,12) Presumptive results were obtained within 32 to 38 hr. Samples that were found to contain Salmonella by conventional cultural procedures produced a massive black precipitate in the presumptive LICNR broth. Even when high levels of coliforms and Proteus species were present, the presence of Salmonella was readily indicated by loss of color and blackening of the medium. In 20% of the samples tested, some difficulty was encountered in detecting Salmonella by conventional cultural procedures. Eighteen different types of samples were evaluated for the presence of viable Salmonella. These samples included chicken parts, feed materials, powdered milk, raw frozen meats, pure culture slants, and environmental swabs. A positive correlation between this presumptive test and cultural results was obtained. (Table 2). Ninety-four percent of the samples found to be presumptively positive by this method was confirmed by cultural methods (AOAC, USDA, BAM, APHA). Of 110 samples tested, 58% were found to be positive for Salmonella by cultural methods as opposed to 62% with LICNR broth. Of the 110 samples tested only 50 were evaluated by all three techniques in parallel: cultural, FA, and the presumptive LICNR method. Salmonellae were found in 76% of those 50 samples by the cultural method and in 82% by both the FA and presumptive LICNR broth methods. The high percentage of samples positive for Salmonella by all techniques was due to the fact that samples were obtained as presumptive positives from previous screening programs. We think the difference between cultural results and those obtained by the presumptive and FA methods may have been due to the inability of the cultural method to detect Salmonella in the presence of high numbers of non-Salmonella organisms. False-positive results have been encountered by the FA method due to the presence of non-Salmonella organisms which share common antigens with Salmonella and consequently fluoresce (8). The presumptive LICNR broth, after hydrogen sulfide development, yielded smears that exhibited extremely bright (+3 to +4 fluorescence) peripheral staining. Many smears also exhibited excellent flagellar staining. FA smears in a few instances contained low numbers of fluorescing cells, indicating that low levels of Salmonella were present in some of the final enrichments. Similar results were observed by Reamer and Hargrove (14). Tetrathionate broth was used in this study because it has been found to be the single most suitable enrichment medium for use in this laboratory. Tetrathionate broth allows the enrichment of most Salmonella while inhibiting many of the unwanted organisms present in foods and food material (13). The combination of mono-and dihydrochlo- rides of L-lysine lowered the pH of LICNR broth and resulted in the ingredient materials going into solution more easily. Therefore, these materials were substituted for the monohydrochloride originally used by Hargrove et al. (6). Further work (pure and mixed culture) showed false-positive reactions (LICNR broth) could occur. These false-positive results were caused by a combination of a coliform and Proteus species that were isolated from eviscerated chicken samples. When grown in combination these organisms produced a false color result in the LICNR broth. None of these organisms alone gave false-positive results. This problem was eliminated by the incorporation of novobiocin at 10 to 15 Ag/ml in the LICNR broth. Jeffries (9) used novobiocin for suppressing competing organisms such as Citrobacter and Proteus. This suppressive effect on competing non-Salmonella organisms was substantiated by testing naturally contaminated samples for the presence of Salmonella. Food materials and ingredients were enriched using procedures currently in use in the quality control laboratory to see if the LICNR broth technique could detect the presence of viable Salmonella. What appeared to be falsepositive results were observed when novobiocin was not used during enrichment. When novobiocin was used in the LICNR broth, identical presumptive and cultural results were obtained (Table 3). Presumptive results from samples enriched with novobiocin present in the tetrathionate (rather than in the LICNR broth) broth enrichment, however, did not correlate as closely with those obtained culturally (Table 4). These results suggest that novobiocin was more effective in eliminating false positives when it was used in the LICNR broth. No false negatives were encountered in this study. It should be noted that presumptive LICNR results were compared with cultural results and that further cultural isolation might have resulted in the detection of Salmonella in sam- ples reported as negative. The assumption inherent in this approach is that conventional cultural procedures yield correct and absolute results, and this is not necessarily true. Using this new medium (LICNR broth), it was possible to detect Salmonella in food samples within 3 days which is 1 day faster than standard Salmonella detection procedures (AOAC, USDA, BAM, APHA) permit. This procedure, using modified LICNR broth, requires no special equipment or antisera. Its use as a routine quality control screening procedure should be of value in shortening the holding time for foods and food ingredients while awaiting cultural results. Presumptive results can be further evaluated by any of several presently recognized confirmatory procedures with no delays or increase in time beyond that presently required. The number of such samples requiring the more extensive confirmatory procedures should be substantially reduced through the use of this screening procedure. The savings from this reduction in sample volume are obvious.

v3-fos

2018-04-03T04:19:45.093Z

1973-09-01T00:00:00.000Z

38616169

s2

Sand beach bacteria: enumeration and characterization. Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans. Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans. Bacteria are considered to be an important component of the sand beach community (5-7, 12, 14). The productivity of the sand beach is ultimately limited by nutrient input. From laboratory studies, it appears that nutrients (carbon) pass first through the bacterial community and then into the protozoan and metazoan community (12). To initiate studies on sand beach bacteria in Lebanon, a tideless, fully exposed marine beach was chosen: Sindbad beach, 30 km south of Beirut. The beach has no obvious signs of pollution. Small recreational swimming areas are located on the north and south quarters of its 1.5-km length. In the center part, samples were taken repeatedly, to enumerate bacteria and also to isolate several hundred individual strains to determine their biochemical, morphological, and cultural characteristics. MATERIALS AND METHODS Enumeration of bacteria in sand. The total number of bacteria per gram of sand was obtained by: (i) collecting a small sand sample in the field (kept chilled until return to the laboratory); (ii) aseptically adding 6 to 7 g of the wet sand to a pretared 18-by 250-mm sterile screw-cap tube for obtaining the wet weight of sample; (iii) adding sterile 0.1% peptone (Difco) in sea water or distilled water to the 5.0-ml mark of the tube; (iv) vortex shaking for 60 s (longer shaking did not increase the number of bacteria detected); and (v) taking samples from this tube for further dilution and plating on peptone-yeast extract medium (PYE). PYE contained: peptone (Difco), 0.5%; yeast extract, 0.05%; and agar (Difco), 1.5% made with either sea or distilled water. Other media (increasing or decreasing the peptone concentration 1Present address: Biology Department, University of Jordan, Amman, Jordan. with the exclusion of yeast extract, or replacing the yeast extract with glucose or phosphate) supported the growth of fewer bacteria. Interstitial bacteria (not attached to sand grains) were collected with an interstitial water sample (13) which had been thoroughly rinsed with the sample. For enumeration, 1, 5, or 25 ml of the interstitial water was then suctioned through HA-Millipore membranes (pore size, 0.45 Am, in Millipore field monitors). PYE broth was then aseptically added to the absorbant pad below the membrane. All incubations were done at room temperature (25 C). The number of colonies on PYE agar plates were counted after 4 days of incubation, and those on Millipore membranes were counted after 36 h of incubation, the latter with the aid of a binocular dissecting microscope. Total (direct microscope) counts were made from untreated interstitial water and sand samples that had been shaken in 0.004 N NaOH. Samples of either were suctioned through Millipore membranes, and 2% erythrosin in 5% aqueous phenol was then added to the absorbant pad to fix and stain the bacteria present on the membrane. After removal of excess stain, the bacteria retained on the filters were counted with the aid of an oil immersion lens. Sites. During 1971 the wave wash zone (WWZ) was sampled at the highest point that the waves kept the sand saturated with water. Also, the water table (WT) was sampled at the point inward from the WWZ where there was 40 cm of sand covering the water table. The WT was usually 3 to 6 m from the WWZ. In 1972, samples were taken from the WWZ, 10 and 20 m inwards on a transect perpendicular to it. Bacterial isolates. All pure cultures were obtained by repetitive streaking on PYE-sea water agar plates and were stored on PYE-sea water agar slants at 4 C. Biochemical and cultural determinations. Sugar fermentation was determined by the method of Hugh and Leifson (9); presence of deoxyribonuclease was determined by the method of Lachica et al. (11); hydrolysis of gelatin and starch was determined as suggested by Skerman (16); the Voges-Proskauer KHIYAMI AND MAKEMSON reaction was determined by the method of Barrit (1); indole production, motility test, catalase, and oxidase tests were determined by the methods of Skerman (16); and the flagella stain was done by the Leifson procedure (4). Sea water was used to prepare all media except for the Hugh-Leifson medium, which contained 3.0% NaCl, 0.3% K2HPO4, and 0.1% MgSO4 in place of sea water. The ability to grow aerobically on single carbon sources was tested by a modification of the method of Stanier et al. (17), in which sea water was used in place of distilled water. Control plates included no added carbon source besides agar and whatever soluble carbon was present in the sea water. No growth was observed on these plates. The ability to grow at various temperatures (50, 37, 25, 15, 5, and 0 C) and at various pH values (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) was checked on PYE-sea water. All test plates were inoculated by the replica plate method from normal PYE-sea water agar master plates. RESULTS AND DISCUSSION The number of bacteria per milliliter of interstitial water accounted for 22 to 46% of the total number of bacteria per gram of wet sand (Fig. 1). The determination of the total number of bacteria in the interstitial water did not involve shaking, and therefore aggregates may have been counted as one cell. Therefore, this number may be underestimated and could be twice as high (8). The numbers of viable bacteria were 10-4 that of total bacteria (Fig. 1). These results suggest that a large proportion of the bacteria active in the ecosystem are not grown in the culture medium and indicate the need for further studies of the efficacy of various nonselective media for isolation and enumeration of these bacteria. The numbers of viable bacteria per milliliter of interstitial water were lower than the numbers of viable bacteria per gram of wet sand. The latter was determined by a method involving shaking; the former did not. Thus, both the total (direct microscope count) and viable numbers of bacteria per milliliter of interstitial water were probably representative of the number of bacterial aggregates, whereas the numbers of bacteria per gram of wet sand (direct and viable counts) were probably more indicative of the number of individual cells. Microscope study by simple (18) and fluorescent staining (2) revealed no bacteria attached to the sand grains in repeated attempts. Table 1 shows that most of the viable bacteria were evenly distributed in the water-saturated zones of the WWZ and WT. The number of viable bacteria varied by one order of magnitude. It increased 10-fold during a local storm. Marine bacteria seemed to be predominant in the samples, with an exception of the sample taken at on August 19, i.e. higher numbers of bacteria were grown on the medium containing sea water. In the spring and summer of 1971, 362 randomly chosen isolates were cultured and maintained as stock cultures for characterization. Table 2 shows the distribution into six arbitrary, physiological groups based on the ability to ferment various sugars. Group 1 isolates were versatile fermenters and comprised only 17% of the isolates; in group 2, the number of isolates capable of limited fermentational versatility was also small, comprising only 23% of the isolates. Rods were the major morphological type (88% of all isolates) and comprised 96% of the major group, group 3, the oxidative group (lacking the ability to ferment). The fact that oxidative organisms are the dominant group (59%) is consistant with the selective effect on aerobic plates and the presence of oxygen in interstitial water. Makemson (unpublished data) has shown that the percent saturation of oxygen was only once as low as 27% (1.41 ml of 0/liter), at 20 m inwards from the top of the WWZ. The normal values of oxygen saturation were between 50 to 75% in the WWZ as well as in the fresh water area in the water table (10 and 20 m inwards). Although anaerobic pockets can exist in aerated soils (8), it seems that in the Sindbad beach the chance to develop such an anaerobic environment is rare, since the interstitial water is in constant flux. Rapid washing of the WWZ with sea water (15) combines with a substantial fresh water movement to the sea through the WT. The later causes a rather steep salinity gradient: 10 m inwards from the WWZ it was common to find a value of 18 to 30% salinity. Thus, it is doubtful that anaerobic pockets could develop in the sand to make free-living anaerobic (fermentative) bacteria a dominant component of the total bacteria present. However, meiofauna and other metazoans in the beach may provide anaerobic environments in their gut cavities, thereby selecting and enriching for anaerobic or fermentative bacteria. This may account for the significiant number of fermentative bacteria (41%) in our collection. Out of the eight carbon sources tested to support the aerobic growth of the isolates as sole carbon sources, lactate appeared to be the more universal carbon source, which 43% of the isolates could utilize (Table 3). Although this study did not determine what carbon sources these isolates are utilizing in situ, these data show that a large percentage of the versatile fermenters have the ability to utilize lactate. Although the number of sole carbon sources tested was not as extensive as the 146 single carbon sources studied in aerobic and fermenta- Klug and DeMoss (10), in which case the total number of isolates does not apply.°N umber of isolates in each category. Aerobic are groups 3a and 3b, and fermentative are groups la, lb, 2a, and 2b in Table 2. c ND, Not determined. These open marine isolates appear to be more versatile than those from Sindbad beach. For example, 100% of the fermentative and 65.8% of the oxidative marine bacteria used glucose as a sole carbon source, compared to only 30% of the aerobic and 22% of the fermentative beach bacteria. Except for the utilization of acetamide, the open ocean marine bacteria were nutritionally more versatile than beach bacteria. Starch was not tested in the papers by Bauman et al. (3,4). The presence of amylase was more prominent in the aerobic beach bacteria than those from the open sea. The reverse was the case when comparing the fermentative isolates. The apparently higher nutritional versatility of the offshore isolates compared to beach isolates may have been caused by the different modes of isolation. Indole production has been demonstrated from 55 to 78% of bacteria isolated from marine invertebrate guts (10). In comparison, indolepositive organisms seem to be less prevalent in estuarine sediments (30-40%), in estuarine water (14-18%), in open ocean inhabitants (4-5%), and in open ocean water (0.07-1.5%). Over half of the bacteria isolated from Sindbad beach were indole positive ( Table 4). The trait was widespread among the physiological groups (Table 5). If it is correlated with invertebrate gut inhabitation, the data would suggest a symbiotic association of our isolates and the resident invertebrates. The beach isolates were deoxyribonuclease positive, gram negative, motile, and oxidase positive. All had a growth preference for 15 to 37 C and pH 7 to 9. The motile organisms were all polarly flagellated. Only one aerobic isolate was peritrichously flagellated, and none of groups la, lb, 2b, and 3b was motile. Only a few of the isolates liquefied gelatin, reduced nitrate to nitrite, or could grow below pH 5. Roughly 25 to 34% of the isolates could grow on PYE made with distilled water. This test was performed over a year after initial isolation on PYE-sea water media. From fresh sand, only 9.4% of the bacteria which grew on PYE-sea water agar also grew on PYE-distilled water media. The variation in these counts (excluding the August 19th sample) was remarkably consistant at both the WT and WWZ (3.1 to 15.6%). In the August 19th sample, the bacteria which grew on PYEdistilled water agar were 43.8% of those growing on sea water agar in the WWZ and 225% in the WT. Although the number of samples was limited, bacteria capable of growth on fresh water medium seem to be a minor component located on the fresh water side of the beach. The percent of bacteria growing on sea water agtir averaged 70% of the total viable bacterial count in the WT and WWZ. ACKNOWLEDGMENT The logistic and technical assistance of Neil Hulings was greatly appreciated by both authors.

v3-fos

2018-04-03T01:22:39.179Z

1973-06-01T00:00:00.000Z

29229920

s2

Persistence of Aflatoxin During the Fermentation of Soy Sauce Aflatoxin was produced by Aspergillus parasiticus NRRL 2999 but not by A. oryzae during fermentation of soy sauce. Little aflatoxin was degraded within 6 weeks unless Lactobacillus delbrueckii also was present. Sudden outbreaks of "turkey X disease," later considered to be aflatoxicosis, have been attributed to the toxic metabolites of Aspergil- lus flavus Link ex Strains of Aspergillus, distinguish or Sudden outbreaks of "turkey X disease," later considered to be aflatoxicosis, have been attributed to the toxic metabolites of Aspergillus flavus Link ex Fries (1). Strains of Aspergillus, e.g. A. oryzae, are popularly used in Asia in the manufacture of soy sauce as well as other varieties of fermented foods (4). Although a number of strains of A. oryzae have been shown not to produce aflatoxin (5), it is difficult to distinguish A. oryzae from A. flavus or A. parasiticus contaminants in substrates. Because both are widely distributed, either could be involved in an impure fermentation. Another microorganism, Lactobacillus delbrueckii, is commonly used with A. oryzae for producing soy sauce (4). Therefore, the research reported here was undertaken to determine whether an aflatoxin producing A. parasiticus strain could grow with A. oryzae and with L. delbrueckii in soybean koji, whether aflatoxin would be present in the final soy sauce, or whether aflatoxin appeared at some point during preparation, but then was degraded at a later stage in the process. A. parasiticus NRRL 2999, A. oryzae NRRL 1988, and L. delbrueckii NRRL B-445 were obtained from the Northern Utilization Research and Development Div., U.S.D.A., Peoria, Ill.; aflatoxin standards were obtained from the Southern Utilization Research and Development Div., U.S.D.A., New Orleans, La. The procedure for soy sauce production cited by Hesseltine and Wang (6) was adopted, but was modified as indicated in Fig. 1. Fernbach flasks (2.8 liters) were used instead of conventional open "koji" boxes to prevent undesirable contamination, and a clean room was utilized as an incubation chamber. The substrates were inoculated with 8 ml of a I Present address: Bundesanstalt fur Fleischforschung, 8650 Kulmbach, West Germany. culture of A. oryzae NRRL 1988 (1 x 10f spores/ml) and a 5-ml suspension of L. delbrueckii (1 x 108 organisms/ml) into the flasks. Then they were stored at room temperature. To study aflatoxin production during the fermentation of soy sauce, a 2 x 106 spores/ml suspension of A. parasiticus NRRL 2999 was added to the substrate immediately after inoculation with A. oryzae and L. delbrueckii. For the aflatoxin B1 degradation study, an aqueous preparation of aflatoxin B1 (5 gg/kg of substrate) and 0.5 M lactic acid (100 ml/kg of substrate) were introduced immediately after inoculation with A. oryzae and L. delbrueckii. Extraction followed the Ass. of Official Analytical Chemists method suggested by Eppley (3). Soy sauce substrate (50 g) containing wheat, soybeans, and liquid (50 ml) was shaken vigorously with chloroform (50 ml) and decanted. The procedure was twice repeated and A.oryzae and L.delbrueckii the three extracts were combined in a 500-ml Erlenmeyer flask. Aflatoxin determinations were then made using Silica gel-GHR-thinlayer chromatographic (TLC) plates developed in chloroform-acetone (97:3) for 45 min. Each sample was done in triplicate. Production of aflatoxin. Data in Fig. 2 show the amount of aflatoxin in each sample during fermentation and throughout the processing of the koji into soy sauce. The sample containing only A. oryzae and lactic acid bacteria showed no production of aflatoxin during the entire fermentation period (sample I). Aflatoxin (8,500 ag/kg) production by A. parasiticus NRRL 2999 alone peaked after 1 week (sample II). Aflatoxin (7,000 Ag/kg) was produced in the sample inoculated with A. parasiticus NRRL 2999 and L. delbrueckii (sample III). Lower quantities of toxin (2,100 ,g/kg) were detected in sample IV after 1 week, i.e., koji inoculated with A. parasiticus, A. oryzae, and lactic acid bacteria. Persistence of aflatoxin. Observations made with TLC plates indicated that aflatoxin degradation starts immediately after bringing in samples II, III, and IV. The TLC chromatograms of all three samples were nearly identical. No aflatoxin was detected in soy sauce containing only A. oryzae and L. delbrueckii (sample I). Sample II showed only 8% degradation of the aflatoxin in 6 weeks, whereas sample III showed 45% conversion. During this same time interval, there was 55% degradation of the toxin in sample IV. Acid production by L. delbrueckii may have catalyzed the conversion of aflatoxin B, to B2a* Direct addition of aflatoxin B1 and lactic acid (sample V) to the fermentation medium resulted in conversion of aflatoxin B, to derivatives comparable in R. value to aflatoxin B2., a comparatively nontoxic form (2). Lindenfelser and Ciegler (7) found the acid-catalyzed conversion of aflatoxin B, to B2a if a sufficient amount of lactic acid was supplied. The mechanism of the reactions among participating organisms is not clear, but the degradation products from samples III, IV, and V show Rf values lower than those of aflatoxin B1 and B2 on TLC.

v3-fos

2018-04-03T02:15:37.613Z

1973-02-01T00:00:00.000Z

32873992

s2

Application of the most-probable-number procedure to suspensions of Leptospira autumnalis Akiyami A. Various statistical tests are presented as evidence that the most-probable-number (MPN) procedure is a reliable method for estimating the density of washed-cell suspensions of Leptospira autumnalis Akiyami A. Colonization of Leptospira was first reported in 1957 by Cox and Larson (2). There is only one known report of the use of this counting procedure for describing growth (4) and there are no reports of its application to survival studies. According to Bodily et al. (1), not all serotypes of Leptospira will colonize, and results between laboratories have not always been reproducible. My attempts at quantitative recovery of pathogenic serotypes on solid media produced, at best, erratic results. Motivated by a desire to study survival of Leptospira at low cell concentrations, and recognizing that semisolid and liquid media have long been used successfully for maintenance and recovery of Leptospira, I undertook an investigation of the applicability of the most-probable-number (MPN) procedure. Estimation of bacterial density by the MPN procedure is based on two assumptions. (i) The distribution of individual cells is assumed to be random in the suspension with complete independence. This precludes use of the method with any microorganism which tends to aggregate in any way. (ii) It is assumed that growth will ensue with the introduction of one or more cells into a tube of medium. Applicability of the procedure is, therefore, essentially dependent on the ability to recover a single cell. MATERIALS transfer every 5 weeks. Antigenic stability was verified periodically by the slide agglutination test with antiserum provided by the Center for Disease Control. Preparation of cells. Cell cultures were grown at 30 C in a medium containing (per liter): Na2HPO4, 1.0 g; KH1PO4, 0.3 g; NaCl, 1.0 g; NH4Cl, 0.25 g; thiamine, 0.005 g; and rabbit serum, 100 ml. After 86 hr of incubation, the cells were recovered by centrifugation for 30 min at 3,000 rev/min, washed twice in buffer (total phosphate, 5.33 mM; pH 7.6), and resuspended in buffer. Cell concentration was standardized by direct count with a Petroff-Hausser chamber and dark-field microscopy. Dilution blanks. Dilutions for MPN determinations were made in 9.0-ml blanks containing Leptospira medium base EMJH (Difco) with 1% rabbit serum. Tubes of recovery medium were inoculated with 0.1 ml of the appropriate dilutions. RESULTS A suspension of washed cells of L. autumnalis was standardized and then serially diluted to 1.0 cell per ml. Three series of 120 tubes each were inoculated with 0.1 ml of the last three decimal dilutions to give a theoretical MPN of 100 cells per ml. Table 1 compares the observed and theoretical ratios of negative tubes to total tubes for the three cell concentrations. The three series of 120 tubes each represent 24 replicate MPN values with 5 tubes per Table 2. Figure 1 shows the 24 replicate MPN values on log-probability paper. A reasonably straight line results, the consequence of a logarithmically normal distribution. The true cell concentration should lie at the 50% probability intersection. Figure 2 is a graphical representation of the 12 replicate MPN values with their 95% confidence intervals. Each confidence interval includes the true cell concentration of 100 per ml based on direct microscopic count and dilution. MPN determinations were completed on a serie f cell suspensions standardized by direct microscopic count and dilution. The results are compared in Table 3. MPN values are expressed as duplicate determinations with 5 tubes per dilution and one determination with 10 tubes per dilution. To compare observed frequencies of MPN codes with theoretically expected frequencies, MPN values from two separate experiments with six different cell suspensions were chosen (Table 4). These MPN tests were completed over time and, therefore, represent different cell concentrations because death was proceeding. This constitutes a stricter test than selecting codes obtained on a single cell suspension. DISCUSSION Each of the statistical tests applied demonstrates that, with supplemented Fletchers medium used for cell recovery, the MPN procedure is a reliable technique for estimating the density of suspensions of L. autumnalis. The observed frequencies shown in Table 1 are within the theoretical frequency plus or minus the expected error for 10 and 1 cells per tube. This is not true for 0.1 cell per tube, but here the problem becomes one of an increasing expected error with a decreasing number of positive tubes. For reliable results, the percentage of positive tubes should be roughly between 35 and 85 (6), which is the case for one cell per tube. The logarithms of MPN values are distributed normally. The mean of the log distribution of MPN values should equal the true cell concentration. The means of 24 five-tube MPN values (106) and 12 ten-tube MPN values (103) agree well with the true cell concentration of 100 per ml. Replicate MPN values from a normal distribution should plot as a straight line on log-probability paper, which is demonstrated in Fig. 1. The mean cell concentration determined graphically is 104, which compares well with the true concentration of 100 cells per ml based on direct microscopic count and dilution. The MPN is not a precise method of measurement; it is only an estimate of the true cell concentration. The 95% confidence interval expresses the imprecision of the method. This interval will include the true cell concentration 95% of the time. Each of the 95% confidence intervals for 12 replicate 10-tube MPN values includes the true concentration of 100 cells per ml. Two of the 24 confidence intervals for replicate five-tube MPN values do not include the true concentration ( Table 2). It is expected that the confidence interval will not include the true concentration 5% of the time or 1.2 times in every 24 tests. In the case of MPN tests completed on 12 different cell suspensions, 2 of milliliter based on direct microscopic count and dilution. (Table 3). This frequency 41.9-208 is slightly greater than the expected 5%. However, the true cell concentrations are based on 12 separate microscopic counts, a method 21.3-106 which also has a significant error. The comparison of observed and theoretical frequencies for five-tube MPN codes shows 52.1-258 good agreement (Table 4). An unusually large number of improbable codes would indicate 25. either that the basic assumptions for the MPN procedure do not hold or that something is wrong in the technique. 33.3-165 This procedure was subsequently used to describe exponential death rates for L. autumnalis in defined solutions (5).

v3-fos

2020-12-10T09:04:13.107Z

1973-12-01T00:00:00.000Z

237230968

s2

Repair and Enumeration of Injured Coliforms in Frozen Foods Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA. There is increasing evidence that Escherichia coli and other coliforms present in frozen samples may remain undetected by procedures normally used for their enumeration (1-3, 9, 11). Bile salts, deoxycholate, and lauryl sulfate present in the selective media are inhibitory for the repair and subsequent multiplication of the freeze-injured coliforms. When injured cells have repaired they regain their resistance to these compounds; it would appear therefore, that repair of damage is essential if enumeration of all cells by the selective media is to be accomplished. Previously, we reported that the detection of unfrozen E. coli also depends upon methods and media used for plating (10). Included in this study are observations concerning the enumeration of coliforms from naturally contaminated frozen foods. Some of these results have been presented previously (Inst. Food Technol. Annu. Meet., 1973). MATERIALS AND METHODS Freezing, thawing, and injury repair in laboratory strains of E. coli cells. E. coli strains NCSM and K-12, from the department culture collection, were grown in Trypticase soy broth (TSB) for 16 to 20 h at 35 C, harvested by centrifugation, and washed with sterile water. They were suspended either in sterile glass distilled water or in selected sterile foods to a population of about 2 x 108 cells/ml. The samples were frozen in 10-ml portions at -20 C in an ethylene glycol bath; after storage for 20 to 24 h, they were thawed at 10 C in a circulating water bath. Sterile foods used for suspending E. coli were 10% reconstituted nonfat dry milk, veal infusion (5% solids), crab meat blended 1:10 in water, and liquid whole egg. Details of the procedures, including preparation of the sterile foods, have been described elsewhere (9)(10)(11). To study repair of the injured cells frozen in water, 1 ml of the thawed sample was transferred to 9 ml each of sterile TSB and water, incubated at 25 C, and enumerated at intervals on several plating media. When cells were frozen in sterile foods, 1 ml of the thawed sample was transferred to 9 ml each of TSB and the respective sterile foods, incubated, and enumerated as before. Enumeration of frozen E. coli. Samples were serially diluted in sterile distilled water and plated in 0.1-ml portions in triplicate on each of the following: (i) Trypticase soy agar (TSA); (ii) violet red bile agar (VRBA); and (iii) deoxycholate lactose agar (DLA). Samples were plated in each medium by the pour, surface, and surface-overlay methods (10,11). The pour method was the conventional plating procedure. In the surface-overlay method, 0.1 ml of the sample was spread over the dried surface of a prepoured plate, and after about 10 to 15 min at room temperature another 4 ml of the same media (45 C) was layered over the surface. The surface method was done in the same manner except that the overlay was omitted. The plates were incubated at 35 C for 20 to 24 h, and the colonies were counted. TSA poured plates were incubated for 48 h. Enumeration of coliforms from naturally contaminated frozen foods. Samples of commercially produced frozen foods were obtained from the Food Microbiology Laboratory of the North Carolina Department of Agriculture and from the local supermarkets. Unprocessed, processed, and ready-to-serve products from different categories of foods were randomly selected. The frozen food samples were first subjected to screening, and those containing a higher number of coliforms were used for actual tests. For screening, a 10-g portion of the sample was blended for 2 min with 40 ml of sterile water (1:5 dilution), then 10 ml of the blended material was added to 10 ml of single-strength TSB (1: 10 dilution) and incubated at 25 C for 1 h. Samples in 1.0-ml and 0.1-ml portions were then plated on VRBA by the pour plate method. After 24 h at 35 C the coliform colonies were counted. Foods found to contain a relatively large number of coliforms (100/g or more) were sampled randomly from four to five areas. A 25-g sample was then blended for 2 min in 100 ml of sterile water (1: 5); 10 ml was then mixed with 10 ml each of single-strength TSB and water (1:10) and incubated at 25 C. At intervals, samples were plated (0.1 ml/plate) in triplicate by the pour and surface-overlay methods on VRBA and DLA. In tests where the total counts were determined, samples were also plated on TSA. The food sample suspended in water and made to the 1:10 dilution was used for the 0-h enumeration within 2 to 3 min after blending. Food samples also were enumerated for coliforms by the most probable number (MPN) procedure (three tubes, three dilutions) using brilliant green bile broth (BGB) and lauryl sulfate broth (LSB). Samples from TSB and water (1:10 dilution) were used as the first dilution. The tubes were incubated at 35 C for 48 h and checked for gas formation. Samples from all LSB tubes showing the presence of gas were reinoculated into BGB tubes; these were incubated for another 48 h at 35 C and examined for gas formation. MPN estimates were then determined on the basis of BGB tubes showing gas production (1,2). In a number of the samples that contained higher levels of coliforms, presumptive tests were performed. Blended samples suspended in water and TSB were plated on VRBA by the pour and surface-overlay methods. The plates were incubated for 24 h at 35 C, and all red colonies were counted as coliforms. Depending upon the number on the plate, 10 to 100% of the colonies were inoculated into tubes containing 9 ml of BGB and incubated at 35 C for 48 h. Tubes showing gas production were considered coliforms. For the determination of cell death, injury, and repair, conventional methods were used (9,11). RESULTS Effect of plating methods on the detection of frozen E. coli. Previous studies in this laboratory have indicated that recovery of unfrozen and frozen cells of E. coli on selective agar media could be influenced by plating methods (10,11). The results of the present study with two E. coli strains were in agreement with the previous findings (Table 1). Of the two test strains, NCSM appeared to be much more resistant than K-12 to freezing and to pour plating with selective media. In both strains a large number of survivors were unable to form colonies on VRBA and DLA (i.e., the cells were injured). Pour plating on DLA was more inhibitory to the survivors of both strains. Unfrozen cells, especially of strain K-12, were highly susceptible to pour plating on DLA. Repair of freeze-injured E. coli. Rapid repair occurred when thawed cells of E. coli were suspended in TSB and incubated at 25 C ( Fig. 1). This was evidenced by the increase in VRBA counts; both the initial rate of repair and the extent of repair at 60 min were much higher in strain NCSM than in K-12. The lower repair in strain K-12 could be related to the greater inherent sensitivity of this strain to selective media. Cells suspended in water did not repair; rather strain K-12 showed progressive death and injury (reduction in TSA and VRBA counts, respectively). In TSB, both strains started cell multiplication after 90 to 120 min of incubation. E. coli K-12 was frozen in sterile foods; after thawing, a sample was transferred to TSB or to the respective sterile foods and the mixtures were incubated for 3 h at 25 C. At intervals, samples were plated by the surface-overlay method on TSA and VRBA. Results (Fig. 2) showed that in crab meat and veal infusion the injured cells repaired, but in veal infusion more cells repaired when they were in the TSB. Cell multiplication did not start before 2 h in any sample. Since both foods had the same number of cells before freezing, cell death appeared to be higher in veal infusion than in crab meat. Among two other foods tested, the least death and injury and maximum repair occurred in milk. Cell death was maximum in liquid egg which probably was due to the sensitivity of the injured cells to egg white lysozyme (8) (data not presented). Repair and multiplication of coliforms present in naturally contaminated frozen foods. A frozen sample of breaded oyster after blending was suspended in TSB and sterile water (1:10 dilution) and incubated at 4 h at 25 C. At intervals, samples were plated by the pour and surface-overlay methods on VRBA; because of the presence of associated organisms surface plating was not used for testing commercial frozen products. Initially, and up to 60 min, more coliforms were recovered by the surface-overlay method (Fig. 3); samples incubated in TSB permitted higher recovery than the samples incubated in water. Repair of injury Effect of foods on repair and multiplication of frozen-thawed E. coli K-12. E. coli K-12 cells were suspended in foods and frozen to -20 C. After thawing, 1 ml of this was transferred to 9 ml of TSB and 9 ml of respective food suspension and incubated at 25 C. Samples were plated by surface-overlay method on VRBA (up to 60 min) and TSA. For other explanations see Fig. 1 and Materials and Methods. -1x =D CL was very rapid for the first 30 min. Cell multiplication was not evident before 90 min. Similar observations have been made with stuffed flounder, deviled crab, crab cake, and ice cream. However, with some foods, such as deviled crab, incubation of samples in TSB, rather than in water, did not show any additional advantage. In this sample the cells repaired rapidly (within 30 min) and started multiplication after about 2 h (data not presented). Samples from a frozen deviled crab were blended and incubated in TSB and water at 25 C and plated on TSA and VRBA by the surface-overlay method. TSA used as the overlay (on TSA plates) contained triphenyl-tet-APPL. MICROBIOL. razolium-chloride (0.005%) for easier recognition of bacterial colonies. It can be seen (Fig. 4) that coliforms constituted about 5% of the total bacterial population. Associated bacteria did not start multiplication before 2 h; however, early in this interval injured coliforms were repaired. These data suggested that repair of the injured coliforms was not affected by the growth of the associated flora. Similar results were obtained with ice cream (data not presented). Relative recovery of coliforms from frozen foods. Coliforms in several frozen foods were enumerated on VRBA and DLA by the pour and surface-overlay methods and in BGB and LSB by the MPN method. Enumeration was done at 0 h from foods suspended in water and after 1 h of incubation at 25 C from foods suspended in TSB and water. In many samples MPN values were > 1,100/g of food; therefore, average values for a particular type of food are not presented, but results for a single sample of each type of food are given (Table 2). However, a similar trend was observed with the other samples. In general, plating methods appeared to give values which were more consistent than the values obtained by the MPN method in both BGB and LSB; poor detection of coliforms from frozen foods by the MPN method has been observed by others (4, 7); many have preferred enumeration of coliforms in frozen foods by plating or solid media (4,6,7). In all samples, more coliforms were recovered by plating the samples after the 1-h repair in TSB; repair in water was not as extensive. The lowest injury was found in chicken chow mein. Plating on VRBA by the surface-overlay method permitted higher recovery of coliforms from samples incubated in water; however, for samples incubated in TSB, recovery was not as greatly influenced by the method of plating or by the media. Average recovery of coliforms on VRBA and DLA from five kinds of foods indicated that plating the samples after 1 h of incubation at 25 C was superior over that of plating them immediately after thawing. This increase occurred irrespective of media and methods of plating; recovery on VRBA was generally better than on DLA. Recovery from different foods and individual samples varied, but an increase in count of x 21 was observed as a result of the repair (Table 3). Presumptive confirmation in BGB of the coliform colonies obtained on VRBA from frozen breaded oyster, deviled crab, crab cakes, and stuffed flounder were studied ( Table 4). The surface overlay provided about 25% more colonies than the pour method, and presumptive confirmation of colonies by both methods were essentially the same. DISCUSSION There are a number of studies which indicate that many semipreserved foods may contain injured microorganisms (5, 6; M. Warseck, M.S. thesis, N.C. State Univ., Raleigh, 1973). If an accurate evaluation of the bacterial population of such foods is to be obtained, it is essential that current methodology be revised so that such microorganisms can be detected and enumerated. Fortunately, the repair of injury proceeds at a sufficiently rapid rate so that this can be accomplished before cell multiplication takes place. Based on the present study, it would appear that a 60-min repair period at 25 C would be adequate without causing complications by cell multiplication of uninjured microorganisms. To assure the repair of injured microorganisms in different kinds of foods, the repair must be done in a culture medium such as TSB, although it was observed that some foods allow repair per se. The repair of injured cells appears to be essential for their detection regardless of the type of enumeration procedure employed. While the surface-overlay plating method is less stringent on damaged cells, the improved detection and enumeration of cells by this method would seem to warrant its use over the pour plate method. Furthermore, the repair of injured cells prior to MPN determination is also advisable. Fortunately, the associated flora in food samples so far tested do not have an adverse effect on the repair of coliform bacteria. Obviously, the adoption of a repair step in the microbiological examination of foods may have a bearing on the acceptability of current standards for coliform bacteria in many foods. Only as the repair step is used routinely will information be available to evaluate the. impact of repair on current standards. The need for repair of other microorganisms is being studied in our laboratories at the present time. Information available to date indicates that most microorganisms related to food sanitation can exist in an injured state in semipreserved foods. Studies are being conducted to establish repair procedures for such microorganisms.

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