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like 1

2020-12-10T09:04:11.118Z

1971-02-01T00:00:00.000Z

237232525

s2

Incidence of Heat-Resistant Molds in Eastern Orchards and Vineyards Over 70% of the samples of fruit, vegetation, and soil obtained in surveys of New York orchards and vineyards were contaminated with heat-resistant molds. The counts generally were low, under one per gram. Byssochlamys fulva was the most common isolate. Other isolates were identified as B. nivea, Paecilomyces varioti, Aspergillus fischeri, A. fischeri var. spinosus, A. fumigatus, Penicillium vermiculatum, and P. ochro-chloron. The first reported spoilage outbreak of thermally processed fruit caused by Byssochlamys occurred in Great Britain during the early nineteen thirties, and for a numbei of years the mold appeared to be restricted to that country (1). More recently the organism has been isolated from other areas including continental Europe (3,5), Canada (8), and the West Coast of the United States (2). Although spoilage outbreaks have occurred in fruit processed in the Northeast, it has not been known whether Byssochlamys was endemic to eastern orchards or whether it was introduced via food ingredients imported from other regions. The objective of this research was to determine the extent to which Byssochiamys is present in the orchards and vineyards of this region and to learn something about its distribution. This information is important to the food processor because it would appear that the best method for preventing spoilage of certain fruit products is to minimize the opportunity for contamination. MATERIALS AND METHODS The samples were obtained from surveys made in Ontario and Chautauqua counties during the 1968 and 1969 growing seasons. Details of the culturing methods have been published (7). Essentially, the procedure consisted of blending the sample, usually 50 to 100 g, in 16°-Brix Concord grape juice until the mixture was homogeneous. The material then was heated 1 hr at 70 C to select for the heat-resistant molds and to activate dormant spores. The Concord juice enhanced this activation. After heating, the homogenate was distributed into petri dishes, approximately 10 ml per plate. Equal volumes of double-strength potatodextrose-agar (pH 3.5) were then added and the material was mixed. Colonies were counted after incuba-I Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper no. 1841. tion for 48 hr at 32 C. Negative plates were incubated an additional 48 hr before being discarded. Several precautions were taken to assure that the heat-resistant molds did, in fact, originate from the field samples and not our laboratory. The samples were processed in an isolated laboratory that had not been used for other mold studies, and in many surveys the tared, sterile, screw-cap blendor jars were carried to the orchards and vineyards. Confirmation that the counts represented populations of heat-resistant molds was achieved by transferring representative colonial types from each sample into culture tubes containing 5 ml of 15°-Brix Concord grape juice. After incubation for 28 days at 32 C, to assure ample time for ascospore formation (6), the pellicle and broth were blended in a Sorvall Omnimixer homogenizer. The homogenate was then heated for 1 hr at 70 C before the plating of appropriate dilutions on acidified potato-dextrose-agar. Identification of the isolates was based on macroscopic and microscopic observations of cultures propagated at room temperature and 32 C in various media including potato-dextrose, Czapek, and maltextract agars. The homogenates of 28 day grape juice cultures were used in the studies on heat activation of dormant spores. The material was washed three times in distilled water and then suspended in 9570 ethanol for 20 min (one part aqueous spore suspension per nine parts ethanol). This treatment served to destroy the heat-labile structures such as conidia and hyphae without affecting viability and dormancy of the ascospores (7). RESULTS AND DISCUSSION A total of 99 samples of various fruit, vegetation, and soil were obtained from 15 vineyards, 9 orchards, and the receiving platform of 1 grape processor during the two growing seasons. Over 70% were found to be contaminated with heatresistant molds (Table 1). In general, the incidence of the organisms was low, usually under one per gram. The highest populations were in soil and in materials collected from the ground. Apples which gave an average figure of only 4.1 per 100 g were an exception, perhaps because of their relatively large weight-to-surface ratio. Only a limited number of sound fruits other than grapes were cultured, because it was assumed that decayed materials would be more likely to yield the organisms. Of seven sound fruit samples, five were negative, whereas single samples of raspberries and peaches gave counts of 11 and 5.6 per 100 g, respectively. Of the sound grape samples, 13 were collected at the receiving platform of a processor and thus represented commercially harvested fruit. All were contaminated with heat-resistant molds. Five of the samples were of mechanically harvested fruit. There was some indication that this material was the more heavily contaminated in that it gave a geometric mean count of 12 per 100 g compared to 4.8 per 100 g with the handpicked fruit. Although it is not known whether the results were typical, our ability to isolate B. fulva from the soiled surfaces of a harvester that had been idle for several days lent support to the data. It is very possible that harvesters can be a significant source of contamination when their sanitation has been overlooked. B. fulva was the species isolated most frequently (Table 1). Although it was not the predominant contaminant of certain materials such as the decayed plums, it was recovered from all sample types. Other molds that survived the selective treatment at 70 C included B. nivea, Paecilomyces varioti, and species of Aspergillus and Penicillium (Tables 1 and 2). It is of interest that B. nivea, which is the most common contaminant of California grapes (2), did not predominate on any of our samples. Many of the isolates were studied to determine whether spores were responsible for their heat resistance, whether they exhibited a dormancy that could be broken with heat, and whether grape juice enhanced spore activation. This work was I completed before the cultures had been identified, and, therefore, it was reassuring to find later that those grouped as a given species had generally responded in a similar manner to the different tests. Thus, the 10 cultures of B. fulva that were tested were similar to the isolate in Table 2 in that Negative 20 X 107 50 X 101 20 X 102 60 X 102 20 X 102 a Heated suspensions were held in a water hath for 60 min. heating in grape juice produced a significant increase in the viable count and that heating for 1 hr at 80 C afforded counts considerably higher than those obtained at 60 C. The counts at 70 C (not shown) were still higher by a factor of twoto threefold, indicating some spore destruction at 80 C. The fact that the maximal viable count agreed quite well with the microscopic spore count indicated that the individual spores present in the homogenate were mainly ascospores rather than heat-labile conidia. B. nivea and P. varioti responded in a manner similar to B. fulva in that activation was stimulated by heating in grape juice. The B. nivea spores, however, were considerably less heat-resistant as evidenced by the significant reduction in viable count at 80 C. The isolates identified as P. varioti appeared identical to B. fulva except that asci were not observed. We suspect that they actually are poor ascospore-producing strains of B. fulva and that the low viable counts of heated suspensions reflected the ascus-ascospore populations, levels that would not have permitted them to be readily detected under the microscope. The aspergilli and penicillia differed from the above species in that grape juice had little or no effect and viable counts usually were not greatly increased by heating at 60 C. A. fischeri, A. fischeri var. spinosus, and P. vermiculatum exhibited considerable heat resistance in that their homogenates gave viable counts of over 105 per ml after being subjected to heating for 1 hr at 80 C. The three A. fumigatus cultures were also relatively resistant when first isolated and, therefore, are believed to be strains of A. fischeri that later lost the ability to form asci. A. fischeri has been isolated in other searches for heat-resistant molds (4). The structures responsible for the limited resistance of P. ochro-chloron remain to be defined since there appeared to be little correlation between the microscopic spore count and the viable population of heated suspensions. Non-alcohol treated homogenates gave similar low counts when heated at 80 C, indicating that a sensitivity to ethanol was not responsible for the low viable recoveries. The ubiquitousness of Byssochlamys in New York orchards and vineyards raises the question as to why spoilage has not been a more serious problem. Preliminary studies indicate that many of the isolates would survive the thermal processes commonly given fruits and fruit products. One possible explanation is that the initial low incidence of spores on sound fruit, combined with the effect of certain processing steps, results in an extremely low level of contamination by the time the product is ready for the heat exchanger or retort. King et al. (2) have shown that a high percentage of spores are removed by the type of filtration given commercial grape juice. Washing, fluming, blanching, peeling, and pressing are some of the other operations that would be expected to remove spores from contaminated fruit.

v3-fos

2018-04-03T02:37:18.463Z

1971-05-01T00:00:00.000Z

34255170

s2

Initiation of Staphylococcal Growth in Processed Meat Environments The probability of staphylococcal growth initiation in laboratory-made cured meats is investigated and compared with growth initiation in broths. Genigeorgis et al. (4) developed equations to predict the probability that one staphylococcal cell will initiate growth aerobically in Brain Heart Infusion (BHI) broths of various pH values and NaCl concentrations at 30 C. The studies reported here tested the reliability of the developed equations in predicting the probability of staphy- lococcal growth in a variety of laboratory-pre-pared processed meats. The probability of staphylococcal growth initiation in laboratory-made cured meats is investigated and compared with growth initiation in broths. Genigeorgis et al. (4) developed equations to predict the probability that one staphylococcal cell will initiate growth aerobically in Brain Heart Infusion (BHI) broths of various pH values and NaCl concentrations at 30 C. The studies reported here tested the reliability of the developed equations in predicting the probability of staphylococcal growth in a variety of laboratory-prepared processed meats. Thirty-nine processed meats at different pH values and with various concentrations of NaCl and NaNO2 were utilized during the course of four experiments. The meats were prepared by homogenizing defatted ground pork loin or beef sirloin in a meat grinder with desired amounts of NaCl and NaNO2. After 24 hr of refrigeration, the pH of each meat was adjusted to low and high levels by the addition of 1.75% (w/w) glucono-6-lactone powder and NaOH (5 ml of 2% NaOH/100 g), respectively, and the meats were again homogenized. After this, they were cured for an additional 2 days and then pasteurized for 15 min (6 min to 121 C, 7 min at 121 C, and 2 min to room temperature), after which the meat juices were drained off. The tubes containing meats with the same brine concentration were placed in a can along with a solution of NaCl equal in concentration to that of the brine concentration of the meats. The cans were covered with plastic lids, and the cultures were then incubated aerobically for 7 days at 30 C. Samples of the various types of meats were then analyzed for pH, brine concentration, water, and nitrite according to procedures described previously (5). After incubation, the meats were homogenized with 10 to 20 ml of sterile distilled water, and portions were plated on blood-agar (BHI base) for staphylococcal and total plate counts. The remainder of the homogenate was centrifuged, and the supernatant fluid was lyophilized after dialysis and treatment with chloroform (2). The lyophilized samples were rehydrated with 0.6 ml of phosphate-buffered 2.8% saline (pH 7.2, 0.02 M) and then tested by the single-gel diffusion (6) and microslide (3) techniques for the presence and amounts of enterotoxins B and C. The results of the experiments are presented in Tables 1 and 2. Only the minimum numbers of cells which initiated growth have been included. The present findings indicate that the meat environments were more conducive to growth of staphylococci than were the BHI broths. Also, at the same brine concentration and pH, it took fewer cells to initiate growth in meats than in BHI broths. This was particularly evident in meats with high brine concentration and low pH. Staphylococcal growth was initiated in such meats even though the levels of inoculated cells were below the minimum limits predicted by the equations (4) as necessary for initiation of growth. As expected (6,7), elevated yields of enterotoxins B and C were obtained at high pH values coupled with low brine concentrations. All meats supporting enterotoxin production had good staphylococcal growth to over 4 x 107 cells per sample. Yet, 70 samples with 108 cells per sample had no enterotoxin B or C production. The highest yields of enterotoxins B and C were 70 and 20,gg per sample, respectively. The results obtained in these studies indicate that the equations derived from data based on previous studies with BHI broth (4) cannot reliably be used to predict the probability of initiating staphylococcal growth in processed meats. Experiments statistically designed to permit development of predictive equations derived from results obtained by direct staphylococcal inoculation of meats are now in progress.

v3-fos

2020-12-10T09:04:11.830Z

1971-03-01T00:00:00.000Z

237233884

s2

Listeria monocytogenes in Nature Samples from 12 farms were examined during two successive spring and early autumn seasons. L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites. A total of 27 strains were isolated from the 19 sites. The organism was not isolated from any of the autumn collections. The wide geographical distribution of listeric infections involving diverse species of animals (7) suggests common sources of Listeria monocytogenes shared by man and animals. The epidemiology of listeriosis is perplexing, and the habitat of L. inonocytogenes is obscure. Seeliger (15) has commented on the resemblances of the biochemical and cultural characteristic of L. monocytogenes to some plant-soil inhabitants and has speculated "that there may well be a primary saprophytic life of Listeria" in which event the epidemiology and epizootology of many listeric infections would be more comprehensible. The purpose of this investigation was to implicate the soil-plant environment as a reservoir of L. monocytogenes. In previous preliminary studies (18), L. monocytogenes was isolated from vegetation in an agricultural area where listeriosis was rare. In the present study, vegetation at the same sites was sampled during two spring and two autumn seasons of successive years. Soil was cultured from the same sites during the second year. To obtain information on the presence of L. mnonocytogenes in a different setting, uncultivated vegetation was examined from sites within a small area of another county. A nonagricultural, residential, suburban community with no known listeric infection was selected to minimize the possible role of livestock in Listeria dissemination. The aims were: to note possible seasonal variation in the frequency of occurrence of the organism, to observe difference or similarity of strains recovered on repeated sampling, to compare plant isolants with the strains isolated from surrounding soil, and to observe similarity or difference in isolants from nature and L. niionotogenes isolated from infections. MATERIALS AND METHODS Plant samples and soil were obtained in Hanover County, Va., from 12 different farms scattered throughout the county and separated by as much as 50 km. One sample per farm was collected in April 1967 from dead and decayed corn or soybean plants as described elsewhere (18). The same farm was visited during September 1967, April 1968, and September 1968 when plant samples again were obtained as near as possible to the original site. These specimens were collected from portions of the plant 50 cm above the ground. Some of the plants collected in auturnn were still green, some were partially green, and some were completely brown. Also, at the time of the last three collections, a total of about 20 g of surface soil was collected to a depth of 2 to 3 cm obtained near the base of the plants being sampled. Plant material also was collected from a nonagricultural, partially developed, suburban residential location (2 to 3 km2 in area) in Henrico County. With one exception (day-lily leaves), the specimens collected here were wild grasses growing either in or at the edge of a wooded area, along the banks of a brook, or in open lots. Seven sites were selected, and each site was visited in April and again in September 1968. The April samples were dead plants which had remained standing during the winter; the September samples were still green. The vegetation was placed in large jars with Brain Heart Infusion (BHI; Difco) as previously described (18); and held at 4 C over a period of 3.5 months (1967) to 5 months (1968) with samples of the liquor taken every 20 to 25 days (1967) to 30 days (1968). Previous experiences (Welshimer, titipuiblished data) in culturing vegetation without prior and prolonged cold enrichment were unrewarding; therefore, all specimens were held at least 20 days before subculturing for Listeria. The culture of the liquor obtained April 1967 has been described in detail (18) and modified as follows for the 1968 specimens: BHI (5 ml) was inoculated with 0.05 to 0.1 ml of the liquor and incubated overnight at 37 C. This culture was handled in two ways. (i) It was streaked on several plates of blood-agar base containing I % glucose (TBG; Difco) and at the same time streaked on Mc-Bride Listerica agar (14). Each plate was streaked by a different spreading technique to ensure good distribution of colonies. (ii) The BHI ctLlture was placed in the dark at 20 to 25 C. If L. monocytogenes was not recovered from the initial plating of (i), the BHI cultures were held for 2 weeks before subculturing on plates of TBG agar and McBride agar. The TBG plates were incubated 37 C, and colonies were examined microscopically at 24 and 48 hr by the oblique lighting method as described by Gray (6). The Mc-Bride plates were inoculated with 4 loopsful of the BHI cultures, incubated at 37 C for 2 days, placed at 20 to 25 C for 1 to 2 days, and examined by oblique lighting for small intensely blue colonies. Soil samples (15 g) were placed into 100-ml Erlenmyer flasks containing 50 ml of BHI adjusted to pH 7.4. These samples were held at 4 C and subcultured over a period of 3.5 months as described above for vegetation. Colonies suspected of being L. monocytogenes were picked, streaked on TBG agar plates, and incubated for 18 to 24 hr. Typical colonies of catalase-producing, grampositive, evenly staining, small rods, motile at 20 to 25 C, were tested on the following carbohydrates: glucose, salacin, lactose, trehalose, esculin, rhamnose, maltose, melezitose, mannitol, and sucrose. These carbohydrates were held at 37 C and observed for 8 days for reactions characteristic of L. monocytogenes (8,15). Organisms with the morphological, cultural, and biochemical reactions of L. monocytogenes were tested serologically in Richmond by macroscopic tube agglutination tests with rabbit antisera developed against the major serotypes and then forwarded to Utrecht where, in addition to repeating the biochemical and cultural reactions, an extensive analysis was made of each strain by using monospecific antisera developed against the individual H and 0 antigen factors of the various serotypes of L. monocytogenes. Pathogenicity of the isolants was based on the response of mice (Rockland Farm SW) weighing 16 to 18 g, intraperitoneally inoculated with suspensions of 2 X 109 L. monocytogenes. Isolants failing to kill sets of mice in 3 weeks were considered avirulent. At this dose, the mice either died within 5 days or survived the 3-week observation period. The dead mice were autopsied and the liver and spleen were cultured. Isolants were streaked on sheep and rabbit bloodagar with 5% blood in Tryptose-blood-agar base, incubated at 37 C, and observed at 24 and 48 hr. After mixing and holding the soil or vegetation in BHI at 4 C for the first 18 to 24 hr, some of the broth was streaked on eosin-methylene blue plates. RESULTS L. monocytogenes was not recovered from vegetation or soil collected from the agricultural area (Hanover) during September 1967 or September 1968. L. monocytogenes was not recovered from vegetation collected from the smaller suburban area (Henrico) during the single September collection, but it was isolated from the spring collections in both areas. Eight strains of L. monocytogenes were isolated from vegetation at 7 of the 12 sites in the spring of 1967, and 9 strains were isolated from vegetation of 9 of these sites 1 year later. L. monocytogenes was isolated from vegetation at three sites where none was found the previous year. Only at one site were the organisms isolated the first year and not the second year. Specimens simultaneously collected from soil and the adjacent vegetation provided contrasting results: L. monocytogenes was isolated from soil at four sites, three of which also yielded Listeria from the vegetation; however, the soil strain and the vegetation strains were antigenically distinct. A virulent soil strain was isolated from a site where previously no Listeria were isolated. Of the 12 farms examined, L. monocytogenes was recovered from vegetation, soil, or both at each of 11 sites either in April 1967 or 1968. L. monocytogenes was isolated in the spring from six of the seven nonagricultural sites. The usual H and 0 antigens of L. monocytogenes with an additional 0 factor, XV, were found in 17 strains. This factor previously was encountered (Donker-Voet, unpublished data and designated as "XV" in L. monocytogenes W-Li 93/65 received by one of us (D-V.) from H. Seeliger, Wurzburg, originally isolated by H. E. Larsen, Copenhagen, from chicken feces. Strains with factor XV closely resembled subtypes of type 4 L. monocytogenes; e.g., in addition to H factors A, B, C and 0 factors III and XV, seven strains contained 0 factors V, VII, and IX as does type 4a; five of the strains contained factors VI and IX; and five strains contained factors V, VI, and IX, thus resembling 4ab and 4e but lacking factor VII or VIII associated with these serotypes. One strain, with the unusual combination of factors I and IX, was indeed anomalous. The nine remaining Listeria strains were type la or lb; two strains were avirulent. Factor XV strains were all avirulent. Mice inoculated with the virulent strains presented the hepatic lesions and overwhelming septicemic response characteristic of mice inoculated with human animal strains of Listeria. All strains were tested on both rabbit and sheep blood-agar. The virulent strains hemolyzed sheep blood-agar, but the avirulent strains did not. Rabbit blood was hemolyzed by all of the virulent strains and six of the avirulent strains. The avirulent strains which hemolyzed rabbit blood produced a darkening or alpha-like hemolysis on sheep blood. The method of subculturing the refrigerated specimens collected in 1968 resulted in earlier detection of L. monocytogenes than previously accomplished (18). Of the 19 positive isolations of L. monocytogenes in 1968 from the different specimens, 11 were obtained from specimens cultured after refrigerating for 1 month, and 8 isolations were obtained after refrigerating the specimens 518 WELSHIMER AND DONKER-VOET for 2 to 5 months. The four soil strains were isolated after 1 month of refrigeration. Nine of the isolations were obtained from BHI subculture of the refrigerated liquor held at 20 to 25 C for 2 weeks after the initial overnight incubation at 37 C (method ii). AU of the plant specimens obtained in the agricultural area during the spring gave lactosepositive colonies on eosin-methylene blue-agar. The number varied from 3 to 4 colonies per plate to uncountable numbers. No Escherichia coli was observed, for these were all Klebsiella-Enterobacter types of colonies. Only one sample of soil from the same 12 sites contained any lactose-positive colonies. At the nonagricultural sites, lactosepositive colonies were found in two of the seven plant samples. Autumn specimens were comparable to spring specimens with respect to the numbers and distribution of Klebsiella-Enterobacter type colonies. No E. coli colonies were observed in any of the 52 specimens examined during 2 years. DISCUSSION Decaying moist vegetation favors the support of L. monocytogenes, for none was isolated from the green or recently dead vegetation collected in early September although the same plant growth yielded Listeria in the spring after standing over winter. The dryness of the surface soil may explain the absence of organisms from that source in the autumn collection; experimental studies (13,17) have shown that survival of Listeria in soil is influenced by moisture content. The morphology, colony appearance, biochemical reactions, growth characteristics, antigenic composition, and mouse response to inoculation with the virulent plant-soil strains of Listeria were indistinguishable from L. monocytogenes strains isolated from infected humans and animals. The avirulent strains likewise were similar except for the presence of factor XV in many of the strains and the absence of beta-hemolytic activity on sheep blood-agar. These properties should not exclude the strains from the species, for the organisms do possess other H and 0 factors associated with L. monocytogenes (5). In the absence of less definitive antigenic analysis, some of these strains might have been placed within the group 4 serotypes as was the case in an earlier report (18). Bojsen-M0ller (2) found that 12% of the strains of L. monocytogenes which he isolated from human feces grew without hemolysis on a variety of blood-agar media. infection is important, with the animal gut acting as a reservoir of the agent. In our studies, the absence of E. coli indicates that there was no marked fecal contamination of the soil at the time of sampling; however, it does not exclude the possibility of earlier deposition with death of the less hardy E. coli. The isolation of Listeria from the suburban community indicates that the organism is not peculiar to agricultural pursuits nor restricted to association with farm animals in the area, but one can not exclude the possible role of birds and feral animals such as squirrels, chipmunks, and oppossums. The ability of Listeria to multiply at low temperature, its ability to survive for long periods in soil (13,17), and its recovery from decaying vegetation implies a saprophytic existence wherein the plant-soil environment may serve as a reservoir. Rather than attribute the presence ofListeria in nature solely to past contamination with animal feces to the exclusion of an independent role as a free-living organisms, one might liken Listeria to the Klebsiella-Enterobacter organisms which extensively exist as free-living forms on plants and soil yet inhabit the gut of man and animals and under appropriate circumstances produce disease.

v3-fos

2018-04-03T06:21:35.772Z

1971-05-01T00:00:00.000Z

25433724

s2

Effect of Sodium Chloride and pH on Enterotoxin C Production Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 101 cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 101 cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 108 cells per ml and 6.38 to 7.30 for 3.6 x 106 cells per ml. In repeated experiments in which the inoculum contained 101 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concen- trated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts. both broth broth was The was and the broth was the of staphylococcal The application of knowledge gained from studies on the environmental factors that influence the production of staphylococcal enterotoxins is important in the prevention of staphylococcal food poisoning. The effect of two components of the food microenvironment, pH and NaCl, on the production of the enterotoxins has not been studied extensively. A previous communication (6) reported the effect on the production of enterotoxin B in Brain Heart Infusion broth and reviewed the limited literature on the subject. Since then more information has become available, mainly concerning the production of enterotoxins A and B (5,8,11,12,14,15). There is only one report providing data on the effects of pH, medium, and incubation time on the production of enterotoxin C (14). The present study extends our knowledge of the combined effects of NaCl and pH on the growth of staphylococci and on the subsequent production of enterotoxin C. I Present address: School of Veterinary Medicine, Aristotelian University, Thessaloniki, Greece. MATERIALS AND METHODS Enterotoxin C production and purification. Enterotoxin C was produced by growing Staphylococcus aureus strain 137 (ATCC 19095) in a broth medium containing 3% + 3% protein hydrolysate powder (PHP) and N-Z Amine NAK (1). The enterotoxin C was later purified by the method of Borja and Bergdoll (2). A 32-mg amount of highly purified enterotoxin C was obtained from 10 liters of broth. The specific enterotoxin C antiserum to strain 137 and the crude enterotoxin C used during the purification steps were supplied by M. S. Bergdoll of the Food Research Institute of the University of Wisconsin. Production of antisera. Two young New Zealand white rabbits (4 lb) were inoculated with the purified enterotoxin C. At weekly intervals each rabbit received in order, 11, 23, 112, and 575 ,ug of enterotoxin C dissolved in 1 ml of saline and later mixed, with a Vortex mixer, with 1 ml of complete Freund's adjuvant. The antigenic preparation was injected intramuscularly and subcutaneously. Two months after the last injection, the immunity of the rabbits was challenged with 2.3 mg of enterotoxin C in saline injected intramuscularly. Beginning 1 week after the last injection, three 50-ml samples of blood were drawn from the heart over a 3-week period. Additional challenges with 1 10 ,Ag of enterotoxin C per ml. A single line of precipitation was obtained with as much as 2,500 jAg of enterotoxin C per ml, treated by the microslide procedure, even against undiluted antiserum. These results indicated the high purity of the enterotoxin C preparation. Growth media. The 3% + 3% PHP-NAK medium (1) was used throughout this study as the basic medium to which different amounts of NaCl were added and in which the pH was adjusted to various values. Thirty grams of N-Z Amine NAK (Sheffield Chemical, Norwich, N.Y.) and 30 g of protein hydrolysate powder (Mead Johnson and Co., Evansville, Ind.) were mixed with distilled water to make 600 ml. The powders were dissolved with mild heating. The medium was cooled and then distributed, in volumes of 30 ml, to 20 beakers. Sodium chloride in amounts of 0, 2, 4, 5, or 6 g was dissolved in each beaker, and the pH of each broth was adjusted to the desired value with 1 N NaOH or 1 N HCI. Next, each broth was transferred to a 50-ml volumetric flask, autoclaved for 15 min at 121 C, and cooled to room temperature. Then 0.5 ml of a solution containing 500 ,Ag of both thiamine and niacin, and sterilized by filtration, was added. The volume of the broth in each flask was brought to 50 ml by adding sterile distilled water. A 9-ml sample of each broth was placed individually in a 25-ml Erlenmeyer flask (micro-Fernbach style). The flask was then covered with a glass cap, and later the broth was inoculated with the desired number of staphylococcal cells. Inoculation and incubation. Applying the antiserumagar plate technique of Sugiyama et al. (16), colonies of strain 137 producing large amounts of enterotoxin C were selected on PHP-NAK agar (pH 6.2) containing 1:40 enterotoxin C antiserum and lyophilized on porcelain beads (9). One bead was added to a test tube containing PHP-NAK broth (pH 6.2, 0% salt), and the broth was incubated for 24 hr. A flask containing 9 ml of the same medium was later inoculated with freshly grown cells and incubated overnight. The next morning the broth was centrifuged, and the supernatant fluid was kept for enterotoxin analysis. The cells were washed twice in saline and then resuspended in saline to a desired optical density (OD) at 650 nm by using a Spectronic-20 colorimeter (Bausch & Lomb). Each experimental flask was inoculated with 0.1 ml of the cell suspension, placed in a waterbath shaker (Precision Scientific Co., Chicago, Ill., model 66900) at 37 C, and incubated for as long as 8 days at a speed of 160 rev/min. Control flasks were inoculated with 0.1 ml of distilled water. The number of cells in each flask was estimated by plating in duplicate on blood-agar. The initial OD and pH of the broths were determined and recorded at zero time. The pH was measured with a Beckman Expandomatic pH meter, by using a Q15 Thomas combination electrode. Analysis of broths. Samples of broth, usually 2.5 ml, were taken at 2, 6, and 8 days of incubation. Of the 2.5 ml, 0.5 ml was used for determination of OD after dilution with similar broth. After its pH was determined, the remainder of the sample was placed in a cellophane tube and dialyzed at 2 to 4 C against 20 volumes of distilled water. The water was changed once. After dialysis, the samples were lyophilized in a VirTis lyophilizer (model 10-145-MRBA). To test for the presence and amount of enterotoxin C, the lyophilized samples were first rehydrated with 0.6 ml of phosphate-buffered saline (pH 7.2, 0.02 M) plus 2% NaCl. Next the amount of enterotoxin C was measured by the single-gel, diffusion-tube test (6,7), with an antiserum diluted 1:60 with gel. The gel diffusion tubes were incubated for 24 hr at 30 C, and diffusion band measurements were evaluated for the amount of enterotoxin C by using a previously constructed, standard enterotoxin C curve. The curve was prepared from data obtained by using various amounts of pure enterotoxin C dissolved in the phosphate-buffered saline. The specificity of the diffusion bands in the gel diffusion tubes as due to enterotoxin-antienterotoxin C precipitation was tested by the microslide, double-gel diffusion test (3). Leftover sample material was heated at 100 C for 15 min and then tested for the presence of heatresistant nuclease (Lachica and Genigeorgis, unpublished data). RESULTS Both NaCl and pH affected growth of staphylococci and production of enterotoxin C. The rate of growth of strain 137 decreased as the NaCl concentration was increased gradually to 12% (Fig. 1B). Optimum pH for growth of this strain appeared to be between 5.00 and 6.50. In the presence of 0, 4, 8, and 12% NaCl, the respective pH limits for initiation of growth were 4.00 and 9.83, 4.20 and 9.43, 4.40 and 8.55 (Table 1). When broths with 10% NaCl were inoculated with 3.5 X 106 cells per ml, growth was permitted, as this was demonstrated by plating when the pH range was 4.50 to 8.50. The effects of NaCl and pH on the production of enterotoxin C by strain 137 are indicated in Fig. 1A. Enterotoxin C was produced in broths inoculated with more than 108 cells per ml and having an initial pH range of 4.00 to 9.83, 4.20 to 9.43, 4.50 to 8.55 and NaCl concentrations of 0, 4, and 8 %, respectively (Fig. 2). In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for the inoculum level of 108 cells per ml and 6.38 to 7.30 for the level of 3.5 X 10 cells per ml. In repeated experiments in which the inoculum contained 108 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The optimum pH for the production of enterotoxin C appeared to be between 5.50 and 6.5 (Fig. IA). When the OD of the broth and the yield of Downloaded from enterotoxin C after 48 hr of incubation were plotted as ordinates on semilog paper and the NaCi concentration was plotted as the abscissa, an essentially linear relationship was evident between concentration of NaCl in the broth and both OD and yield of enterotoxin C. Both OD and yields decreased with increases in the NaCl concentration. This relationship was true at least for pH 5.50, 6.50, and 7.50 and 0 to 8% NaCl. Data for higher NaCi concentrations and pH values outside the range of 5.50 to 7.50 were not sufficient to permit evaluation. DISCUSSION At first glance the present findings indicate that production of enterotoxin C in foods heavily contaminated with staphylococci cannot be prevented by the proper manipulation of pH, NaCl concentration, or combinations of the two within levels acceptable by the consumer. Yet in view of the ideal laboratory conditions used, it is difficult to extrapolate the findings in broths to food products or environmental situations that might exist in food processing plants. The medium used for these experiments was favorable for the growth of staphylococci and free from natural inhibitors and competing microorganisms that may be present in food. The broths were heavily inoculated with staphylococci and incubated at or near optimal temperature with heavy aeration. Consequently the limiting pH values and NaCl concentrations represent the minima and maxima below or above which there is no enterotoxin C production. Therefore, a processor could feel safe in the knowledge that no enterotoxin is produced in a given product if it is more acid, alkaline, or salty than the limiting pH and NaCl concentration values presented above. Initiation of staphylococcal growth at a pH as low as 4 has been observed for the first time. This is not surprising in view of the heavy inoculum used and the recent reports on the probability of initiating staphylococcal growth in various environments (4; Genigeorgis, Abstr. 131, 3rd Int. Congr. Food Sci. Technol.). These studies indicated that, for each combination of pH and NaCl, there is a minimum number of cells required for growth in a new population. The inocula with greater numbers of cells thus have a higher probability of initiating growth. Whether the growth at such a low pH was due to inadvertent selection of a few acid-tolerant cells, or to the shifting of the pH to higher values by metabolic products of nonreproductive living cells or decomposition products of dead cells, has not been determined. Shifting of the pH to higher values will eventually initiate growth and production of enterotoxin. Markus and Silverman (13) have demonstrated release of about 600 ,ug of protein per ml, including enterotoxin B, by 3 X 10's nonreplicating staphylococcal cells per ml in a nitrogen-free medium incubated for 10 hr at 37 C. In the present study, there was an inverse linear relationship between growth of staphylococci and concentration of NaCl at pH levels of 5.50, 6.50, and 7.50. In other studies, statistical analysis of the data indicated a direct linear relationship between concentration of NaCl and the probability of initiating growth by five staphylococcal strains in broths with different pH. When statistically untested data for each strain were plotted, the relationship varied from linear to sigmoid, depending on the pH of the broth (4; Genigeorgis, et al., unpublished data). The inhibitory effect of NaCl on the total viable population, regardless of the pH and temperature of incubation of the medium, has been reported previously (10). Production of any of the enterotoxins has not been reported before in media, including foods, with pH values as low as 4 or as high as 9.83. Tatini et al. (Bacteriol. Proc., p. 10,1969) reported production of enterotoxin A in sterile, reconstituted, nonfat milk solids with an initial pH of 4.50. Detectable amounts of enterotoxin A were associated with 3 to 5 million cells per ml regardless of the initial inoculum (10U to 105 cells per ml). We have demonstrated enterotoxin B production in Brain Heart Infusions at pH 5.05 (6). Reiser and Weiss (14) recently reported on the production of enterotoxins A, B, and C in four media at three pH levels (5.30, 6.00, and 6.80). With strains 137 and 483, they obtained maximum yields of enterotoxin C at pH 6.00 and 6.80, respectively, in the medium we used in this study. They found that neither the medium nor the pH in the range used materially affected production of enterotoxin A by strains 100 and 196 but that there was considerable effect of pH and medium on production of enterotoxins B and C. Kato et al. (11) studied the effects of pH, composition of broth (various protein hydrolysates), and aeration on production of enterotoxin A. The pH of their broths ranged from 5.0 to 8.0 and the toxin yields ranged from 2.9 to 4.2 ,ug/ml. In contrast to its effect on production of enterotoxins B and C, pH did not appreciably affect production of enterotoxin A. The effect of NaCl on production of enterotoxin C aerobically appears to follow the same pattern as with enterotoxin B (6,8,12,15). As the concentration of the salt increases from 0 to 10%, the yields of enterotoxins B and C decrease VOL. 21, 1971 865 on March 18, 2020 by guest http://aem.asm.org/ Downloaded from to undetectable amounts. Enterotoxin C was also produced aerobically in cured meats having up to 10% NaCI in the brine (Genigeorgis et al., unpublished data). We have obtained similar results with enterotoxin B (5). The minimum final OD of a culture positive for enterotoxin C was 1.35. Although high yields of toxin are always accompanied by high OD values, there were broths containing 10 and 12% NaCl with OD values as high as 8.7 but no enterotoxin. Such results indicate that the mechanism of enterotoxin production is more sensitive to osmotic changes than the mechanism controlling cell multiplication. Similar findings have been reported for enterotoxin B (5,6,8,12). Adjustment of pH and addition of NaCl after sterilization of the basic broth did not significantly affect the growth of staphylococci and the production of enterotoxin C. Therefore, for practical reasons this adjustment was done before sterilization.

v3-fos

2017-07-29T04:24:53.247Z

1971-01-01T00:00:00.000Z

31879597

s2

Comparative studies on progeny test results from stations and field data ween genotype and feeding level (ad libitiim versus restricted) in Diitelz Landrace pigs. Each replication consisted of 120 or 128 animals. These animals were sired, per replication, by 9-11 boars. From each sire 2 or 3 littcrgroups of 4 animals were usually taken. From each littergroup 1 gilt and 1 castrate were fattened ad tibitum, and 1 gilt and 1 castrate were fattened restricted (twice a day using a feeding schema according to age). All animals were housed and fed in single pens, and the fattening period was from 25 to 100 kgs. live weight. The average daily food intake of the restricted fed gilts was about 79 per cent of the daily intake of the art libitum fed gilts. For the castrates, the corresponding figure was 71 per cent. The data of the first two replications were analysed at the time of reporting. In none of Butterfat records from 270 Red j)ullish bulls tested on the basis of about 30 000 daughters, partly in farmer herds and partly at the special progeny testing stations in Denmark, were analysed. Only bulls whose sires had at least four tested sons were included in the study. The material was analysed in two different ways. In the first analysis, the progeny test results of the same bull from field data and from stations were considered to be records of separate characters and the actual and the expected correlations between those were calculated. In the calculation of the expected correlations heritabilities of 0.20 in the field and of 0.75 at the stations were used. The correlation between field and station results was 0.46 with an expected value of 0.81 and that between two independent field tests was 0.65 with an expected value of 0.69. In doing so, the accuracy of the test in question can be calculated as four times the intraclass correlation between paternal half brothers. As regards the station tests the accuracy became very low as compaired to the expected. In contrast to that the accuracy of the field tests was in good agreement with expectation. It was concluded that the large variance component between bulls at the stations and also the low repeatability of a station test in commercial herds are caused by a non-genetic correlation between daughters within groups. This accounts for about 2/3 of the intraclass correlation between half sibs at the stations. Les corrélations entre les performances de reproduction (taille de la portée à la naissance et au sevrage, poids de la portée à 60 jours) de 1 140 truies de race Large White et les performances d'engraissement (gain moyen quotidien de 30 à 100 kilogrammes et indice de transformation) et de carcasse (longueur, épaisseur du lard dorsal, % de morceaux nobles et % de morceaux gras) de 3 de leurs descendants issus de la même portée et contrôlés dans les stations de contrôle de la descendance ont été estimées. Les corrélations phénotypiques intra-verrat et station sont très faibles dans l'ensemble et non significatives. Trois coefficients dépassent cependant légèrement le seuil de signification (p < 0,05) : entre la longueur de la carcasse et la taille de la portée à la naissance (r = -0,09) et au sevrage (r = -0,10) et entre le pourcentage de morceaux nobles et le poids de la portée à 60 jours (r = 0,11). Les corrélations génétiques accentuent ces tendances sans qu'il soit possible de leur attribuer une valeur définitive. In planning the comparison of several breeds of cattle for use as beef crossing sire lines it is necessary to decide how many bulls (N) per breed and how many progeny (n) per bull to measure. Assuming that the breeds are to be compared for a single major trait, the following seven factors need to be taken into account : h' : the intrabreed heritability of the trait. r : the repeatability or accuracy of the progeny test. a : the phenotypic standard deviation for the trait. d : the true difference between breeds that it is desired to detect. Y : the probability that a true difference of d will be detected. p i the probability level at which it is desired to test the significance of a difference. B : the number of breeds to be compared. If h' is presumed known, and a value is specified for r, n becomes fixed. It then becomes possible to use standard experimental design theory to find the minimum value of N which satisfies a given difference, phenotypic standard deviation, probabilities and number of breeds.

v3-fos

2018-04-03T03:17:35.726Z

1971-10-01T00:00:00.000Z

1677132

s2

Effect of controlled atmosphere on growth of mold on synthetic media and fruit. Growth of seven spoilage molds on agar plates at several temperatures in both controlled atmosphere (CA) and in air was studied. Each mold responded somewhat differently to CA at each temperature; however, there were some general tendencies. The lag phase was generally increased by CA and, in some cases, was substantially extended when incubation was just above the minimum growth temperature. The mycelial structure of molds seems to be different when grown in CA than when grown in air. With only two exceptions of 24 holding conditions, the maximum amount of mycelia was always less in CA than in air. Spore development varied with each mold at each temperature; generally, it was considerably less in CA than in air. CA storage of cherries above 34 F (1 C) did not retard mold infection to any extent; at 34 F, mold growth was inhibited and storage life was extended several days as compared to air storage. CA storage of strawberries at 34 F resulted in a mold-free product after 7 days of incubation, whereas the air-stored berries were slightly infected. However, when mishandled berries showing some mold growth were stored at 34 F, CA did not stop further mold growth. Controlled atmosphere (CA) is defined as an environment with elevated carbon dioxide and decreased oxygen levels as compared with air. It has been used in conjunction with refrigeration to extend the storage life of certain fresh food products (6). CA gas composition may be adjusted by several means. The earliest method took advantage of the natural respiration of the commodity in storage (3). However, several weeks were generally required to develop the desired atmosphere. Faster methods of obtaining the desired CA composition involved the use of dry ice or compressed CO2. In recent years, CA generators (Whirlpool Corp., St. Joseph, Mich.) have been developed which burn natural gas and instantly deliver a CA of the desired composition. The extension of storage life of food held in CA is partially attributed to inhibition of the microbial flora (2). There have been a number of studies on growth of bacteria and fungi in CA (6), but few studies have been made on mold growth in CA developed by generators (1, 5; C. W. Hastings, M.S. Thesis, Univ. of Illinois, Urbana, 1967). The purpose of this research was to compare the growth of selected spoilage molds in CA with that in air on a synthetic medium and on cherries and strawberries at several temperatures. MATERIALS AND The synthetic agar medium was of the following composition: dextrose (anhydrous), 4.0 g; peptone (Difco), 1.0 g; agar (Difco), 1.5 g; water, 100 ml. Agar plates were inoculated by dispersing mold from a stock culture in 5 ml of sterile 0.01% Triton X-100 solution. Then 0.1 ml of inoculum was spread over the poured agar surface with a bent glass rod. Each inoculated plate was placed, uncovered, within an individual plastic box to prevent cross-contamination. The plastic box, 4.25 inches (10.8 cm) by 4.25 inches by 1.5 inches (3.8 cm), was covered and sealed except for inlet and outlet ports (19/64 inches in diameter) for gas flow at opposite ends. The boxes were placed in refrigerated incubators and connected to a gas manifold by vinyl tubing. The gas, either CA or air, was pumped in order through a cotton sterilizing filter, a copper tube for cooling to incubation temperature, and a water trap to remove condensed water from the saturated (100% relative humidity) atmosphere. Air had been humidified before the pump, and CA was obtained with a high relative humidity. The gas was then forced through another cotton filter before passing through the manifold to each plastic box. Outlet gas was led outside the incubator via another manifold. Mold growth measurements. The time required for appearance of definite growth was noted. This growth was considered to be aerial mycelia rather than thin, thread-like mycelia on the surface. Storage was terminated when there seemed to be no apparent increase or change in the mold. The number of spores on the plate at termination was determined by plate count. The entire mycelial mat, including the agar, was blended with 100 ml of 0.01% Triton X-100 solution in a 300-ml cup (Omni-Mixer, Sorvall, Inc., Norwalk, Conn.). Dilutions were pour-plated on the synthetic medium described above and incubated at 75 to 80 F (24 to 27 C). The counts were expressed as the number of spores per plate. This expression assumed that outgrowth of mycelial fragments would not make a significant increase in the number of colonies. For those molds (Mucor, Rhizopus, Penicillimn and Aspergillus) whose spores could be counted directly with an improved Neubauer AO Spencer Bright Line counting chamber, this was shown to be true at the 95% confidence level. The dry weight of the mycelia was determined from the homogenate used to make the spore count. All of the homogenate, except for the 0.5% that had been removed for plating, was melted in boiling water and vacuum-filtered through a tared Whatman no. 1 ifiter paper on a Buchner funnel. After rinsing with hot water, the paper and mycelia were air-dried overnight at 150 F (66 C) before weighing. Fruit storage. Bing cherries of good quality were purchased from local stores. The cherries were placed in sealed boxes (same as used for the plates) and continuously flushed with CA or air. The boxes were held at 60, 45, and 34 F (16, 7, and 1 C). They were examined for degree of infection and breakdown. Strawberries were shipped directly from California. The berries were ripe and quite soft upon arrival, with many exhibiting gray mold growth. The poorest berries were discarded. As one treatment, the strawberries were placed in plastic boxes for storage at 60, 40 (4 C), and 34 F and continuously flushed with CA or air. As .a second treatment, the berries were held overnight at warm room temperature so that the mold growth was barely evident before the experiment was started. Then the experiment was performed as the first treatment, except that storage temperatures were 40 and 34 F. Replication. In every experiment, there were two plastic boxes of each inoculated plate or fruit at each condition. All synthetic media experiments were duplicated and some were replicated as many as four times. The fruit experiments represent one trial. RESULTS AND DISCUSSION Pure culture study. The time at which definite growth occurred and the extent of final growth as indicated by spore count per plate and total mycelial weight are given for each mold at several temperatures in Table 1. P. expansum. As shown in Fig. 1, CA holding caused a lag in the appearance of mold growth as compared with air storage. This lag period increased with decreasing storage temperature. Spore counts at termination of growth were (Fig. 2). However, as storage temperatures approached 34 F, the number of spores that developed in CA was about one-twentieth that found in air. Mycelial development (Table 1) showed little difference between air and CA over the temperature range of 70 to 45 F. However, growth at 34 F in CA was two-thirds that in air, and the submerged mycelia at 34 F in CA had an intense orange coloration. A. niger. CA holding of A. niger caused a delay in initial appearance of growth by 2.5 days over the temperature range of 70 to 55 F (13 C; Table 1). The mold did not grow in either air or CA at 50 F (10 C). The spore count at termination of growth in air at 70 F was nearly 1,000 times more than in CA, but at 55 F the spore counts were equal. Mycelial weights in air and CA were equal at 70 F, but at 60 and 55 F growth in air was twice as great as in CA. M. hiemalis. The time for initial appearance of growth at 70 F was only 1 day in both CA and air ( Table 1). The times were also equal at 60 F. However, at 45 and 34 F, it took about 1 to 2 days longer in CA. Spore counts were similar in air and CA from 70 to 45 F, but at 34 F there appeared to be a greater count in the CA sample Mycelial weight was greater from 70 to 45 F in air than in CA (Table 1). However, at 34 F there was better growth in CA than in air. Microscopic examination showed that the aerial mycelia in CA were deformed, very thick, and highly branched as compared with those in air storage. R. oryzae. The increase in time for initial appearance of growth resulting from CA holding was about 1 day in the temperature range 70 to 55 F ( Table 1). The mold did not grow at 50 F. At storage temperatures of 70 and 60 F, there were about 1,000 times more spores produced in air than in CA. At 55 F, which was near the minimum growth temperature, there was a greater development of spores in CA than air. The dry mycelial weight was always a little greater in air than in CA at the several storage temperatures (Table 1). Microscopic examination showed that the mycelia produced in CA were thicker and rougher, more deformed, and more highly branched than those in air. Many "immature" sporangia were noted in the CA samples. C. herbarum. This mold showed high sensitivity to CA storage. Growth in air gave a dense mycelial mat with typical dark green color. Growth in CA was poor with only slight green color. Initial appearance of growth was delayed in CA, and the delay was increased with decrease in storage temperature (Table 1). At 45 F, the lag in CA was at least 6 days, and, in several replications, the mold did not grow at all at this condition. At termination of holding at 50 and 45 F, there were about 50 times more spores and 10 times more mycelia in air than in CA. Alternaria sp. CA holding caused a delay in the initial appearance of growth, and the delay increased with decrease in storage temperature. After 50 days of incubation, the mold had not grown in CA storage at 34 F. At 70 F, there was little difference in spore count between air and CA storage. At temperatures below 70 F, there was about 10 times more spore development in air than in CA. Mycelial weight development at 70 F was nearly equal for air and CA storage. At 45 F, mycelial development in air was about 10 times greater than in CA, and, at 34 F, the mold did not grow in CA (Table 1). F. bulbigenum. CA holding caused a delay in appearance of growth by several days compared with that caused by air storage. Spore counts were equal for air and CA samples at 60, 50, and 45 F ( Table 1). The mold did not grow at 34 F in either air or CA, even with extended incubation time. Mycelial weight determination showed that slightly greater development occurred in air than in CA. Fruit storage. Cherries held in air at 60 F showed mold within 3 days, but the CA samples were still mold-free; after 5 days, several molds as well as yeasts were growing on the CA-stored cherries. After 12 days at 45 F, the air-stored cherries had a musty off-odor and several cherries were slightly infected with Penicillium and yeast; however, in CA there was little infection and the cherries were of firmer body, better odor, and brighter color. After 3 weeks at 34 F, storage in air resulted in some mold development but in CA there was none. Although CA cherries showed some bacterial and yeast growth, they had better color and odor than the air-stored cherries. In treatment 1, strawberries at 60 F were highly infected within 1.5 days in either air or CA storage. At 40 F, the air-stored berries were visually infected by gray mold within 4 days and the CA berries within 5 days. After 7 days of storage at 34 F, berries in air were infected to a slight degree, whereas the CA-stored berries were free from mold, of brighter red color, and had greener caps. In treatment 2, the objective was to observe the degree to which CA could stop or inhibit growth after it had been initiated. At 40 F, mold continued to grow well for about 1.5 days and then the growth rate decreased in both air and CA. After 7 days at 40 F, the berries in both atmospheres were spoiled but the CA samples showed less mold growth. After 7 days at 34 F in both air and CA, mold growth was greatly reduced as compared with storage at 40 F but neither sample was acceptable. This study shows that the effect of CA on growth and development of seven spoilage molds is quite variable and, for the most part, is temperature-related. Of most interest in this work is the interaction effect of CA and storage temperature. The effectiveness of the CA was usually increased as storage temperature was reduced. Thus, for control of mold growth, it is evident that CA should generally be used with the lowest storage temperature that would be acceptable for the specific product.

v3-fos

2019-04-02T13:05:27.874Z

1971-01-01T00:00:00.000Z

90604468

s2

COMPARATIVE DIGESTIBILITY STUDIES WITH HENS AND RATS Trials were made to compare the digestibility of feeds in hens and rats. In the comparison particular attention was paid to the true digestibility of the feeds. The digestibility of rusks made of wholewheat meal was compared in hens and rats. The rats digested 62.1 % of the cell wall substances of the whole wheat meal, while the value for the hens was 16 %-units lower. The true digestibility of the other N-free organic matter was also clearly lower in the hen trial than in the rat trial. In the rat trials the digestibility of wholewheat meal was compared with that of bark meal. The true digestibility of the organic matter of the bark meal was only 23.6 %, while that of the wholewheat meal was 92.4 %. The digestibility of the cell wall substances of the bark meal was 18.5 % and of the wholewheat meal 62.1 %. It is rather difficult to compare the digestibility of feeds in hens and rats, owing to the great differences in the digestive tract and excreta of these animals. If the comparison is based on the apparent digestibility values, the results can be deceptive. This is due to the fact that the quantity and quality of the endogenous and bacterial materials of faeces vary considerably in the different animal species, and also in the same species when different rations are given. With poultry the uric acid of the excreta also leads to inaccurate digestibility results, if these are based only on the apparent digestibility. If the efficiency of digestion in different animal species is to be compared, the true digestibility of the feeds of these animals has to be known. The same applies if widely differing feeds are given to the same animal species (Syrjälä 1967). In this paper the word faeces means also the excreta of the hens. The purpose of this study was to investigate and compare the efficiency of digestion in hens and rats by estimating the true digestibility of their feeds. The feeds used in the digestibility trials were wholewheat meal and bark meal. The latter is made from the inner bark of pine. Experimental procedure Feeds. The digestibility of wholewheat meal was studied in hens and rats; the digestibility of bark meal only in rats. It is rather difficult to compare the digestibility of feeds in hens and rats, owing to the great differences in the digestive tract and excreta of these animals. If the comparison is based on the apparent digestibility values, the results can be deceptive. This is due to the fact that the quantity and quality of the endogenous and bacterial materials of faeces vary considerably in the different animal species, and also in the same species when different rations are given. With poultry the uric acid of the excreta also leads to inaccurate digestibility results, if these are based only on the apparent digestibility. If the efficiency of digestion in different animal species is to be compared, the true digestibility of the feeds of these animals has to be known. The same applies if widely differing feeds are given to the same animal species (Syrjälä 1967). In this paper the word faeces means also the excreta of the hens. The purpose of this study was to investigate and compare the efficiency of digestion in hens and rats by estimating the true digestibility of their feeds. The feeds used in the digestibility trials were wholewheat meal and bark meal. The latter is made from the inner bark of pine. Experimental procedure Feeds. The digestibility of wholewheat meal was studied in hens and rats; the digestibility of bark meal only in rats. Cr 2 0 3 , sugar and salt were first mixed together carefully, and then mixed thoroughly with the meal. After baking the bread was sliced and dried to rusks. These were ground for the hens and given whole to the rats. The bark bread for the rats was made according to the following recipe bark meal Bark meal was made by drying and grinding inner pine bark. The bread was not dried. The rats received it fresh. The animals ate the bread almost greedily. Trial with hens. Four healthy hens that had finished laying were used for the experiment. The hens were kept in the same 2x2 m 2 net-covered pen. Before the experiment the animals were accustomed to the new conditions on normal feeding. During the experiment they were fed twice a day. Chicken lime and water were given ad libitum. The faeces were collected twice a day. They were each time collected very carefully so that the proportion of the caecum and other excreta in the samples remained correct. The animals were given the experimental feed for 8 days, but the analyses were made only from the faeces excreted in the last two days. Trials with rats. The experimental procedures in the wholewheat meal and bark meal trials were the same. Four rats of the same sex were used in both trials. They were kept in the same cage. The rats were weighed at the beginning and end of the trial. They had food and water ad libitum during the day-time and water at night. In the evening the rats were brushed lightly and moved to two net cages, under which was another closer net for the collection of faeces. Only the faeces excreted in the night, which were free from hair and food, were used for the analyses. Both digestibility trials lasted 9 days, but faeces were collected only in the last 5 days. In both trials the average weights of the rats decreased: 17 g when the animals were fed with wholewheat meal rusks and 22 g with bark bread. Analytical methods. From the meals and ground rusks determinations were made of dry matter, ash and crude protein. Plant cell wall substances were also determined by the methods of Salo (1965 a) and Paloheimo and Vainio (1965), and Cr 2 0 3 from the rusks (Paloheimo and Paloheimo 1935). Most of the stones were removed from the faeces of the hens before the analyses. All the analyses were made from fresh faeces samples. The faeces of the rats were very dry, their dry matter content being 60 %. They were prepared for the analyses by pounding in a porcelain mortar with two parts of water to one part of faeces. From the faeces samples determinations were made of dry matter, ash, Cr 2 0 3 , crude protein and plant cell wall complex. The last-mentioned was determined from the faeces samples in the wholewheat meal trials of the hens and rats by the »sinter method» and in the bark meal trial of the rats by the »incubation method». Both methods have been developed by Paloheimo and Syrjälä, but are still unpublished. In the sinter method (Pulliainen, Paloheimo and Syrjälä, 1968 p. 8) the faecal exogenous material is separated from the faecal endogenous and bacterial material (Paloheimo 1962, Paloheimo 1966, Paloheimo, Syrjälä and Vainio 1968 by ultrasonic treatment and sinter filtration. In the incubation method the faecal endogenous and bacterial material is dispersed with the aid of enzymes derived from the faecal bacteria by incubating a suspension of the faeces for about 20 hours at 40°C. The method also involves washing and sedimentation. The incubation method was used instead of the sinter method in the bark meal trial with the rats, because the faeces contained a large proportion of mucus, which disturbed the sinter filtration. The cell wall complex is obtained in both methods from a certain crude fraction (F-fraction) by extraction with 80 % (v/v) ethanol and using ash and crude protein corrections. Table 1 shows the chemical composition of the wholewheat meal, the bark meal and the faeces samples, and their digestibility values. Table 2 shows the amounts of the digestible nutrients per 100 g of dry matter of meals. In calculating the digestibility percentages of the meals it was supposed that all the indigestible materials in the rusk and bread were derived from the wholewheat and bark meal. This supposition is not quite correct, however, because 36.5 % of the bark bread dry matter consisted of white wheat flour. However, since only 3.7 % of the white flour consisted of cell wall substances, the major part of it being composed of more easily digestible cell enclosure substances, the error caused by this supposition cannot be great. The negative apparent digestibility of the dry matter of the bark meal, 1.3 %, shows, however, that the indigestible dry matter of the faeces was derived from other constituents of the bark bread besides the bark meal. Results In compiling the four columns in the right-hand part of the tables, it was supposed that the exogenous organic matter of the faeces equals the organic matter of the F-fraction. This being so, the exogenous crude protein of the faeces equals the crude protein of the F-fraction. Further, the exogenous N-free organic cell enclosure substances equal the exogenous N-free organic matter minus the cell wall complex. The digestibility values in the left-hand part of the tables show the apparent digestibility of the feeds, with the exception of the values of the cell wall complex, which are true. The values in the right-hand part show the true digestibility. Discussion When comparing the digestibility values of the whole-wheat meal trials with the hens and rats, it is advisable to consider only the values which do not include ash or crude protein. Hen excreta contain small stones and sand which are not derived from the food and these remain in the ash after incineration. The ash also contains minerals excreted through the kidneys. In addition, hen excreta contain uric acid, which accounts for the values for crude protein being too high. Another factor which makes the figures for crude protein excessive is the high N content of the uric acid, which is 33.3 %. This means that the N content of the crude protein is over 16 % and that the coefficient of 6.25, used in calculating the crude protein, is consequently too high. The plant cell wall complex fraction of the faeces determined by the sinter method is relatively free from endogenous material, including uric acid. The values of the cell wall complex are also protein-corrected and ash-free. It can thus be seen that only the values for plant cell wall substances and the other figures showing the true digestibility can be used when comparing the efficiency of digestion in hens and rats. Table 1 shows that the digestibility of the cell wall substances of the wholewheat meal is 16 %-units higher in the rats than in the hens. In the case of the exogenous organic and the N-free organic matter, the values are about 3 %-units higher in the rats, while the digestibility values of the exogenous N-free organic cell enclosure substances are about same in both species and very high, about 99 %. When the hens consume 100 g of the dry matter of the rusks, they loose 11.6 g of organic matter in the endogenous fraction of the faeces. The corresponding figures for the rats is 6.8 g. A comparison of the digestibility of the wholewheat meal and the bark meal in the rats shows that they digested 62.1 % of the former and only 24.4 % of the latter. This is due to differences in the composition of the cell wall substance fraction (Salo 1965 b. Salo and Kotilainen 1969), which are shown as percentages in the following tabulation: The value for the »other cell wall substances» is calculated by subtracting from the cell wall complex determined by the method ofPaloheimo and Vainio (1965) the fractions of the cellulose, hemicellulose and crude lignin (Salo 1965 a). The remaining fraction includes uronic acid anhydrides. The value can be calculated in this way because the cell wall substance fractions determined by the methods of Paloheimo and Vainio and of Salo do not differ very much from each other. Lignin, which is practically indigestible, protects other cell wall substances and cell enclosure substances against the digestive enzymes. This is probably why the digestibility of the exogenous N-free organic cell enclosure substances is 99.5 % in the wholewheat meal, and only 82.5 % in the bark. It also explains why the digestibility of the exogenous organic matter is high, 92.4 %, in the wholewheat meal, and low, 23.6 %, in the bark meal. It may seem surprising that the digestibility value of the exogenous crude protein of the bark meal is negative. This is apparently partly due to the protective effect of the cell wall constituents, but also to the fact that a part of the correction for protein made to the cell wall fraction relates to endogenous and N-rich mucus, which was very abundant in the faeces obtained in the bark meal trials. When the rats consumed 100 g of the dry matter of bark bread they lost 8.4 g of endogenous organic matter, while in the case of the rusks made of wholewheat meal the corresponding loss was 6.8 g. The chemical composition of bark meal made from the inner bark of pine is different from that of pine wood (Salo 1965 b, Salo andKotilainen 1969). The following tabulation shows the percentage composition of the dry matter of bark meal and pine wood. The above figures show that pine wood contains much larger amounts of poorly digestible crude lignin and xylose anhydrides (Salo 1965 a) than bark meal, while the more easily digestible arabinose, glucose and galactose anhydrides constitute 85 % of the hemicellulose of bark meal. In pine wood the corresponding percentage is only 30. This probably explains why man is able to utilize bread made of bark. Tigerstedt (1919) has shown that the apparent digestibility of dry matter in man is 9.3 %.

v3-fos

2020-12-10T09:04:11.477Z

1971-02-01T00:00:00.000Z

237232803

s2

Statistical Evaluation of Diluents and Automatic Diluting and Pipetting Machines in Influenza Serology The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased. The application of microtechniques to serological investigations in virology, i.e., hemagglutination (HA), hemagglutination-inhibition (HI), and complement-fixation (CF) tests, is well known. In recent years, automation of these microtechniques has enabled laboratories to increase their volume of testing. Few investigators, however, have reported on the standardization and comparative reproducibility of the HA and HI tests utilizing these newer methods (1)(2)(3). Because of this, and because standardized procedures are necessary for the correct interpretation of serological test results, we investigated the reproducibility of the HA and HI tests for influenza by comparing the standard tube test with the microtechniques performed by hand (7) and by use of semiautomatic equipment (6,10). This report deals with a statistical analysis of our results. Antibody. The antibody reagents used in the HI tests consisted of sera from chickens immune to Al/FM/I/47 and A2/Japan/170/62 virus strains. The sera were treated with receptor-destroying enzyme to remove nonspecific inhibitors. [A detailed description of the receptor-destroying enzyme procedure is the subject of a separate report (Tauraso et Hirata et al. (3), all loops were presoa the same diluent used in the test. Dilutions wer formed by manual twirling of eight loops simu ously, and all reagents (diluent and RBC s5 sion) were delivered by hand. To 0.025 ml of twofold dilutions of antigen were added 0.0, of the same diluent and 0.05 ml of a 0.5% erel RBC suspension. After being sealed with tic tape, the plates were shaken vigorously incubated at room temperature until the RB' tled. The procedure for the semiautomatic micr nique (AUTO) was similar to that described f manual procedure except that all dilutions were with a semiautomatic autodiluter (Fig. 1 HI tests. Two homologous antigen/antibody systems were employed (i.e., Al/FM/l/47 and A2/ Japan/170/62) in evaluating the different diluents and titration procedures. The HI tests were performed as previously reported (9) except for modifications necessary to adapt to the three different procedures described above. Essentially, to one volume (0.25 ml for tube test, 0.025 ml for microtechnique procedures) of serial twofold dilutions (starting at 1:8 and 1:12) of serum was added one volume of diluent containing 4 to 8 antigen units. After incubation of this mixture at room temperature for 1 hr, two volumes of 0.5% cockerel RBC suspension was added. The tubes or plates were sealed, shaken vigorously, and incubated at room temperature until the RBC settled. The HI end point was the highest serum dilution completely inhibiting hemagglutination as determined visually. Other test details (such as quadruplicate titrations, recording of end points, etc.) were essentially similar to those described for the HA tests. RESULTS LIJLyp' Evaluation of the reproducibility of diluting prosearch cedures and diluents to measure HA antigen titer. hesda Two-way analysis of variance (8) was performed on the observed variation within each diluting were method and each diluent (Table 1). No significant e two difference in the reproducibility of the HA tests lution was observed when different diluents were used. were A significant difference (P < 0.01) was observed itigen, among the various methods used. When the ie day means of the various methods from Table 1 were i.ph-s analyzed by Tukey's honestly significant differduced ence (HSD) test (4), the TUBE and MANUAL derent methods differed significantly (P < 0.01), and the s. The AUTO and the MANUAL procedures differed ausing significantlv (P < 0.05); however, no significant oattern difference between the TUBE and AUTO methods was detected. When the within-cell variances of the different methods in Table 1 were compared (Table 2), the TUBE procedure appeared to be the most reproducible method and the MANUAL microtechnique to be the least reproducible. However, the between-cell variances ( Table 2) of the individual cell means of the different methods used to measure HA antigen titer show the AUTO microtiter system to be the most reproducible from test to test and the MANUAL microtiter method again to be the least reproducible. Evaluation of the reproducibility of diluting procedures and diluents to measure Hl antibody. Two-way analysis of variance (8) was performed ;a. on the observed variation within each method and each diluent (Table 3). No significant difference in the reproducibility of the diluents was observed. However, the analysis of variance showed a difference among the three methods. When the means a Abbreviations: PBS = phosphate-buffered saline; GEL = gelatin; BPA = bovine plasma albumin; TUBE = standard macro-tube test; AUTO = semiautomatic microtechnique; MAN-UAL = manual microtechnique; F = F ratio; P = probability; NS = not significant. b The value in each cell was calculated as follows: for practicability in mathematical calculations, the four variances obtained from the HA titers (see Materials and Methods) were multiplied by 1,000 and summed; the logio of that value was calculated, and then its square root was taken. The values for that particular cell for each replication were summed. Mathematically, the values can be expressed as: of the various methods from Table 3 were analyzed by Tukey's HSD test (4), both the MANUAL and AUTO microtiter procedures approached borderline significance at the 5% level but did not differ significantly from the TUBE procedure. Neither the MANUAL nor the AUTO microtiter methods differed significantly from each other. Both the within-and betweencell variances ( Table 4) of individual cell means of the different methods in Table 3 show the TUBE method to be the most reproducible, and the MANUAL microtiter procedure is only slightly more reproducible than the AUTO microtiter method. Evaluation of differences in HA titer with different diluents and different diluting procedures. Sincethe two-way analysis of variance (8; Table 5) of the sums of the mean HA titers from four replications for each cell revealed significant interactior (P < 0.01) between the methods and the diluents, the means of the sums of the mean HA titers of individual cells for each particular diluent and method were compared by Tukey's HSD test. Analysis of these means revealed the AUTO microtechnique and PBS/GEL to be the method and diluent of choice for accuracy in the determination of HA titers. Evaluation of differences in HI titer with different diluents and different diluting procedures. Two-way analysis of variance (8) of the sums of the mean HI titers of two replications for each cell showed no significant differences among the three methods or the three diluents. Another instrument, the Autopipetter (Fig. 2), which will automatically add either 0.025 ml of diluent or 0.05 ml of RBC suspension, was evaluated with three different diluents (PBS, PBS/ GEL, and PBS/BPA) by our laboratory to determine whether antigen carryover with the row of droppers attached to the Autopipetter occurred. diluent in the first row of eight wells was removed, and 0.025 ml of undiluted antigen was added by dropper. In the first of two procedures, the second 0.025 ml ofdiluent was added by the Autopipetter, and the 0.05 ml of 0.5% RBC suspension was added manually by dropper. In the second procedure, the second 0.025 ml of diluent was added manually by dropper, and the 0.05 ml of 0.5% RBC suspension, maintained in suspension by a magnetic stirrer attached to the Autopipetter, was added by the Autopipetter. The remainder of the HA test was performed as described in Materials and Methods. With both procedures, the average carryover after eight replications of the antigens with PBS/GEL and PBS/BPA as diluents was one well into the second row. With PBS, the average carryover was seven of eight wells in the second row. DISCUSSION Comparison of the reproducibility of HA titers by analyses of between-cell variances of the different methods showed the AUTO microtiter method to be more reproducible, by a factor of 1.84, than the standard TUBE method. The MANUAL microtiter method was the least reproducible. In the HI test, both the withinand between-cell variances showed the TUBE method to be the most reproducible. Although the TUBE method was the most reproducible, only a borderline significant difference between the MANUAL and AUTO microtiter methods and the TUBE method was observed. The additional number of replications one is able to perform with the MANUAL method and especially the AUTO microtiter method would rapidly negate the reproducibility ratio differences observed in the comparison of withinand between-cell variances in the HI test. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter method and PBS /GEL diluent appeared to be the method and diluent of choice. Although other investigators (5) observed lower HI titers with the microtechnique as compared to the standard TUBE test, no significant difference in HI titers could be observed when all methods and diluents were statistically compared. As emphasized by Leiderman and Mogabgab (5), the savings gained by the use of microtechniques should not justify their use in place of the standard TUBE method unless sufficient statistical data on the reproducibility and sensitivity of these newer methods warrant it. Although only a limited number of replications were performed, our statistical analyses indicate that the AUTO microtechnique is a highly reproducible technique for the determination of HA and HI titers. Furthermore, this reproducibility can be increased with the use of the Autopipetter which standardizes the volume of diluent in each well, reducing errors in performing the serial twofold dilutions. Our tests with the Autopipetter revealed minimal antigen carryover when PBS/GEL and PBS /BPA were the diluents. Statistical analyses on the three diluents in the HA and HI tests showed PBS/ GEL, closely followed by PBS/BPA, to give the most accurate and reproducible results in the determination of both HA and HI titers. The conservation of time and reagents, as well as the practicability and reproducibility of the AUTO microtechnique, particularly when used in conjunction with the Autopipetter for large-scale influenza HA and HI testing, as well as its applica-tion for CF tests, supports its use. However, since the results obtained by using the MANUAL microtechnique are the least reproducible, the conservation of time and reagents may not justify the use of this procedure in place of the standard TUBE test.

v3-fos

2019-03-10T16:04:30.662Z

1971-12-01T00:00:00.000Z

83728439

s2

Wheat mutants permitting homoeologous meiotic chromosome pairing Plants of Triticum aestivum (2n = 6x = 42) ditelocentric 5B L were treated with EMS in order to produce mutations in the 5B system by which meiotic pairing between homoeologous chromosomes is normally prevented. To check for the occurrence of mutation T. aestivum ditelo-5B L plants were pollinated with rye (Secale cereale 2n = 14) and meiosis was examined in the resulting hybrids. Wheat-rye hybrids were scored for the presence of mutants when the wheat parents were either the EMS-treated wheat plants, or their selfed derivatives, or their progenies obtained after pollination with untreated euploid individuals. Mutants were detected by each of these procedures and mutant gametes were produced by the treated ditelocentric plants with frequencies between 1-5 and 2-5 %, but there were differences between the mutants in the extent to which homoeologous pairing occurred in the derived wheat-rye hybrids. The differences may have resulted from the occurrence of mutation at different loci or to different extents at the same locus. Two mutants, Mutant 10/13 and Mutant 61, were fixed in the homozygous condition. Mutant 10/13 was made homozygous both in the 5B L ditelocentric and in the euploid conditions but these genotypes regularly formed 21 bivalents at meiosis, and there was no indication of homoeologous pairing although the mutant 10/13 gave rise to homoeologous pairing in wheat-rye hybrids. INTRODUCTION A genetic activity of chromosome 5B of the bread wheat (Triticum aestivum 2n = 6x = 42) is responsible for the restriction of chromosome pairing at meiosis to fully homologous partners (Riley & Chapman, 1958;Riley, 1960;Riley & Law, 1965). When this activity is removed, or suppressed genetically, homoeologous chromosomes, which are genetically and evolutionarily related chromosomes of different genomes, will pair with each other. The effect of chromosome 5B on meiotic chromosome pairing is, therefore, responsible for the meiotic stability and disomic inheritance of hexaploid wheat. The long arm alone of the chromosome (5B L ) is responsible for the prevention of homoeologous pairing. This was shown from the behaviour of haploid plants which had either the complete euhaploid complement of 21 chromosomes or which were deficient for chromosome 5B or for its long arm or its short arm separately (Riley & Law, 1965). Chromosome pairing at meiosis in the deficiency of the short arm A. M. WALL, RALPH RILEY AND VICTOE CHAPMAN resembled that in the euhaploid, whereas pairing in the deficiency of the long arm resembled that in 20-chromosome haploids lacking the entire chromosome. A major restriction in the study and exploitation of this sytem is that the pattern of pairing can only be modified by the presence or absence of the entire chromosome or its long arm. Although it has been assumed that the effect derives from the activity of a single locus, allelic variation is very rare in nature. Nakajima (1952Nakajima ( , 1956 has described the meiotic behaviour of a T. aestivum x Secale cereale hybrid with a much higher level of chromosome pairing than its sibs. It seems reasonable to conjecture that this hybrid arose from a fertilization in which the wheat gamete carried a mutant condition permitting homoelogous pairing. Apparently, therefore, allelic variation can occur in the system controlling such pairing. An additional example of the spontaneous origin of allelic variation occurred in this laboratory in a progeny of ten hybrids from the cross T. aestivum var. Chinese Spring and S. cereale. The hybrids from a single pollination segregated to give the ratio 5 with to 5 without homoeologous pairing, indicating that the wheat parent apparently had been heterozygous at a locus affecting pairing. Unfortunately it was not possible to recover the mutant allele and no controlled study of allelic variation in this system has yet been made. Attempts have therefore been made to isolate induced mutants so that the analysis of the formal genetics of the 5B system could be completed. Such mutants would also provide genotypes for use in practical work on wheat improvement and for the investigation of the causal basis of the alternative functional conditions leading to entirely homologous or to homologous and homoeologous meiotic pairing. This paper describes work directed towards the isolations of mutants and a subsequent paper (Wall, Riley & Gale, 1971) will describe the genetics of one mutant more fully. The mutants for which selection was practised were those in which the genetic regulation of meiotic chromosome pairing had been modified to permit homoeologues to pair. MATERIAL The wheatparental plants used in this work were all derivatives ofTriticum aestivum L. emend. Thell ssp. vulgare Mackey variety Chinese Spring (2n = 6x = 42) in which either chromosome 5B was complete and disomic or in which this chromosome was represented disomically by the telocentric for its long arm (ditelo (5B L ), the short arm being entirely absent (Fig. la)). The rye parental plants were Secale cereale L. variety Petkus. Aegilops longissima Schwein. and Musch. (2n =14) was also used. METHODS The mutagen used was ethyl methanesulphonate (EMS). Samples of 50 dry seeds of T. aestivum ditelo-5B L were soaked in 50 ml of 1-0 or 0-5% aqueous solutions of EMS for periods of either 16 or 24 h at room temperature. Immediately following this treatment the seeds were washed in running tap water for 15 min. The treated seeds were then planted out in seed trays in conditions of continuous light and at a constant temperature of 20 °C. The aim was to use treatments that resulted in 50 % A. M. WALL, RALPH RILEY AND VICTOR CHAPMAN lethality before or immediately following germination, since this was expected to give the highest rate of mutation. The initial scoring, to detect the occurrence of mutation, was carried out on T. aestivum x S. cereale hybrids using temporary aceto-carmine squashes of anthers with pollen mother cells at first metaphase of meiosis. Normally in these hybrids there is a very low level of chromosome pairing at meiosis (Fig. lc). By contrast, in otherwise similar hybrids, but lacking chromosome 5B, pairing is considerably increased (Riley, 1960). The distinction in the meiotic behaviour of wheat-rye hybrids with and without chromosome 5B is so obvious that it can be detected at low-power (x 60) on the light microscope. The purpose of the initial sieve of potential mutants was to detect hybrids that, although still carrying 5B L , resembled hybrids lacking chromosome 5B in meiotic pairing. This system removed the need to search for homoeologous meiotic pairing at the 42-chromosome level in T. aestivum where detection would have been made much more difficult. When hybrids appeared to have higher than normal levels of chromosome pairing other anthers from the same plants were fixed in acetic-alcohol and stained by the Feulgen procedure. First metaphase of meiosis was then scored on permanent slides of anther squashes. The same methods were used for the staining and squashing of all the materials from which scores were taken to record meiotic behaviour. Determinations of somatic chromosome constitutions were made from squashes of root-tips that had been pretreated with mono-bromonaphthalene and stained by the Feulgen procedure. SPONTANEOUS VARIATION IN THE 5B SYSTEM Before an attempt was made to induce mutation in the system restricting meiotic chromosome pairing to full homologues, an assessment was made of the frequency of such variants in an untreated wheat stock. One spike on each of 230 plants of T. aestivum Chinese Spring euploid was pollinated with 8. cereale Petkus and a second spike was bagged to ensure self-pollination. The progeny obtained by self-pollination were intended to be used in the recovery of any variants with unusual meiotic pairing that might be revealed from the behaviour of T. aestivum x 8. cereale hybrids. Altogether 990 wheat-rye hybrids derived from the 230 wheat parents were examined at meiosis and of these 988 had extremely low levels of chromosome pairing. The mean chromosome pairing of two plants typical of this majority is shown in Table 1 (plants 26/3 and 82/5). This is the pattern of pairing normally to be expected in wheat-rye hybrids. In the other two hybrids, 92/3 and 179/2 (Table 1), bivalents ranged from one to six per cell, trivalents from zero to three per cell and there was an occasional quadrivalent. This pattern of pairing resembles that in wheat-rye hybrids lacking chromosome 5B (Riley, 1960) so that it may be inferred that modification had occurred in the 5B system contributed by the wheat gamete to the hybrids. One of these hybrids (179/2) had only 27 chromosomes and, although chromosome 5B was not structurally recognizable in this material, it was assumed that deviation from the usual pattern of meiotic pairing arose from the absence of this chromo-some. The other hybrid (92/3) with higher meiotic pairing had 28 chromosomes but the presence of chromosome 5B could not be confirmed. The wheat gamete involved in the fertilization from which this hybrid arose, therefore, either carried a mutant allele removing the restriction on homoeologous pairing or was simultaneously deficient for chromosome 5B and disomic for a compensating homoeologue. The two deviant hybrids were derived from different parental plants of T. aestivum. There were fourteen sibs of the deviant hybrid 92/3 derived from the same cross but all had the normal low level of pairing. There was one hybrid sib of hybrid 179/2 and this also had the normal low pairing. In addition it was not possible to detect a genetic condition leading to higher levels of meiotic pairing when the selfed progeny of the T. aestivum parents of these hybrids were checked in crosses with 8. cereale. The breakdown in the mechanism preventing homoeologous pairing that is assumed to have occurred was therefore a property only of hybrids 92/3 and 179/2 and possibly of the wheat gametes from which they arose. Apparently the abnormal genetic condition was not transmitted from the wheat parents concerned, so that in two of 990 wheat eggs or in the derived zygotes, genetic changes occurred affecting homoeologous pairing, that could be detected in wheat-rye hybrids. One of these events certainly involved the origin of aneuploidy and the other could have been mutation involving allelic substitution or structural change too slight to be detected cytologically. This survey confirmed that spontaneous change can occur in the genetic system by which meiotic chromosome pairing is normally restricted in wheat. However, such change is relatively infrequent and to improve the probability of isolating genetic variants in the 5B system it was necessary to induce mutation. DIRECT DETECTION OF MUTATION Seeds of T. aestivum ditelocentric 5B L were treated with EMS and survivors of those treatments that resulted in about 50 % lethality were grown on and pollinated with S. cereale pollen. Altogether, 75 wheat female parents gave rise to 206 T. aestivum x S. cereale hybrids which were grown in a glasshouse at about 20 °C. Chromosome pairing in the 28-chromosome, 5B L telocentric hybrids was checked at first metaphase of meiosis. There was a low level of pairing with only rare bivalents in 199 hybrids (Table 2). Three 27-chromosome hybrids (9/1, 72/17 and 83/11), all 316 A. M. W A L L , R A L P H R I L E Y AND VICTOR CHAPMAN lacking the telocentric marking chromosome 5B L , had much higher levels of pairing, with from one to eight bivalents and zero to six trivalents per cell (Table 2). This pattern is characteristic of that of T. aestivum x S. cereale hybrids lacking chromosome 5B, and the absence of the telocentric indicated that chromosome 5B was not present in these three hybrids. One hybrid (9/2) had 28 chromosomes but the 5B L telocentric was replaced by a small centric fragment which was presumed to be a breakage product of the telocentric. The hybrid had a level of pairing at meiosis close to that of some 5Bdeficient hybrids. From this it is concluded that the region of chromosome 5B normally responsible for the prevention of homoeologous pairing had been deleted and if this were so it would suggest that the effective region is not immediately adjacent to the centromere. Three hybrids (5/12, 8/12 and 81/15) had 28-chromosomes, including an apparently normal telocentric 5B L , and yet displayed higher than normal levels of meiotic pairing (Table 2). Pairing was similar to that in hybrids deficient for chromosome 5B and the breakdown in the normal limitation of pairing was probably due to a mutation in the 5B system of pairing regulation. Mutation may have occurred at a critical locus on 5B or at an inhibitor locus anywhere in the genotype that interacted with the 5B activity. Irrespective of their precise nature, the mutants detected in this limited experiment suggested that, with the EMS treatment employed, mutation rate in the overall 5B system was quite high at about 1-5 %. Of course it is recognized that small deletions may have been misinterpreted as gene mutations in these instances but in the present material these alternatives would be indistinguishable. Since EMS is less likely to cause deletion it is probable that the changes observed arose by allelic substitution. All three mutant hybrids, 5/12, 8/12 and 81/13, were derived from different wheat parents and were therefore of independent origin. The wheat-rye hybrids were sterile so that the mutant condition could not be recovered from them. Con-sequently an attempt was made to recover the mutants from the progenies obtained by self-pollinating the T. aestivum parents of the mutant hybrids. Eight plants were grown from each of these three progenies. All 24 plants were checked at meiosis and found to have regular bivalent formation and all were pollinated with S. cereale pollen to give 24 wheat-rye hybrid families all of which contained at least five plants. Altogether 159 wheat-rye hybrid plants were checked at meiosis and all had low levels of pairing like normal wheat-rye hybrids. The mutant conditions had not, therefore, been transmitted to the selfed progenies and it was presumed that the EMS-treated parental plants had been sectorial for the mutant states which had been transmitted only to the hybrids in which they were detected. In spite of the failure to recover and retain a mutant the initial exploration provided a guide to the methods that could be used in the isolation of mutants. Clearly the EMS treatment was highly mutagenic and a different breeding procedure would increase the probability of holding mutants in the 5B system. INDIRECT DETECTION OF MUTATION The inflorescences of the ditelocentric 5B L parents, from which wheat-rye hybrids 5/12, 8/12 and 81/15, were derived, were apparently sectorial for mutant conditions affecting meiotic pairing since only one hybrid was mutant in the progeny of each. The progeny sizes were respectively 6, 6 and 4, and, of course, the occurrence of other mutant sectors might have gone undetected in wheat-rye hybrid families derived from other EMS-treated parents. To examine this possibility progenies were grown, each of six M 2 plants, that had been derived by selfing eight EMS-treated M x parents. All 48 M 2 plants were pollinated with rye and 42 families of wheat-rye hybrids resulted. There was very low pairing at meiosis in all 209 hybrids examined in these families and no evidence that their ditelocentric 5B wheat parents had been mutant in the 5B system nor that the EMS treated plants had carried mutant sectors. A larger progeny of 27 M 2 plants derived, by selfing, from EMS-treated ditelocentric 5B plant 10, was also grown and each plant was pollinated by rye to give 662 hybrids. Meiotic chromosome pairing was scored in 133 of these with the results indicated in Table 3. There were high-pairing hybrids, presumably with homoeologous pairing, in 12 families, while 14 families contained low-pairing hybrids only. One family (10/1) contained hybrids with high and with low pairing, but those with high pairing all had 27-chromosomes and were deficient for the 5B L telocentric so that the M 2 wheat parent was apparently monotelocentric 5B L . Consequently this plant and its family of hybrids with rye will be discounted from any further consideration. The occurrence of 28-chromosome, 5B L telocentric, wheat-rye hybrids with homoeologous pairing indicates that mutation had apparently occurred in EMStreated, 5B L ditelocentric, plant 10. Its selfed M 2 progeny contained plants which, on the basis of the sometimes limited families derived from rye pollinations, were 318 A. M. W A L L , RALPH RILEY AND VICTOR CHAPMAN diagnosed as having been homozygous non-mutant, heterozygous, or homozygous mutant. Where segregation occurred the heterozygous status of the M 2 parent was clearly determined, assuming no misclassification, but the family size was often too small for homozygosity in the M 2 parents to be diagnosed with certainty. Nevertheless there was an excess of homozygous non-mutant M 2 parents. The selfed spike from which the M 2 progeny of EMS-treated plant 10 were derived was probably sectorial for the mutant condition, but even so the deficit of mutant homozygotes relative to heterozygotes cannot readily be explained although it may have arisen from a selective advantage of the non-mutant condition. A major difficulty was the possibility of misclassification in this generation because of the effects of temperature variation in the glasshouse. Only the wheat-rye family 10/13, containing 11 hybrids, suggested that the M 2 parent from which it was derived might have been homozygous for the mutant condition and there was uncertainty about this because of environmental variation. In wheafc-rye family 10/13 there were differences between individual hybrids in the level of meiotic pairing (Table 4). Some hybrids (10/13/12, 10/13/5 and 10/13/6) had chiasma frequencies approaching those in hybrids deficient for 5B, while in others pairing was considerably lower. This variation raised doubts about the homozygosity of M 2 plant 10/13 so it was necessary to test its progeny derived by self-pollination. For this purpose 35 M 3 plants derived from 10/13 were grown and pollinated with rye. A total of 350 wheat-rye hybrids derived in this way were grown and 176 of these were examined at meiosis mostly in a controlled environment chamber held at 20 °0 with continuous light (Table 5). The majority of hybrids displayed high pairing approaching that of wheat-rye hybrids lacking chromosome 5B but there were also some with much lower pairing. Some family sizes were too small for judgements to be made about the status of their wheat parents, but, ignoring those with less than four hybrid plants, the segregation indicated that the M 3 had segregated into homozygous mutant and non-mutant and into heterozygous categories. Apparently, therefore, the M 2 plant 10/13 was heterozygous. A. M. W A L L , RALPH R I L E Y AND VICTOR CHAPMAN The wheat-rye hybrid family derived from M 3 plant 10/13/8 consisted of nine plants all of which had high pairing ( Fig. Id; Table 6). Subsequent testing of the selfed progeny of M 3 plant 10/13/8 showed it to have been homozygous for the mutant condition affecting meiotic pairing. A homozygous mutant line was thus derived and was designated Mutant 10/13 (Fig. 2). Although there was distinctly higher meiotic chromosome pairing in hybrids between Mutant 10/13 and S. cereale than in normal wheat-rye hybrids it was considered necessary to check that the use of Mutant 10/13 genuinely permitted chromosomes to pair that were normally precluded from doing so in such hybrids. It should be realized that S. cereale had been used in the recognition of mutants primarily because it crossed readily with wheat and at the same time gave rise to hybrids in which disturbances could be detected in the normal restriction of chromosome pairing, but the differences between mutant and non-mutant forms was not large. Consequently a test was needed in which the distinction between the normal operation, and ineffectiveness, of the 5B L system would be readily apparent. For this purpose comparisons were made between hybrids of T. aestivum ditelocentric 5B L non-mutant, or mutant 10/13, with Aegilops longissima (2n = 14). This test was chosen because of the large differences in the level of meiotic chromosome pairing in hybrids of T. aestivum x Ae. longissima with and without chromosome 5B (Riley, Chapman & Kimber, 1959 Non-mutant plants of T. aestivum ditelocentric 5B L and plants of Mutant 10/13 also ditelocentric 5B L were therefore crossed with Ae. longissima and the resulting hybrids examined at meiosis. There was very little chromosome pairing in hybrids derived from non-mutant parents, indeed there was a mean of no more than 1*16 chiasmata per cell (Fig. le). Whereas in hybrids derived from Mutant 10/13 pairing was much higher with mean chiasma frequencies ranging from 5-40 to 12-05 per cell (Fig. 1/; Table 7). The difference between the two types of hybrid with Ae. longissima emphasized the marked effect on meiotic pairing of Mutant 10/13. This effect, like that arising from chromosome 5B L deficiency, is more readily demonstrated in hybrids with Ae. longissima than with S. cereale. Table 8 together with the score for a non-mutant control. Mutant 10/13 plants never had any multivalents in the way that might have been expected in genotypes in which there was reduced meiotic isolation of homoeologues (Fig. 16). There may have been some reduction in chiasma frequency in Mutant 10/13 but the experiment was inadequate for this to be determined unequivocally. The short arm of chromosome 5B (5B S ) promotes meiotic chromosome pairing and was absent from the ditelocentric 5B L Mutant 10/13 examined. It seemed possible that the presence of 5B S in Mutant 10/13 might increase pairing to a level at which multivalents would occur due to the association of homoeologues, so the mutant condition was made homozygous in a euploid chromosome complement. MUTANT 10/13 IN A EUPLOID CHROMOSOME COMPLEMENT T. aestivum ditelocentric 5B L and homozygous for the Mutant 10/13 condition was pollinated by T. aestivum euploid to give derivatives heteromorphic (telocentric 5B L and complete) in the 5B pair and heterozygous for the mutant state. These F 1 derivatives were self-pollinated and an F 2 progeny was grown in which plants with euploid chromosome constitutions -that is, without 5B L telocentricswere selected. The genotypes of these F 2 plants were tested in hybrids obtained by pollinating them with rye. Ten wheat-rye families were grown and 112 hybrids in these families were scored at meiosis for the presence of the normal or mutant phenotype. Eight famines segregated, containing hybrids with low and others with high levels of pairing, so their T. aestivum parents were diagnosed as having been heterozygous. Two families, one containing 15 hybrids and the other 13 hybrids, were entirely high pairing and their T. aestivum parents were diagnosed as having been homozygous for the mutant 10/13 condition. Selfed progeny were available from these plants so Mutant 10/13 was established in the euploid chromosome complement. Euploid Mutant 10/13 homozygotes were entirely regular at meiosis. Many plants of this genotype were examined but there were no multivalents although these might have been expected in a genotype with a reduced barrier to homoeologous pairing (Table 9). Apparently the effect of Mutant 10/13 on meiotic pairing was only detectable in hybrids like those with S. cereale or Ae. longissima in which there were no fully homologous partner chromosomes. In terms of its effect on meiosis in T. aestivum, Mutant 10/13 could be regarded as being neutral to selection. However, such alleles are not common in nature so it may be that there are other disadvantageous phenotypic effects that either do not disturb meiosis or do so under environmental conditions different from those used in the present work. When EMS-treated plants were crossed with rye to detect the occurrence of pairing mutations the mutant condition was lost because of the sterility of the resulting wheat-rye hybrids. Searching for pairing mutants in the progeny obtained by selfing EMS-treated plants increased the labour because of segregation. A further breeding system was consequently examined in an attempt to avoid these difficulties. In this system plants of T. aestivum ditelocentric 5B L were treated with EMS and subsequently pollinated with untreated euploid T. aestivum. The products of this cross were potentially heterozygous for mutant conditions and heteromorphic for chromosome 5B, with one complete and one telocentric member. These plants were pollinated with rye and the resulting hybrid families examined at meiosis for deviation from the usual very low level of chromosome pairing indicating the occurrence of mutation. Of course single locus heterozygotes would be expected to produce wheat-rye hybrid families segregating in a 1:1 ratio. The progeny derived by selfing plants whose heterozygous status had been detected in wheat-rye hybrid families could then be searched for homozygotes. Applying this system, 233 heteromorphic plants were obtained from the cross EMS-treated ditelocentrics x euploid. All had 42 chromosomes with one telocentric and 118 of them were pollinated with rye to give hybrid families. From these 118 families 854 wheat-rye hybrids were scored for the level of chromosome pairing at first metaphase of meiosis. In all, except four, of the famines the hybrids had the very low level of pairing 'expected in normal wheat-rye hybrids with only occasionally a bivalent, and with mean chiasma frequencies never exceeding 0-80 per cell in the plants scored in detail. In the other four families there were plants with unusually high pairing, with from one to six bivalents, up to two trivalents, and occasionally a quadrivalent. One of these families (112) contained six plants 324 A. M. WALL, RALPH RILEY AND VICTOR CHAPMAN all with 27 chromosomes and it was assumed that chromosome 5B was deficient (Table 10). Family 53 contained four 28-chromosome wheat-rye hybrids two of which had the usual low level of pairing while the other two had distinctly higher pairing. Although the pairing in these plants, which had mean chiasma frequencies of 2-75 and 3*45 cell, was not as high as that in hybrids deficient for chromosome 5B it seems likely that 5B heteromorphic plant 53 was heterozygous for a mutant condition (Table 10). Family 6 had six plants, all with 28 chromosomes, including 5B L telocentric in two and 5B complete in four plants. All six hybrids had a level of meiotic chromosome pairing higher than that in normal hybrids, but lower than that expected in the deficiency of chromosome 5B (Table 10). Consequently heteromorphic plant 6 was probably heterozygous for a mutant condition affecting meiotic pairing, but like that carried by plant 53 its effect on pairing was less profound than that arising from the absence of chromosome 5B or from the presence of Mutant 10/13. The fourth variant family (61) contained hybrids with levels of pairing comparable with standard wheat-rye hybrids and others with chiasma frequencies close to, but still somewhat below, those of 5B-deficient hybrids (Table 11). It seems probable that the heteromorphic plant 61 was heterozygous for a mutant in the system regulating homoeologous pairing and that the variation in pairing in the wheat-rye family arose from environmental variation in a mutant genotype in which there was an incomplete breakdown in the isolation of homoeologues. Only two of the hybrids in family 61 carried the 5B L telocentric and both of these were in the low pairing category. In an .attempt to retain the mutant condition present in the apparently heterozygous plant 61, its progeny obtained by selfing was grown and pollinated by rye. Wheat-rye hybrid progenies were obtained from 12 selfed derivatives of plant 61. A total of 96 hybrids in these families was examined at meiosis. Segregation occurred into high-and low-pairing categories which had chiasma frequencies like those of the corresponding segregants among the hybrids of plant 61. The low-pairing hybrids had mean chiasma frequencies less than 0-76 and the high pairing hybrids had mean chiasma frequencies between 2-23 and 4-70, in the plants scored. The segregation of hybrids, with high or low pairing and with the complete chromosome 5B or telocentric 5B L , is shown in Table 12. Considered overall the ratio was 75 low:21 high pairing and 73 complete:23 telocentric 5B. Apparently, although a high-pairing mutant condition was transmitted to the selfed progeny of plant 61 it was not inherited in the 1:1 ratio expected if mutation had occurred A. M. WALL, RALPH R I L E Y AND VICTOR CHAPMAN at a single locus. Also, as expected because of the poorer competitive ability of pollen carrying the telocentric relative to that with the complete 5B, the telocentric was less frequent than the complete chromosome in the progeny of plant 61. Because of the confirmed mutant status of this material it was subsequently named 'Mutant 61'. The status of the selfed derivatives of plant 61 can be inferred from the status of wheat-rye hybrid progenies to which they gave rise, although some progenies contained too few individuals for them to be an accurate guide to the genotypes of their wheat parents. Seven wheat-rye progenies were entirely non-segregating and low pairing, eight segregated and one was non-segregating and high pairing. If plant 61 had been heterozygous at a single locus and the alleles had segregated at random into its selfed progeny a 1:2:1 ratio would have been expected in these three categories of wheat-rye families. The observed segregation was consistent with plant 61 having been heterozygous at two loci with duplicate effects, such that only wheat-rye hybrids carrying mutant alleles at both loci had higher than normal pairing. However the segregation in the initial wheat-rye family derived from plant 61 was not consistent with this hypothesis. On the basis of the series of wheat-rye families only plant 61/17 appeared to have been homozygous for the mutant 61 condition but tests on the selfed progeny of this plant demonstrated that it too was heterozygous. Subsequently, although the mutant condition of family 61 was confirmed by the higher than normal pairing of T. aestivum x Ae. longissima hybrids (Table 13), further work was concentrated on Mutant 10/13 because the lack of clarity in the inheritance of Mutant 61 and because of its lesser effect as judged by the level of meiotic chromosome pairing in Mutant 61 x rye hybrids. The three mutants recognized in plants from the cross EMS-treated ditelocentric 5B L x euploid wheat had increases in the level of pairing in wheat-rye hybrids but the level did not attain that of 5B-deficient hybrids. Nevertheless three mutants were observed among the 118 gametes examined, which is a mutation rate of about 2-5 % compared with a rate of about 1-5 % in the work described earlier. 11. DISCUSSION Okamoto (1962Okamoto ( , 1966 has already shown that mutants can be recognized in T. aestivum that disrupt the normal isolation of homoeologous chromosomes at meiosis. These mutants were detected following X-ray treatments but none was retained so that no evidence was provided on the chromosomal location of the loci involved. Okamoto's investigation of the SB problem by induced mutation was, apparently, the first attempt to study meiotic chromosome pairing in this way. Most other induced mutants affecting meiotic pairing have been isolated fortuitously in general studies of mutation. In the present work, mutants with increased levels of homoeologous pairing were isolated with frequencies of between 1-5 and 2-5% among the gametes examined from EMS-treated germinating seeds. Although this frequency is high it is lower than that observed by Okamoto (1962) following X-irradiation of almost mature wheat. This higher frequency may have resulted from the greater numbers of deletions resulting from X-ray treatment or from the reduced somatic competition to which mutant cell lines were exposed, when mutation occurred immediately before meiosis. From the present work it is clear that mutants affecting homoeologous meiotic pairing can be isolated and retained in wheat. However, somewhat tedious systems are required for their recognition and for the progeny-testing required to establish homozygosity. The need to use wheat-rye hybrids to determine the genotypes of the wheat parents makes this work extremely laborious. In the present work, at all stages of the programme, meiosis was examined in 2935 wheat-rye hybrids, which is probably a larger number of hybrids than has been reported on in the entire literature on the cytogenetics of the wheat-rye combination. The chromosomal location of Mutant 10/13 is discussed in a subsequent paper (Wall, Riley & Gale, 1971) but it is appropriate at this stage to allude to one characteristic of this genotype. Although in hybrids between Mutant 10/13 and either S. cereale or Ae. longissima there is considerable homoeologous chromosome pairing at meiosis, there is no evidence that homoeologues pair in homozygotes for the mutant condition in T. aestivum irrespective of whether 5B is represented by the long telocentric or the complete chromosome. Apparently, therefore, Mutant 10/13 had no effect on the meiosis of T. aestivum. Consequently it might be expected that such genetic variants would suffer no selective disadvantage in wheat and would therefore be found in normal wheat stocks. However, if they occur at all they are not common, so it may be that there are pleiotropic effects which place them at a selective disadvantage. There were several reasons for attempting to select and fix mutants giving homoeologus pairing. One was to determine whether a single locus on 5B L is implicated in the normal restriction of this pairing. Another use for the variants was to be in breeding exercises of several kinds. Both of these aims have been or are being realized with the exploitation of Mutant 10/13.

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2018-04-03T00:44:55.948Z

1971-09-01T00:00:00.000Z

26561557

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Microbial Penetration of Muslin- and Paper-Wrapped Sterile Packs Stored on Open Shelves and in Closed Cabinets Microbial penetration of sterile packs was studied using single-wrap (two layers) muslin, double-wrap (four layers) muslin, and two-way crepe paper (single layer) to wrap 20 gauze sponges (2 by 2 inch). These packs were stored in the central ster- ile supply departments of two hospitals and processed for sterility at predetermined intervals. Microorganisms penetrated single-wrap muslin as early as 3 days and double-wrap muslin and single-wrap two-way crepe paper in 21 to 28 days stored in open shelves. The time required for microbial penetration was at least twice as long when closed cabinets were used. Single-wrap muslin packs stored in sealed, impervious plastic bags remained sterile for at least 9 months. All sterile materials in pervious wrappers should be handled as little as possible and then only with extreme care and caution. Closed cabinets offer more protection than open shelves, and single wrappers are not recommended. There are reports in the technical literature describing the length of time sterile goods can be stored and still be considered sterile, but the safe storage times reported range from as short as 1 week to indefinitely (10,11,14,15,17). To add to the confusion, some reports discuss neither the wrapping material used nor conditions for storage in relation to the safe storage periods (10,17). Dyer et al. (8) reported the shelf life to be at least 60 days for cotton applicators wrapped in parchment and muslin when stored in closed cabinets. Alder and Alder (1) found that crepe and bleached kraft paper were more effective than calico or balloon cloth. Also, they found that approximately 50 and 100% of the test swabs wrapped in double and single layers of muslin, respectively, were contaminated after 13 to 14 days of storage; 10 and 30%, respectively, were contaminated within 1 to 2 days. These investigators concluded that packs wrapped in two layers of paper and enclosed in cartons should have a shelf life of at least 3 weeks. Nichols (13) found that packs double-wrapped in muslin stored and sealed in plastic bags remained sterile for 18 months. Fitzwater (9) compared the number of viable microbial particulates settling into open petri dishes on open shelves and in a normally closed cabinet in an operating room during set up and an operative procedure. Doors of the closed cabinet were opened 24 times during the evaluation. Fewer than one-tenth as many viable particulates settled in the closed cabinet than on the open shelves. The exterior surfaces of sterile packages become contaminated during storage. Several investigators have commented on the probability of transfer of this contamination to the contents when single-wrapped sterile packages are opened (2,5,12,15,18). Speers and Shooter (16) demonstrated that sequential unwrapping of doublewrapped packages substantially reduces contamination during removal of the sterile contents. Central supply personnel have no firm scientific data on which to base selection of the most effective wrapping material or establishment of conditions for storing sterile packs; hospital central sterile supply departments (CSSD) use many types of wrappers and storage conditions vary widely. Sterile packs wrapped in muslin are considered by most hospitals in the United States to be safe if used within 1 month after sterilization, an assumption probably based on the findings and recommendations of Perkins (14). In previous studies on length of uncontaminated storage of sterile packs, the materials in the packs examined for microbial contamination were generally cotton applicators or small metal or glass objects (1,4,8,13,14). A deficiency of all of these studies was that only small portions of material inside the sterile packs were assayed for microbial contamination. The present study was designed to determine how long sterile packs of a size widely used in hospitals (15) would remain sterile when wrapped in single-wrap muslin, double-wrap muslin, and single-wrap two-way crepe paper (4,15) when stored on open shelves and in closed cabinets. MATERIALS AND METHODS Standard packs were used for studies of microbial penetration into the pack. Twenty [2 by 2 inch (5.08 by 5.08 cm)] 12-ply gauze sponges were arranged to form a pack with a surface area of 8 by 10 inches. The packs were wrapped with single-wrap muslin (two layers), double-wrap muslin (each two layers), or single-wrap (single layer) two-way crepe paper. A Kilit Ampule containing resistant bacterial spores was placed inside each pack as a check on the effectiveness of sterilization. All packs were autoclaved in a conventional steam sterilizer for 1 hr at 121 C. Cloth wrappers used for the standard packs were 140 thread-count muslin, unbleached, dyed green, laundered, and ironed at least 1 to 10 times before use. The paper wrappers used were commercially available two-way crepe paper (Dennison Wrap). Both types of wrappers were approximately 24 by 24 inches (61 by 61 cm). After sterilizing and drying, the packs were held overnight in the autoclave with the steam supply turned off to allow the packs to cool. The packs were then removed and placed in sealed, sterile 8-mil polyvinyl chloride (PVC) bags [25 by 35 inches (63 by 89 cm)]; transported to the CSSD of the two hospitals; removed from the PVC bags; and placed on shelves in the same areas that the hospitals' sterile supplies were stored. On the same day, three packs wrapped in each type of wrapper were chosen at random and transported back from the hospitals to the laboratory for an initial control assay to confirm that the packs were not contaminated during transportation. The remaining test packs were picked up in pairs at selected time intervals and transported back to the laboratory for microbiological assay. All packs returned from the hospitals were transported in sealed, sterile, 3-mil polyethylene bags [9.5 by 18 inches (20 by 46 cm)]. Relative humidity and temperature were monitored in the two CSSD throughout the study by using 7-day recording hygrothermographs. Calibration for accuracy of these instruments was checked at weekly intervals by using a sling psychrometer. Hospitals were used for this study to provide locations for storage of test packs under actual institutional rather than laboratory conditions. Hospital no. 1 is a 100-bed pediatric hospital; the CSSD of the hospital has four employees on the day shift on weekdays and one on each of the other shifts. Hospital no. 2 is a 350-bed hospital; the CSSD had 14 employees on the day shift on weekdays and at least one on all other shifts. Storage shelves with enclosed backs and sides, but with the fronts open, were used in hospital no. 1. The distance between shelves ranged from 11 to 14 inches (28 by 36 cm). No packs were less than 16 inches (31 cm) from the floor. Some shelving completely open on all sides and some shelving with enclosed backs were used in hospital no. 2. The shelves were spaced 10 to 13 inches (25 by 33 cm) apart, and no packs were less than 24 inches (61 cm) from the floor. In addition, closed metal cabinets measuring 36 by 78 by 18 inches (91 by 198 by 46; width by height by depth) also were set up in hospital no. 2 for this study. The shelves were arranged 9 inches (23 cm) apart; no packs were less than 13 inches (33 cm) to the floor. A silent electrical counter was installed to record the number of times the cabinets were opened and closed. Test packs were picked up at various times during the first week of the study: days 1, 2, 3, and 4 for study series 1 (hospital 1); days 1 and 6 for study series 2 and 3 (hospital 2); days 1 and 3 for study series 4 (hospital 1); and day 3 for study series 5 (hospital 2). Thereafter, weekly pick-ups were made in all study series. The first pick-up for study series 6 (hospital 2) was on day 7. At each pick-up, two sterile packs used as transportation controls were wrapped in singlewrap (two layers) muslin, transported to the hospital in sterile plastic bags, and then returned to the laboratory along with and in the same manner as the test packs. Series 1, 2, and 3 were done during cold months, and series 4, 5, and 6 were done during hot months. Immediately on arrival at the laboratory, all packs were processed inside a closed laminar-flow hood (40 by 20 by 40 inch (102 by 51 by 102 cm) ]. The hood was decontaminated before each daily use with 70% ethanol and purged and dried for at least 30 min at a rate of three air changes per minute (ac/min) through an ultra-high-efficiency air filter (6). While packs were being processed, the airflow was set at a rate of 1 ac/ min. The hood was purged for at least 2 min at a rate of 3 ac/min between processings of each pack. After the packs were opened, seven of the sponges were placed in each of two screw-cap serum bottles containing 100 ml of Trypticase soy broth (TSB, BBL) and incubated aerobically at 37 C. The remaining six sponges were placed into another serum bottle containing 100 ml of TSB and incubated under anaerobic conditions in a Brewer jar at 37 C. All cultures were incubated for 21 days before being considered negative for viable microorganisms. An estimate of the amount of viable surface contamination that may have collected on the outside of the packs was made by using stainless-steel strips [1 by 2 inches (2.54 by 5.08 cm)]. Using these strips, two evaluations were made of open shelves versus closed cabinets in hospital 2: one was carried out in the cold months (series 2 and 3), and the other was carried out during the hot months (series 5 and 6). Strips were placed on a stainless-steel tray wrapped in doublethick aluminum foil and sterilized in a hot-air oven at 150 C for 3 hr. The trays with strips were transported to the hospital with the test packs. The trays of strips were placed on the same shelves with the packs and opened. Five randomly selected strips were collected for microbial assay when each set of packs was picked up for examination. Each strip was aseptically placed into a sterile 4-oz specimen jar and returned to the laboratory. Upon return to the laboratory, 50 ml of TSB was aseptically added to each jar, and the jars were vigorously shaken for 5 min on a wrist-action shaker. Just before assay, each jar also was handshaken 50 times. Sets of two pour plates each for aerobic and for anaerobic incubation were prepared by using 5-ml sample amounts mixed with 10 to 15 ml of Trypticase soy agar (TSA). The remaining sample in the jars was then heat-shocked for 15 min at 80 C, and four additional pour plates were prepared, as described above, again for aerobic and anaerobic incubation. Aerobic incubation was carried out in a waterjacketed incubator at 37 C, and anaerobic incubation was carried out in Brewer jars at 37 C. Colonies on the plates were enumerated after 48 hr and after 7 days of aerobic incubation and after 7 days of anaerobic incubation. Concurrent with these studies, single-wrap (two layers) muslin packs prepared and sterilized as previously described were sealed in sterile 3-mil polyethylene bags (0.5 by 18 inch). Eight packs were stored in each hospital CSSD on shelves that collected the most dust. Two packs were examined at 1, 3, 6, and 9 months for assay as previously described. In an attempt to determine depth of penetration of contamination, six single-wrap (two layers) muslin packs containing nine stacks of 15 sponges per stack, sterilized as previously described, were placed on open shelves with other sterile supplies in the CSSD of hospital no. 2 for a period of 5 weeks. The first nine sponges were placed in separate 2-oz screw-cap bottles containing 25 ml of TSB. These specimens were incubated aerobically at 37 C for 21 days before being considered negative for viable microorganisms. RESULTS Microbial contamination was determined from single-wrap (two layers) muslin packs, doublewrap (each two layers) muslin packs, and singlewrap (single layer) two-way crepe paper packs. Table 1 shows the first time in days at which each type of pack was found contaminated in the openshelf study in both hospitals; contamination occurred as early as 3 days after initiation of storage with single-wrap muslin and in 21 and 28 days (first noted at 28 days) with double-wrap muslin or single-wrap two-way crepe paper. Results from the closed-cabinet studies (series 3 and 6) are shown in Table 2. Contamination first became apparent at 14 days for single-wrap muslin and at 56 days for double-wrap muslin, and no contamination was found for at least 63 days with two-way crepe paper. Series 1, 2, and 3 were done during cold months, and series 4, 5, and 6 were done during hot months. Series 1 and 4 were done in hospital no. 1; 2, 3, 5, and 6 were done in hospital no. 2. The time interval in which contamination first became apparent in single-wrap muslin packs was shorter in hot months than in c3ld months; however, the double-wrap muslin and single-wrap two-way crepe did not appear to follow this pattern. Averages of weekly average indoor temperatures in the CSSD did not vary significantly between the hot (25 C) and the cold (25.5 C) months and only slightly between hospital 1 (27.2 C) and hospital 2 (24.4 C). The CSSD in hospital 1 was more humid in the summer and less humid in the winter than the CSSD of hospital 2. Averages of weekly average indoor relative humidities were about 35% in the studies during the cold months and about 48% in the studies during the hot months. Table 3 shows the total microbial counts from stainless-steel strips exposed in two evaluations of open shelves versus closed cabinets in hospital 2. The microbial counts were calculated on the basis of an area measuring 80 square inches, equal to the surface area of the packs used in the study. The doors on the closed cabinets were opened (and closed) an average of 28 times per day during the investigation. The results of these evaluations showed that only about one-tenth as much viable microbial contamination settled onto horizontal surfaces in the closed cabinets as on the open shelves. About 50 % of the settled microorganisms were aerobic and not heat-shocked, about 20% were anaerobic and unshocked, over 10% were molds, and less than 10% each were aerobic and anaerobic, heat-shocked organisms. bNo packs or strips processed. Table 4 shows the types and frequencies of organisms isolated from contaminated packs. About half of the organisms isolated were grampositive rods; the next most frequently isolated organisms were staphylococci, Aspergillus spp., and Micrococcus spp. The single-wrap (two layers) muslin packs sealed in 3-mil polyethylene bags remained sterile throughout the 9-month study. Examination of the six single-wrap muslin packs used to study depth of microbial contamination after 5 weeks of open-shelf storage showed penetration down to the fifth sponge in one pack. Two to seven of the nine surface sponges per pack examined were found to be contaminated. A total of 195 control packs were utilized throughout the study. Only two (1.0%), both single-wrap (two layers) muslin, were found to be contaminated. No growth was found in any of the ampoules of resistant spores placed in the packs to check the effectiveness of sterilization. DISCUSSION Many wrapping materials are commercially available to hospital CSSD today, but almost no data are available on how well these materials maintain sterility. Basically, bacteria penetrate muslin draping material much more rapidlf than they penetrate water-repellent paper drape fabrics (7). It has been shown that bacteria penetrate a surgeon's intact gown more readily and in greater numbers during surgery requiring unusual physical effort than during simple procedures (3). The same may be true of the more porous materials used to wrap sterile supplies. That is, the less the materials are handled and moved, the less likely it is for contamination to occur. It has been suggested that a change in atmospheric conditions may cause a breathing effect in packs and thus contribute to penetration of microorganisms (14). In the present study, neither the temperature nor the relative humidity varied greatly, and there were few rapid changes in either of these variables; thus, the frequency of contamination could not 435 VOL. 22, 1971 on December 9, 2020 by guest http://aem.asm.org/ Downloaded from be shown to be directly related to atmospheric conditions. Gradual increases in settled contamination on the outside of packs together with handling or vibration and possibly atmospheric changes were responsible for ultimate contamination of packs. This study utilized the entire contents of the sterile package as the assay system. Using this system, single-wrap (two layers) muslin packages become contaminated as early as 3 days, and double-wrap (each two layers) muslin and single-wrap (single layer) two-way crepe paper maintained sterility for at least 3 weeks (contamination was first found at 4 weeks) stored on open shelves. Packs stored in closed cabinets remained sterile for at least twice as long as those held on open shelves; however, even in closed cabinets, the time of sterile storage for single-wrap muslin (less than 14 days) was too short to be practical. In an effort to show a visual comparison of a single thickness of muslin and a single thickness of two-way crepe paper, a photograph at approximately 40 X magnification was made of each. Figure 1 shows the muslin; there is a visible opening through the material at almost every thread junction. Figure 2 shows the two-way crepe paper; in this figure, three dark areas near the center indicate very thin areas in the paper fiber. These photographs help demonstrate the possibility of rapid recontamination of sterile objects wrapped in a single wrap of muslin. The results of the study on the amount of microbial contamination settling on stainless-steel strips corroborate the findings of Fitzwater (9); only about one-tenth as many viable microorganisms settled in the closed cabinets as on the open shelves. Thus, the results of our studies clearly show the advantages of closed-cabinet over openshelf storage of sterile packs. Of course, cabinet doors must be kept closed except for replacement or removal of contents. Although a single thickness of two-way crepe paper was observed to maintain sterility as long as double-wrap muslin, it is not recommended that any sterile pack be wrapped with only a single thickness of material. With a single wrapper, the possibilities for contamination are greatly increased via contamination from the outside surface of the pack (16). Impervious plastic wrappers have been observed to maintain sterility for as long as 18 months (13). The 9-month period of sterile storage obtained from this study of materials sealed in polyethylene bags at least in part confirms these earlier observations. With proper rotation of stocks, no material should be held for as long as 9 months. All sterile materials in pervious wrappers should be handled as little as possible and then only with extreme care and caution. Closed cabinets offer more protection than open shelves, and single wrappers are not recommended. LrITRATURE CITED

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2018-04-03T04:15:51.920Z

1971-12-01T00:00:00.000Z

38441501

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Minimal growth temperature, sodium chloride tolerance, pH sensitivity, and toxin production of marine and terrestrial strains of Clostridium botulinum type C. Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 x 10(6) viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 x 10(6) unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin. Michener and Elliott (13), who reviewed the minimal growth temperatures of various microorganisms, found no evidence of growth of Clostridiwn botulinum type C at temperatures below 10 C. Tanner and Oglesby (20) observed growth from a vegetative-cell inoculum in a laboratory culture medium at 15 C, but not at 10 C. From a spore inoculum, they noted growth and toxin production at 20 C, but not at 15 C. However, Tanner, Beamer, and Rickher (18) obtained growth from a spore inoculum in peas and in asparagus at 10 C, but they were unable to detect growth at 5 C, the next lower temperature tested. Castell (5) obtained type C growth in ground lobster and ground cod fillets at 25 C, although he was unable to demonstrate growth in either substrate at 3 C during 3 months of incubation. Beerens, Sugama, and Tahon-Castel (2) failed to obtain growth of two type C strains in a laboratory culture medium at 6 C. Although definite proof is lacking, it appears that all C. botulinum type C strains tested for psychrophilic ability have been isolated from terrestrial sources. Because type C exists in marine environments with botulinum types known to possess low-temperature growth ability, and because of cultural similarities to those types, it was considered that marine strains might show psychrophilic growth characteristics (16). Therefore, the principal objective of this research was to compare the minimal growth temperatures of marine and terrestrial strains of C. botulinum type C. In addition, work was conducted to determine their sodium chloride tolerance, pH sensitivity, and toxin-producing ability in various culture media. MATERIALS AND METHODS Type C strains. The origin of the terrestrial and the marine strains was previously described (16). The terrestrial strains are numbered 468 and 571; the marine strains are designated 6812, 6813, 6814, and 6816. Sporulation and standardization of suspensions. Spores of each strain were produced in FEM medium (Difco Egg Meat Medium fortified with additions of 1% yeast extract, ammonium sulfate, and glucose). SEGNER, SCHMIDT, AND BOLTZ As already described (16), aqueous suspensions were prepared and standardized by a deep-tube technique with beef infusion agar (BIA). The recovery medium was supplemented with 0.1% L-cysteine hydrochloride and 0.14% sodium bicarbonate by aseptic additions immediately before use. Colony counts were made after 24 to 48 hr of incubation at 30 C. Low-temperature growth studies. FEM, autoclaved at 121 C for 15 min, was used in 20-g quantities in screw-cap tubes (20 by 150 mm). A fish substrate was prepared from fresh haddock fillets (Melanogramus aeglefinus). The fillets were finely ground and distributed in 10to 12-g amounts in screw-cap tubes (16 by 125 mm). Air pockets in the substrate produced during filling were removed by centrifuging at a low speed. Tubes for incubation at 10 C and below were heated in flowing steam for 30 min; those for incubation above 10 C were autoclaved at 121 C for 15 min. After heating and cooling in cold tap water, the tubes were dried and held at 2 C for inoculation the next day. The inoculum level used equaled 0.1 ml/tube or 2 X 106 viable spores. Prior to inoculation, the spores were preheated at 71 C for 15 min. Five-tube replicate sets were inoculated per variable. The inoculated tubes and corresponding uninoculated controls were Vaspar-sealed and incubated at 15.6 C (60F), 12.8 C (55 F), lOC (50F), and7.8 C (46F). Incubated tubes of FEM medium and ground haddock were examined for gas production on a Monday, Wednesday, and Friday time schedule. After approximately 8 months of incubation at the highest temperatures showing no gas production, each replicate set was assayed for type C toxin and examined for growth by phase-contrast microscopy. The Vaspar was removed from each tube with sterilized cotton-tipped swabs. For the haddock substrate, the contents of each tube were blended with 90 ml of sterilized, prechilled sodium acetate buffer (0.5 M, pH 5.4). About 6 ml of the blend was transfered to a sterilized tube and held overnight at about 3 C; toxin was assayed the next day. For the FEM medium, 1 ml of the liquid portion was diluted with 9 ml of gelatin-phosphate buffer and the assays were run immediately. Duplicate white mice each were injected intraperitoneally with 0.5 ml of a sample. The mice were observed for botulinum symptoms up to 4 days before a sample was considered nontoxic. Sodium chloride and pH studies. The basal medium used in the sodium chloride and pH studies consisted of beef infusion (BI) broth. Disodium phosphate normally added to the medium was deleted. In the salt experiments, various concentrations of sodium chloride were dissolved in the basal medium on a weight basis. The medium was dispensed in 200-ml quantities in screw-cap bottles and autoclaved at 121 C for 15 min. Before use, sterile L-cysteine hydrochloride was added to the medium to a final concentration of 0.1%. Sterile 1 N sodium hydroxide was added to readjust the medium to pH 7.0 to 7.2. The balance. of the loss in weight due to autoclaving was restored to the nearest gram by the addition of sterile distilled water. In the pH experiments, the basal medium was adjusted from pH 6.0 to 5.0 at 0.2 pH unit intervals by the addition of dilute HCI. The medium was bottled and autoclaved as described above. The pH values cited in the tables represent determinations made after the incorporation of 0.1% L-cysteine hydrochloride. Five-tube replicate sets were inoculated per variable with 0.1 ml of unheated spore suspension (2 X 106 spores) per tube. About 10 ml of medium was poured aseptically into each inoculated tube. Each set of replicates was sealed with Vaspar and incubated at 30 C. Examinations for growth (gas and turbidity) were made daily for the first 2 weeks of incubation and then at less frequent times. Uninoculated control tubes, poured at the beginning of the experiment, were used to detect a possible change in pH of the medium during incubation; no pH changes occurred. Toxin studies. Cardella's medium (4) consisted of 40 g of Proteose Peptone (Difco), 20 g of Trypticase (BBL), and 10 g of glucose per liter; it was adjusted to pH 7.0 before sterilization. Jensen's medium (12) was slightly modified, consisting of 30 g of Lactalysate (BBL), 20 g of yeast extract (Difco), 3.5 g of sodium citrate, and 10 g of glucose. BI broth and FEM medium were described earlier. Jensen's medium, BI broth, and FEM medium were adjusted to pH 7.2 to 7.4, bottled, and autoclaved at 121 C for 15 min along with Cardella's medium. Toxin titrations were made at intervals during 1 month of incubation at 30 C. Serial 10-fold dilutions were prepared in gelatin-phosphate buffer. Duplicate white mice, weighing 18 to 20 g each, were intraperitoneally injected with 0.5-, 0.2-, and 0.1-ml volumes of the dilutions to obtain a minimal lethal dose (MLD) end point. Trypsin digestion. To determine the effect of trypsin on type C toxin, single tubes of FEM medium were inoculated for each type C strain and incubated at 30 C for 3 days. For trypsin digestion, 1 ml of the culture was mixed with an equal volume of 1.0% trypsin (Difco, 1:250) and incubated at 37 C for 1 hr. Serial 10-fold dilutions of the trypsinized and an untrypsinized culture were prepared and injected into mice as described above. RESULTS Tables 1 and 2 summarize the results of the miniimial growth temperature studies. FEM and ground haddock media gave similar results. Both the marine and terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. Type C toxin was verified in all of the tubes showing gas. The marine strains failed to grow at 12.8 C, and the terrestrial strains did not grow at 10 C or below during prolonged incubation. All inoculated tubes of FEM medium and ground haddock at the highest temperature showing no gas formation were examined microscopically for growth and assayed for botuLnum toxin by the injection of white mice. None of the tubes showed growth or. detectable toxin. The minimal growth temperature of strain 6816 in ground haddock was not determined, because the results in FEM medium showed that it had Table 1. When fewer than five tubes showed growth, the time of growth for each tube is given; where no range is shown, all five replicates showed gas production at the same time. a limiting temperature for growth similar to those of the other marine strains tested. The sodium chloride sensitivity of the marine and terrestrial strains is shown in Table 3. Vegetative-cell growth from a spore inoculum was shown by the development of turbidity and gas. Each strain grew in the presence of 2.0% salt, but only 468 and 6814 grew in 2.5% salt. None of the strains grew in 3.0% salt. Recovery of spores of each strain at the end of incubation from the 3.0% salt medium showed that some spore germination had occurred, although many spores were still refractile by phase-contrast microscopy. The limiting pH for type C growth is presented in Table 4. The terrestrial strains failed to grow belo'* pH 5.62. In contrast, three of the marine strains grew as low as pH 5.10, but not below. Strain 6812 was somewhat less pH-tolerant than the other marine strains. The effect of various media formulations on type C toxin production is shown in Table 5. In general, FEM medium gave the highest levels of toxin, although Cardella's medium was almost as good. During 1 month of incubation, the toxin remained relatively stable in each case. The pH of each medium at 3 days showed little change during 30 days of incubation. Jensen's and Cardella's media ranged from pH 5.6 to 5.8 at 3 days depending on the strain; BI broth showed pH 5.2 to 5.6, and FEM ranged from pH 5.8 to 6.5. The marine strains commonly gave a somewhat lower final pH than the terrestrial strains. The effect of trypsin on type C toxin was determined. The results were based on an MLD comparison; hence, a two-to threefold difference between a trypsinized and an untrypsinized titer was not considered significant. There was no conclusive evidence of activation with trypsin; with toxin from 468, an indication of possible inactivation with trypsin was observed (fourfold reduction). Mention should be made of the rapidity of mouse death after intraperitoneal injection with type C toxin. At high toxin levels, i.e., 100 MLD/ml and higher, deaths invariably occurred in less than 24 hr; at low toxin levels, deaths often occurred after 24 hr and up to 72 hr. No deaths beyond 72 hr were ever recorded. DISCUSSION Nonproteolytic strains of C. botulinum type B, type E, and type F are capable of growth and toxin production down to about 3 C (7,8,14). In contrast, it is generally accepted that type A and proteolytic type B are unable to grow at 10 C or below (13). Although type C coexists in a Based on an unheated inoculum of 2 X 106 spores per replicate and five replicates per variable. Range is shown in parentheses; whete no range is shown, all five replicates showed gas production and turbidity on the same day. Where fewer than five replicates showed growth, the time of growth for each tube is presented. Table 3. marine environments with the nonproteolytic botulinum types, neither marine nor terrestrial strains apparently possess psychrophilic growth characteristics. The minimal temperature for growth of C. botulinum type C is very near that accepted as limiting for growth of type A and proteolytic type B. The usual pattern that nonproteolytic strains of C. botulinum exhibit lowtemperature growth characteristics does not appear to be applicable to type C. It is usually recognized that about 10% sodium chloride (calculated as per cent brine concentration) is necessary to inhibit growth and toxin production of C. botulinum type A and proteolytic type B (1, 9,19). In contrast, type E growth is inhibited by a brine concentration of about 5.0% (15). Type C appears to be even less salt-tolerant than type E, its growth being inhibited by 3.0% salt. The lowest pH permitting growth and toxin production of C. botulinum is 4.7 to 5.0, based on numerous strains, inoculum levels, and culture media (11,15,21). Judged by the data presented here, there is no evidence to show that type C can grow at any lower pH than the other botulinum types. Cardella et al. (4) and Skulberg (17) reported that a proteose peptone-Trypticase-yeast extractglucose medium permitted the production of high levels of botulinum toxin by type C. Work presented here shows that both marine and terrestrial strains produce appreciable quantities of toxin in either Cardella's medium or FEM medium. Of course, Cardella's medium has the distinct advantage of being an aparticulate medium, in contrast to FEM. C. botulinum includes both proteolytic and nonproteolytic strains based largely on their ability or inability to digest coagulated egg albumin or meat particles. All available type A strains are reportedly proteolytic, whereas all type C, D, and E strains are nonproteolytic. Both proteolytic and nonproteolytic strains of types B and F are known, but most of the nonproteolytic strains are weakly proteolytic, as shown by the fact that they often hydrolyze gelatin. Until recently, it was generally accepted that only the nonproteolytic strains produced botulinum toxin that could be activated upon treatment with the proteolytic enzyme trypsin. The phenomenon of trypsin activation of botulinum toxin has been studied thoroughly since the first report by Duff, Wright, and Yarinsky (6) on type E toxin. It it commonly presumed that type E toxin is elaborated by the organism as a prototoxin (17). Since type E, unlike types A and B, lack strongly active proteolytic enzymes, the toxin is released by the organism principally in the prototoxin form that is commonly activated by trypsin. In contrast, toxin from the proteolytic strains usually does not show activation with trypsin except in very young cultures (3). The organism's cellular proteases presumably cause activation of the toxin in the same manner as trypsin causes activation of toxin of many nonproteolytic strains. It was recently shown that the proteolytic enzymes of some botulinum strains do not always activate the prototoxin; similarly, toxins produced by some nonproteolytic strains do not always show activation with trypsin. Iida (10) observed both situations in his studies. Two type C strains studied by Iida produced toxin that showed no significant indications of activation by trypsin. In this work, toxin produced by the marine and the terrestrial strains also showed no conclusive signs of activation when incubated with trypsin.

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2020-12-10T09:04:11.225Z

1971-01-01T00:00:00.000Z

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Modification of the American Public Health Association Procedure for Counting Yeast and Mold in Cottage Cheese The American Public Health Association method for counting low numbers of yeast and mold in cottage cheese was unsatisfactory due to altered pH of the culture medium. A modification of this method is presented. Problems in enumerating yeast and mold were encountered during military subsistence testing of cottage cheese. These problems have resulted from the changes made in the American Public Health Association (APHA) 12th edition of Standard Methods for the Examination of Dairy Products (2). Military Subsistence Testing Laboratories must comply with these changes since the APHA method is cited in Military Specification MIL-C-43274A (3). The APHA method (2) for counting yeast and mold in cottage cheese calls for diluting the cheese 1:10 with 2% sodium citrate which has been preheated to 40 C. Where the count is expected to be low, a 10-ml amount is distributed evenly among three petri plates and poured with acidified (pH 3.5) Potato Dextrose Agar (PDA, Difco). This procedure raises the pH of PDA sufficiently to permit bacterial growth, so that it is difficult to distinguish yeast from bacterial colonies. Previous to the 12th edition, the APHA method (1) prescribed a 1:2 dilution of cottage cheese in 2% sodium citrate; 1 ml was then distributed into each of two petri plates. Although the PDA remained inhibitory to bacterial growth, the heavy suspension clouded the medium and made the petri plates difficult to read. The present investigation was undertaken to improve the APHA method for counting yeasts and molds in cottage cheese. Table 1 shows the effect of plating 1 ml and 3.3 ml of a 1:10 dilution of cottage cheese in 2% sodium citrate on the pH and bacterial inhibition of acidified PDA. Bacterial growth occurred only when 3.3 ml of the cheese was cultured. This was due to the increase in pH of PDA from 1 Defense Subsistence Testing Laboratory, Defense Personnel Support Center, Chicago, Ill. 3.5 to 5.6 when 10 ml of the medium was poured as recommended by APHA (2). Even when 20 ml of PDA was poured, the pH of the medium went as high as 4.9, which also permitted bacterial growth. The 1:10 cheese-citrate dilution had a pH of 6.6. Consequently, 3.3 ml cannot be plated without adversely affecting the pH of the medium, upon which the selectivity of the PDA a Cheese was diluted 1:10 with sterile 2% sodium citrate at 40 C. b-, Negative; +, positive. c All plates incubated at 23 i 2 C for 5 days. is dependent. Plating 1 ml into each of five plates resulted in a clear medium which inhibited bacterial growth and yielded a mold count per gram comparable to that obtained by platng 3.3 ml in each of three plates. The samples of cottage cheese examined were negative for yeast. Three bacterial species inoculated into cottage cheese were recovered quantitatively in acidified PDA (Table 2) by the above APHA method (2). None of the bacteria was inhibited by acidified PDA when 3.3 ml of the citrate-cheese slurry was 12 No growth 5 a Cottage cheese was diluted 1:10 with sterile 2% sodium citrate at 40 C. b Based on counts obtained in plate count agar after incubation for 96 hr at 36 C. c Counts in 20 ml of Potato Dextrose Agar were determined after incubation at 23 4 C for 5 days. plated. However, the medium did remain inhibitory to these bacteria when only 1 ml of the cheese-citrate slurry was plated, since the pH of PDA only increased to 4.0 or 4.5 depending on the volume of PDA poured (Table 1). It is apparent that the APHA method (2) for counting low numbers of yeast and mold in cottage cheese should be modified. The method must be one which inhibits bacterial growth but allows yeast and mold to form colonies. The following modification of the APHA method accomplishes this objective. Prepare a 1:10 dilution of cottage cheese in sterile 2% sodium citrate preheated to 40 C, as stated in the APHA method (2). Instead of distributing 10 ml evenly among three plates, transfer 1 ml into each of five petri plates and pour with 10 to 20 ml of PDA acidified to pH 3.5. Multiply the total number of colonies on the five plates by 2 to obtain the count per gram of cheese. The accuracy may be increased by plating 1 ml into each of 10 petri plates, in which case the total number of colonies will represent the count per gram of cheese. Except for these changes, the microbiological testing of cottage cheese remains as given by APHA.

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2014-10-01T00:00:00.000Z

1971-03-01T00:00:00.000Z

2820049

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Do unique proteins exist in taste buds? Proteins in papillae on the bovine tongue were analyzed by semi-micro, polyacrylamide gel electrophoresis. All the proteins in the papillae with taste buds were observed to be common to proteins in the surrounding epithelium without taste buds. The protein band which was reported to form a weak complex with compounds called sweet by man was also found in all parts of the tongue epithelium. The receptor molecules for chemical stimuli may be distributed in all the cells of the tongue epithelium or the content of receptor molecules in taste bud papillae may be extremely low. INTRODUCTION It has been postulated that the initial event in taste stimulation is the formation of a weak complex of the receptor molecule with the stimulus compound (1). Recently, proteins which form complexes in vitro with compounds called sweet and bitter by man were extracted from the epithelium of bovine (2,3), porcine (4), and rat tongues (5,6), and it was claimed that these extracted proteins were the receptors for sweet and bitter compounds. On the other hand, Hansen insisted that the primary process of sugar reception in the blowfly, Phormia regina, is identical with the formation of a sugar-glucosidase complex similar to the enzyme-substrate complex of Michaelis and Menten (7). In mammals, taste stimulation is induced in taste buds which occur in fungiform, circumvallate, and foliate papillae on the surface of the tongue. An early question that arose was whether or not receptor molecules for the stimulus compounds are localized only in the papillae with taste buds. Since the content of taste buds in a single papilla of the bovine tongue is fairly high (for example, a single circumvallate papilla of bovine tongue contains about 1,500 taste buds [8]), we suggest that an appreciable portion of proteins from the papillae seems to be accounted for by proteins from taste buds. In the present study, we aimed to compare the number of protein bands of the papillae containing taste buds with those of the surrounding epithelium without taste buds. The technique of polyacrylamide gel electrophoresis was 297 chosen to compare protein patterns because of its high resolution of complex protein mixtures. Since the papillae are fairly small, a semimicrotechnique was devised for the protein preparation and electrophoresis. EXPERIMENTAL 26 circumvallate papillae and about 200 fungiform papillae were seen on the epithelium of a bovine tongue used in this study. Circumvallate papillae, fungiform papillae, or the surrounding epithelium including filiform papillae which contain no taste buds were cut off with a small scalpel from the bovine tongue, and homogenized with a small motor-driven glass homogenizer in 0.3 ml of a 0.06 M buffer solution (Tris-HC1, pH 8.7, or acetate-KOH, pH 4.5). The homogenate was centrifuged in a microcentrifuge for 30 min at 10,000 X g. The protein concentration of the supernatant was determined on an aliquot of the supernatant by the method of Gornall et al. (9). Half volume of the supernatant (0.13 ml) was subjected to polyacrylamide gel electrophoresis with a 3 (I.D.) X 70 mm glass tubing. Gels for electrophoresis at pH 8.7 and those at pH 4.5 were prepared according to the method of Davis (10) and that of Reisfeld et al. (11), respectively. Electrophoresiswas carried out at 1 ma/tube for 80 min at pH 8.7 and at 1 ma/tube for 150 min at pH 4.5. Gels were stained with Coomassie blue (12) which affords high sensitivity detection of protein bands. The minimum amount of protein detected by the electrophoresis technique used in the present study was determined to be 0.1 4 0.05 g per gel by using serum albumin (Nutritional Biochemical Corp., Cleveland, Ohio) as a standard protein sample. For precise comparison of protein bands in gels, electrophoresis was also carried out by running two different samples on a single gel column (referred to as a "split gel" [13]). All the experimental operations were performed at 4°C. After extraction with the buffer the sediment was subjected to further extraction by a 8 M urea solution containing 1 % sodium dodecyl sulfate (SDS). After 1 hr of extraction at room temperature, the extract was centrifuged and the supernatant was subjected to electrophoresis on 8 M urea gels. In this case, gels were stained with Amido Schwarz (10). The protein reported by Dastoli et al. was prepared according to their method (3) from the whole epithelium of bovine tongues by ammonium sulfate fractionation, followed by gel filtration with a column of Bio-Gel P-150 (Bio-Rad Laboratories, Richmond, Calif.). RESULTS AND DISCUSSION Electrophoretic patterns at pH 8.7 of proteins extracted with the buffer are shown in Fig. 1. The photographs of A, B, and C represent the patterns of proteins from fungiform papillae, circumvallate papillae, and the surrounding epithelium, respectively. These patterns are also shown schematically in Fig. 2. The widths of the bands in the figure are indicative of relative widths of the staining bands and dark-, cross-, and parallel-hatching indicate the relative staining intensity of the bands. As seen from the figures, all the protein bands from the fungiform and circumvallate papillae are common to those from the FIGURE 1. Electrophoretic patterns of the proteins extracted with the buffer from fungiform papillae (A), circumvallate papillae (B), and the surrounding epithelium without taste buds (C). The amounts of protein applied to a gel were: A, 170 jig from seven fungiform papillae (wet weight, 14 mg); B, 160 MAg from two circumvallate papillae (12 mg); C, 160 ug from the surrounding epithelium (13 mg). Electrophoresis was carried out at pH 8.7. All gels were stained with Coomassie blue. surrounding epithelium. Although the protein of AB-16 (Fig. 2) is more abundant in the papillae with taste buds, the protein seems to have no direct relation to taste reception, because the protein was confirmed to be serum albumin by electrophoresis with purified serum albumin in split gel. It is known that in man the sweet taste is most easily sensed at the tip of the tongue, the bitter at the back, the sour at the edge, and the salt both on the tip and at the edge. However, a distinct difference was not found among the protein patterns from the fungiform papillae on different sides of the tongue. Since most basic proteins do not migrate anodically on electrophoresis at pH 8.7, a protein solution extracted with the buffer was also subjected to electrophoresis at pH 4.5. Some of the proteins which migrated at pH 8.7 also 3oo00 THE JOURNAL OF GENERAL PHYSIOLOGY -VOLUME 57 . Schematic representation of polyacrylamide gels of proteins from fungifbrm papillae and the surrounding epithelium without taste buds. A, fungiform papillae, electrophoresis at pH 8.7 (same as A in Fig. 1); B, epithelium, electrophoresis at pH 8.7 (same as C in Fig. 1); C, fungiform papillae, electrophoresis at pH 4.5; D, epithelium, electrophoresis at pH 4.5; E, proteins extracted with a 8 M urea solution containing I % SDS from fungiform papillae, electrophoresis at pH 8.7; F, the same proteins as E, electrophoresis at pH 4.5. Amounts of protein applied to a gel were: C, 170 g from 7 fungiform papillae (13 mg); D, 180 g from the epithelium (14 mg); E, 50 /ig from 20 fungiform papillae (42 mg); F, 4 5ug from 20 fungiform papillae (38 mg). A, B, C, and D were stained with Coomassie blue; E and F stained with Amido Schwarz. migrated at pH 4.5. For example, the band of CD-6 which is more abundant in papillae with taste buds was confirmed again to be that of serum albumin by split gel. The diagrams C and D represent the protein patterns of fungiform papillae and the surrounding epithelium, respectively. As in the electrophoretic patterns at the alkaline pH, all the protein bands from the fungiform papillae were found in the surrounding epithelium, while two bands (D-2 and D-3) which were lacking in the fungiform papillae were found in the surrounding epithelium. Since the protein reported by Dastoli et al. (3) was reported to be a basic protein, the band of this protein must be contained in the electrophoretic pattern at the acidic pH. In order to find the band of the protein in the gel, the protein was prepared from the whole epithelium of bovine tongues according to the method of Dastoli et al. (3). The protein preparation obtained gave one major band which was found to be CD-14 in split gel, accompanied by five minor bands (CD-1, 2, 4, 5, 7). The results indicated that the content of the proteins reported by Dastoli et al. in the epithelium is sufficiently high to be detected by electrophoresis and also that the proteins are homogeneously distributed in the whole epithelium of the bovine tongue surface. Since all the protein bands from fungiform papillae are common to those from the surrounding epithelium, the protein which was reported to form a weak complex with bitter compounds (4) must also be homogeneously distributed in the whole epithelium of the tongue surface. Some proteins may be firmly bound to the cell membrane and thus not solubilized with buffer solution. Therefore, after extraction with buffer the sediment was subjected to extraction by a 8 M urea solution containing detergents such as Triton X-100, deoxycholate, and SDS. Electrophoresis of the extract was carried out at pH 8.7 and at pH 4.5. Since gels containing detergents often became opaque in the process of staining with Coomassie blue, gels were stained with Amido Schwarz, although the sensitivity of this stain was less than that of Coomassie blue. The electrophoretic patterns of the extraction by a 8 M urea solution containing 1% SDS, which was found to solubilize most strongly the proteins in the sediment, are shown in diagrams E (pH 8.7) and F (pH 4.5). Again, no difference was found between the protein pattern of the papillae with taste buds and that of the surrounding epithelium. It was concluded from the present study that all the proteins in the papillae with taste buds are common to those in the surrounding epithelium without taste buds. It is unlikely that the proteins from the epithelium without taste buds would happen to show the same mobilities as those from the papillae with taste buds, since polyacrylamide gel electrophoresis affords a very high resolution of protein bands. Various interpretations may be placed on the present conclusion. One of these interpretations is as follows. The receptor molecules for chemical stimuli are distributed in all parts of the tongue epithelium. Although the stimuli may bind to the receptor molecules at all parts of the epithelium, only the binding of the stimuli at taste buds produces effective taste stimulation. An alternative interpretation is that the content of receptor molecules in the papillae is too low to be detected by polyacrylamide gel electrophoresis. Any new protein band in addition to the bands of diagrams A and C in Fig. 2 was not detected even when 260 ug of protein extracted from 10 fungiform papillae (wet weight, 21 mg) was applied to a single gel without urea for the electrophoresis at pH 8.7 and pH 4.5, respectively. The minimum amount of protein detectable by the electrophoretic technique used in the present study was determined to be around 0.1 ug per gel by using serum albumin as a standard protein sample; therefore, the content of receptor molecules in a single fungiform papilla may be lower than 0.01 Mg, if a receptor molecule is a protein unique to taste buds and is extractable in aqueous buffer. Since the content of the proteins reported by Dastoli et al. (2,3) in the papillae and the surrounding epithelium was sufficiently high to be detected by electrophoresis, we are forced to deny that the proteins are true receptor molecules, if we admit the above interpretation. Further study will be needed to clarify the actual nature of taste receptor molecules.

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2020-12-10T09:04:10.761Z

1971-10-01T00:00:00.000Z

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Lack of Toxicity of Helminthosporium maydis-Invaded Corn and Culture Filtrates to Chicks and Micel Six isolates of Helminthosporium maydis, obtained from southern leaf blight- damaged corn, were grown separately on autoclaved corn and fed to chicks and mice to evaluate their toxigenicity. Two-, three-, and four-week-old culture filtrates from three pathogenic isolates grown separately on modified Fries medium were also evaluated for toxigenicity. None of the invaded corn samples or culture filtrates affected the weights of chicks or mice when compared to controls. Postmortem examinations did not reveal significant gross lesions. H. maydis was not toxigenic under our experimental conditions. Southern corn leaf blight reached epidemic proportions in the Southeast and the Midwest corn belt areas of the United States in 1970. Although Helminthosporium maydis Nisik. and Miyake (Cochliobolus heterostrophus Drechs.), the causal agent of this disease, had never been reported to produce a mycotoxin, livestock and poultry producers were concerned about feeding blight-damaged corn. This concern was greatly magnified by rumors of livestock losses attributed to feeding blight-damaged corn. Cattle, swine, and poultry feeding studies were quickly initiated in several states, making use of naturally blightdamaged corn silage and grain to assess the toxicity and feeding quality. Studies at the University of Florida showed that blight-damaged corn silage had no effect on gains and conversion rates in beef and dairy calves (5) and that blightdamaged corn grain had essentially the same nutritive value as nonblighted grain with regard to gains and conversion rates in swine (2). Feeding studies with chickens at the University of Georgia revealed that blight-damaged corn grain had no effect on mortality, growth rate, feed efficiency, or blood clotting time in broilers (9) and no effect on mortality, egg production, or egg quality in layers (1). These studies indicated that there was no toxicity associated with feeding naturally blightdamaged corn in 1970. In addition, no cases of mycotoxicoses associated with blighted corn have been reported to the Diagnostic and Research Laboratories (Tifton, Georgia) as of June 1971. 1 Published as journal series paper no. 1004 and Institute of Comparative Medicine manuscript 850. In view of a recent report on mycotoxin production on corn by H. maydis (A. Ciegler et al., Bacteriol. Proc., p. 6,1971), however, we wanted to assess the toxicity of autoclaved corn invaded by pure cultures of known pathogenic ("race T") isolates of H. maydis and of culture filtrates to chicks and mice. The isolates of H. maydis used in this study were isolated from blight-damaged corn in 1970, three from leaves and three from grain. One of the isolates from grain was obtained from E. S. Luttrell (University of Georgia, Athens). The isolates were tested for pathogenicity by using the method described by Smith et al. for single spore isolates (8) and for pathotoxin production by using the root inhibition method described by Luke and Wheeler (7) on two corn hybrids, Coker 52 [Texas male-sterile cytoplasm (Tms)] and Coker 71 (normal cytoplasm). Test diets, each containing 60% autoclaved corn heavily invaded by a single isolate of H. maydis, were prepared as previously described (3,4). Each diet was fed ad libitum for 2 weeks to a group of 10 1-day-old Babcock B-300 cockerels and for 3 weeks to a group of three randomly bred male white mice (minimum age 30 days). Control chicks received a commercial chick starter diet, and control mice received a commercial laboratory diet. Each pathogenic ("race T") isolate also was grown separately on modified Fries medium similarly to the method described by Luke and Wheeler for the production of victorin, the pathotoxin produced by H. victoriae (7). Culture filtrates were collected from each of these isolates on March 22, 2020 by guest http://aem.asm.org/ Downloaded from VOL. 22, 1971 after 2, 3, and 4 weeks of incubation. One milliliter of each culture filtrate was given orally, via a plastic tube attached to a tuberculin syringe, to each of three 1-day-old chicks, and 1 ml of each filtrate was injected intraperitoneally into each of three 25-g male white mice. The chicks were then fed a commercial chick starter diet for 1 week, and the mice were fed a commercial laboratory diet for 3 weeks, both ad libitum. Control chicks and mice each received 1 ml of modified Fries medium. Water was provided ad libitum for all groups in both the feeding and filtrate tests. Initial and weekly body weights and daily observations were recorded for each group of chicks and mice. All chicks and mice were killed at the end of the test periods and examined for gross lesions. Tissues were collected in 10%S neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for future examinations by microscope. By the classification scheme of Hooker et al. (6), the six isolates were tentatively classified as follows: two produced a susceptible reaction on both hybrids and were designated as "race 0"; three produced a susceptible reaction on Coker 52 (Tms) and were designated as "race T"; and one produced a resistant reaction on both hybrids and was given no race designation. Only the ''race T" isolates produced a positive pathotoxin reaction. Fifty-fold dilutions of "race T" culture filtrates reduced the growth of Coker 52 (Tms) roots more than 50%. Growth suppression of 10% or more, as compared to controls, was not recorded for any groups of chicks or mice fed H. maydis-invaded corn. One mortality was recorded in one group of chicks on the second day of the test. The cause of death was omphalitis and was thought to be unrelated to the invaded corn. Growth suppression was not recorded for any of the groups of chicks or mice given H. maydis culture filtrates; however, growth stimulation of 10% or more, as compared to controls, was recorded for three of the nine groups of chicks. No mortalities were recorded during the culture filtrate test period. Postmortem examinations of the chicks and mice at the end of the test periods did not reveal lesions associated with the test diets or culture filtrates. One chick fed fungus-invaded corn had severe fibrinosuppurative epicarditis and localized peritonitis in the umbilical region. These lesions were characteristic of a bacterial infection and were thought to be unrelated to the test. Our data do not support the findings of Ciegler et al. (Bacteriol. Proc., p. 6,1971) that H. maydis produces a mycotoxin on corn. They gave no details on the race of H. maydis used or on the extraction procedures and concentration factors involved in obtaining their solvent extracts before intraperitoneal injection into mice, rats, and a pig. It is possible that their experimental conditions preclude analogy to situations involving naturally infected corn. Under our experimental conditions, H. maydisinvaded corn and culture filtrates were not toxic to chicks or mice. If H. maydis does produce a toxin which affects animals, a much higher concentration of toxin should occur in corn which is inoculated with a pure culture and incubated than in naturally infected corn. Our data add support to the accumulating evidence that H. maydis, per se, is not toxic to animals.

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Lysine Decarboxylase Activity in Broth and Agar Media Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. TIhe nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. Amino acid decarboxylase activities are useful tests in determinative microbiology. Since Moeller (7) in 1955 described practical tests for determining decarboxylases among the Enterobacteriaceae, microbiologist have used Moeller's methods, or modifications, with increasing frequency. Today, these are standard tests in clinical and public health microbiology laboratories. Unfortunately, some of the modifications that were aimed at making the test easier and more convenient are not as accurate as Moeller's methods. This is true whether one is attempting to demonstrate lysine, ornithine, or arginine decarboxylase. The modified Moeller medium as described by Difco was simplified by having one indicator, and the technique required no extraction or addition of reagents, in contrast to some other methods. Johnson's (6) semisolid medium (Moeller base) was modified by the addition of 0.3% agar, which avoids the necessity of the oil overlay. Bachrach (1) incorporated lactose into the Moeller base medium to permit differentiation of the Salmonella-Arizona strains from Escherichia, Klebsiella, and certain Enterobacter strains, which also decarboxylate lysine. The lysine-lactose-agar medium was a modification of the medium of Edwards and Fife (3) because of the addition of lactose and the omission of iron salts. It was useful in differentiating most Salmonella, nonlactosefermenting Arizona, Edwardsiella, Serratia, and certain Enterobacter from Shigella, Escherichia, Klebsiella, Enterobacter aerogenes, and Citrobacter. In the course of identifying a number of microorganisms isolated from biological specimens in the clinical laboratory, procedures for determining lysine decarboxylase activity were evaluated. The problem of interpreting results developed when inconsistent readings from lysine decarboxylase broth medium were obtained. Sometimes these results were positive, and at other times they were negative. At times, no change in indicator (purple color) can be seen. This can be confused with a positive result (purple color), when a change of indicator from yellow to purple is not noticed. This is partially due to the rapid pH shift resulting from the breakdown of glucose substrate and low hydrogen ion concentration. Also, a change ofindicator from purple to yellow in the fermentation of glucose may be missed during the overnight incubation period. The biggest problem seems to involve nonlactose-fermenting gram-negative bacilli that fail to ferment glucose. Although published reports indicate the advantage of using a lysine-agar medium (3,6), decarboxylase broth is more commonly used. It therefore seemed worth while to examine four lysine decarboxylase methods in the hope of arriving at some conclusion regarding a method of choice. MATERIALS AND METHODS The bacterial strains included recent isolates from 149 urines, 104 sputa, 25 wounds, 6 body fluids, 4 ear swabs, and 2 fecal specimens as well as strains taken from our culture library. Original isolations from biological specimens were made on human blood-agar and MacConkey agar. An individual isolate was planted on Kligler iron agar (KIA). When a question of purity arose, one half of the colony was transferred to MacConkey agar for purification. Tests for hydrogen sulfide; urease; phenylalanine deaminase production; indole formation; citrate utilization; lysine decarboxylase activity; glucose, dulcitol, and lactose fermentation; gelatinase; and motility were made from KIA as described by Edwards and Ewing (2). With nonfermenters, the carbohydrate oxidative-fermentative (0-F) test medium of Hugh and Leifson (5), the nitrate test, Kovacs oxidase test (4), and the gluconate test as described by Haynes (4) were added. These biochemical tests were used for identification of the wild strains. Occasionally, a more elaborate series of tests was necessaryto identify wild strains. Identified cultures were inoculated into four lysine decarboxylase test media. The four test media employed here were as follows. Lysine decarboxylase medium (Moeller base). This medium contained decarboxylase basal medium (Difco) with 1% L-lysine and bromocresol purple indicator. An inoculated control made up of the above ingredients minus L-lysine was included with each strain tested. The results were read after incubation for 24 hr and for as long as 4 days. Johnson's seniisolid medium. This medium consisted of decarboxylase basal medium (Difco) with 1% Llysine. Agar (0.3%) was added as indicated by Johnson (6) in the ornithine decarboxylase semisolid medium. The indicator was bromocresol purple. The semisolid medium was inoculated by a single stab with a straight wire to the bottom of the tube. The inoculated control was made up of decarboxylase basal medium in 0.3% agar without L-lysine and was included with each strain tested. Results were read after 24 hr of incubation. Bachrach's lysine-lactose broth. This medium was that described by Bachrach (1) with L-lysine made up to 1%. The indicator used was bromothymol blue. The inoculated control consisted of decarboxylase basal medium and lactose without L-lysine and was included with each strain tested. Results were read after 16 to 24 hr of incubation. Lysine-lactose-agar slants. The test medium contained lysine decarboxylase basal medium (Difco) with 1 % L-lysine, 1 c lactose, 1.5% agar, and bromocresol purple as the indicator. An inoculated control made up of the above ingredients minus L-lysine was included with each strain tested. Results were read at 24 and 48 hr. These media were dispensed in 5-ml samples in sterile screw cap tubes (125 by 16 mm) and were sterilized in the autoclave for 15 min at 15 lb of pressure (121 C). RESULTS Initial studies were done with a series of 305 Enterobacteriaceae and 42 nonfermenters. The latter group included 40 Pseudomonas aeruginosa organisms and 2 Herellea strains ( Table 1). The two Alkalescens-Dispar strains were positive in Bachrach's broth after the prescribed 24-hr incubation period. Whether this may represent a very weak decarboxylase manifesting itself late is open to question. In Table 3, these results were recorded as negative. These two strains were positive with all of the other methods except Johnson's semisolid medium. Repeated tests produced the same results. One of the atypical Escherichia strains was negative in Johnson's semisolid agar but positive in lysine decarboxylase broth. Of the seven atypical Escherichia strains, six were positive by the lysine decarboxylase broth and Johnson's semisolid method, although all were negative by the lysine-lactose media. As expected, Bachrach's broth with lactose and the lysine-lactose-agar slants gave negative reactions with most of the Escherichia and Klebsiella strains. Poor results were obtained with Enterobacter aerogenes with the nonlactose medium of Johnson. Of 14 E. haJniae strains which were positive in lysine decarboxylase broth, 7 were positive in Bachrach's broth, 8 in Johnson's semisolid medium, and five in the lysine-lactose-agar slants. When the results of lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, 8 of the 16 (50%0) strains tested in the latter medium were in agreement with the same strains tested in the former medium. When the results of Bachrach's broth and lysine agar slants with lactose were compared, 5 of the 16 (31 %) strains tested in the latter medium were in agreement with the same strains tested in the former medium. The results of the four lysine decarboxylase methods with the Proteus-Providence group were good except for two P. rettgeri strains which gave falsepositives in the lysine-lactose-agar slants. The Salmonella test results with the four methods correlated very well. Two of the Citrobacter strains negative in lysine decarboxylase broth were not tested with the other test media. With the 20 remaining Citrobacter strains, six discrepancies were found between the results obtained in lysine decarboxylase broth and the test medium of Bachrach and Johnson, whereas five were found between lysine decarboxylase broth and the lysineagar slants. These discrepancies represent six of the nine Citrobacter strains positive in lysine decarboxylase broth. One of the six strains was positive in lysine decarboxylase broth in 24 hr, five were positive only after 48 to 72 hr of incubation, whereas all six were negative in the media of Bachrach and Johnson in the prescribed incubation time of 24 hr. One of the latter strains produced a weak reaction in a lysine-lactose-agar slant in 24 hr. Of 40 Pseudomonas aeruginosa strains tested, 7 failed to grow in the lysine media, whereas 31 of 33 strains produced no change in the indicator by all methods used. One of the two remaining strains changed the indicator to yellow (acid) only in Bachrach's broth, whereas the other strain produced an acid reaction in both lysine decarboxylase and Bachrach's broth media. In these two instances, it is probable that the rate of metabolism was increased to such a point that the acid formed changed the color of the media to yellow. Both of these strains produced acid in conventional glucose fermentation tubes. Both of the Herellea strains utilized glucose on 0-F medium and in conventional broth but failed to use it in any of the lysine decarboxylase media. DISCUSSION All four methods were reasonably reliable when dealing with nonlactose fermenters, whereas the lysine decarboxylase broth method and the semisolid method of Johnson were better for all of the types of bacteria studied. A major explanation for the great difference in the test results with lactose fermenters such as Escherichia and Klebsiella has been the incorporation of lactose into the medium (Bachrach's broth and lysine-lactose-agar). The high acidity produced from lactic acid usually cannot be overcome by lysine decarboxylase activity in this type of medium. Lysine-agar slants as originally designed by Edwards and Fife (3) are used mainly to separate Salmonella and Arizona strains from the Citrobacter group. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend further the range of usefulness of this medium to other members of the enteric family. For example, this would in- crease the positive results with lactose-positive Arizona strains. When the results of lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, the latter gave 6% fewer positives ( Table 2). The poor results that were obtained with E. aerogenes strains in Johnson's semisolid medium could not be explained. The results with lysine decarboxylase broth and Johnson's semisolid medium were identical with Klebsiella, except for one strain which produced a positive reaction in lysine decarboxylase broth. False-positives produced by Voges-Proskauerpositive Klebsiella did not occur with our strains after 24 hr of incubation. The addition of agar to Moeller's basal medium did not seem to affect our results, even though the addition was suggested by Johnson et al. (6) to decrease the chances for false-positives amont these strains. The difference in results between lysine decarboxylase broth and Johnson's medium with Citrobacter strains might be accounted for by the longer incubation period afforded the decarboxylase broth method. An advantage to Johnson's semisolid medium is that it does not require a paraffin oil seal added to the medium in the tube; however, a tightly secured screw cap tube works equally well. A 1% difference existed between the results obtained with Bachrach's broth and the lysine-agar slant with lactose (Table 3). Most lactose fermenters will give a negative reaction in lysine decarboxylase medium containing lactose even in the presence of enzyme activity. The physical state of the media (broth versus agar) seems to have no effect on decarboxylase production. In the lysine decarboxylase media with bromocresol purple indicator, the color range extends from a deep purple to yellow to purple. At times, reading was found to be difficult with intermediate degrees of color change, espe- cially with the Proteus group; similar colorchanging problems were also encountered to some extent with our controls. Nonetheless, the controls were generally useful in reading and interpreting results. Occasicnally, the change from purple (alkaline) to yellow (acid) was not discernible. This is mainly due to the fact that Enterobacteriaceae fermented glucose during the overnight growth period, since these bacteria usually utilize glucose in the first 6 to 8 hr of incubation. On occasion, we have experienced negative reactions involving glucose in which the yellow (acid) color was absent or faint and the medium had a colorless appearance (reduction of the indicator). Sometimes this happened in the bottom two-thirds of the tube. In some cases, the decoloration changed very little from 6 to 24 hr. This occurred mostly in Johnson's semisolid medium. An inexperienced individual could fail to recognize the first color change brought about by the utilization of glucose, expecially when weak reactions occur. Determining the pH of the colorless medium with pHydrion (Micro Essential Laboratory, Brooklyn, N.Y.) paper usually revealed a change to acid. One of the discrepancies of Bachrach's broth with bromothymol indicator was that quite often the lysine decarboxylase-positive bacteria changed the color of the indicator from yellow to green rather than blue. Another problem has been the shift in color from pale yellow to purple in the lysine-agar slants from 6 to 48 hr of incubation which may result in erroneous positives. Sometimes this was evident in the lysine-containing medium but not in the controls. When tests depend on pH changes as a result of the activity of bacterial enzymes on a substrate, a high percentage of discrepancies occur. With any of the methods, a positive reaction usually is reliable although false-negatives can occur. A pH change was not seen in the lysine decarboxylase media with the two Herellea strains. The pH of the broth media did not fall appreciably over a 10-hr period. Repeated tests showed the concentration (milligrams/100 ml) of glucose in lysine decarboxylase broth before inoculation and during and after a 24-hr incubation period to be about the same. Several P. aeruginosa strains were checked in the same manner and gave similar results. The inability of P. aeruginosa or Herellea to utilize glucose under the anaerobic conditions of the medium is a feature which makes the lysine decarboxylase test an undesirable procedure for these organisms. In conclusion, of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. A (YLASE ACTIVITY 1095 test method the results of which can be interpreted in 24 hr would be a more suitable one for diagnostic work. LITERATURE CID

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Improved Isolation of Anaerobic Bacteria From the Gingival Crevice Area of Man A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms in the gingival crevice area of man. This technique achieves an anaerobic state by flushing the local environment with oxygen-free gas. Once collected, the crevicular debris was immediately placed into sterile oxygen-free test tubes which were flushed out by the oxygen-free gas. In this manner, the samples were weighed, dispersed, diluted, and cultured in roll tubes and plates. The medium for control (Brewer Jar technique) and Hungate techniques was Heart Infusion Agar fortified with 10% defibrinated horse blood. When the Hungate technique was used, the recovery of viable bacteria, as a percentage of the direct microscopic count, was significantly greater than plates incubated aerobically or utilizing the Brewer Anaerobic technique. Cultural counts by using the Hungate method averaged 41.3% for six samples when 90% nitrogen and 10% hydrogen were used, 70.4% for eight samples when 85% nitrogen, 10% hydrogen, and 5% carbon dioxide were used, and 63.4% for eight samples when 100% carbon dioxide was the gaseous atmosphere. At no time were cultural counts, by using anaerobic plates (Brewer Jar), more than 24% of the direct microscopic count. This suggests that exclusion of oxygen and the presence of carbon dioxide maximized recovery of gingival crevice bacteria. A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms in the gingival crevice area of man. This technique achieves an anaerobic state by flushing the local environment with oxygen-free gas. Once collected, the crevicular debris was immediately placed into sterile oxygenfree test tubes which were flushed out by the oxygen-free gas. In this manner, the samples were weighed, dispersed, diluted, and cultured in roll tubes and plates. The medium for control (Brewer Jar technique) and Hungate techniques was Heart Infusion Agar fortified with 10% defibrinated horse blood. When the Hungate technique was used, the recovery of viable bacteria, as a percentage of the direct microscopic count, was significantly greater than plates incubated aerobically or utilizing the Brewer Anaerobic technique. Cultural counts by using the Hungate method averaged 41.3% for six samples when 90% nitrogen and 10% hydrogen were used, 70.4% for eight samples when 85% nitrogen, 10% hydrogen, and 5% carbon dioxide were used, and 63.4% for eight samples when 100% carbon dioxide was the gaseous atmosphere. At no time were cultural counts, by using anaerobic plates (Brewer Jar), more than 24% of the direct microscopic count. This suggests that exclusion of oxygen and the presence of carbon dioxide maximized recovery of gingival crevice bacteria. The gingival crevice area of man appears to harbor an anaerobic microflora. Thus, when bacteriological samples were removed, dispersed, diluted, and plated on nutritionally complex media, twice as many organisms were recovered when the high dilution plates were incubated in an anaerobic atmosphere than when incubated in air (2,3,9). Most of the aerobic isolates were facultative bacteria, as they would grow also in an anaerobic environment (3). When the viable counts were compared with the total microscopic counts, only 20 to 25% of the organisms present were cultivated (9). The inability to cultivate all of the gingival crevice bacteria was attributed to the presence of nonviable bacteria, the absence of a medium that would be nutritionally adequate for all of the indigenous gingival crevice organisms, or to the failure to disperse completely the clumps of bacteria present in the sample. Re crevice samples by performing the various bacteriological procedures in an anaerobic glove box in an oxygen-free atmosphere. Their work suggested that many of the gingival organisms were killed by oxygen during the manipulative procedures prior to anaerobic incubation. The purpose of the present investigation was to develop a means of increasing the recovery of viable organisms from the human gingival crevice by achieving continuous anaerobiosis in all phases of the experimental procedure. The technique developed by Hungate (4) was used for this purpose. MATERIALS AND METHODS Complete anaerobiosis. Atmospheric oxygen was eliminated or greatly diluted in the vicinity of the bacteriological sample by means of an oxygen-free gas (OFG). Commercial gas mixtures containing (i) 90% nitrogen and 10% hydrogen; (ii) 85% nitrogen, 10% hydrogen, and 5% carbon dioxide, or (iii) 100% carbon dioxide were made oxygen-free by passing the gas through a Pyrex tube containing hot copper turnings (4,8). The OFG was passed through neoprene tubing fitted at the end with a stainless-steel needle. This was used to either displace or prevent air from entering the sample, diluent, and media during collection, preparation, and inoculation. When 100% carbon dioxide was employed, 0.5% sodium bicarbonate (final concentration in medium) was autoclaved separately, equilibrated with C02, and added to the diluent and medium so as to neutralize the acidity created by the dissolved carbon dioxide. The diluent and medium were prepared in 100-ml amounts in 250-ml Erlenmeyer flasks fitted with gauze stoppers. Immediately after autoclaving, these solutions were flushed with OFG until the resazurin was colorless and sterile neoprene stoppers were inserted into the flasks. If it was necessary to add any ingredient after autoclaving, these were also flushed with the OFG. After addition of blood to the medium, a change in the resazurin indicator could not be visualized. The diluent was tubed in 9-ml amounts and portions of the medium were tubed in 4-ml amounts and maintained at 48 C until inoculated. Both the diluent and medium were placed in sterile test tubes (16 by 150 mm) and flushed with OFG. The remainng medium was poured into petri plates which were used in culturing the gingival debris under conventional aerobic and anaerobic techniques. Collection of sample. Periodontally normal individuals (i.e., no periodontal pockets greater than 3 mm and no evidence of gingivitis or bone loss) ranging in age from 21 to 33 yr served as subjects. These volunteers had not eaten for at least 2 hr prior to sampling. They were seated adjacent to the OFG source so that the sample could be collected under a stream of OFG. Samples were removed from sulci which were less than 3 mm in depth and showed no clinical signs of inflammation. Crevicular debris was placed immediately on a piece of sterile aluminum foil contained in a preweighed tube. The tube was flushed with OFG, stoppered, and quickly weighed to obtain the wet weight of the crevicular debris. Dispersion, dilution, and plating of sample. A 9-kc Raytheon sonic oscillator was used to disperse the gingival crevice debris. The oscillator chamber was fitted with a neoprene stopper containing two open glass tubes, one of which was connected to the OFG and the other served as a vent. Reduced diluent (15 ml) was placed in the chamber. The small piece of aluminum foil containing the crevicular debris was then carefully removed from the tube under coverage of the OFG and placed in the oscillator. The sample was dispersed for 30 sec under a continuous flow of OFG. Samples were removed from the oscillator and serially diluted. It was possible to execute this procedure without oxidation of the resazurin indicator in the diluent. Samples (0.1 ml) of the appropriate dilution were plated and incubated as follows. (i) Roll tubes, complete anaerobiosis (4). Tubes were prepared in triplicate by inoculating the reduced blood-agar medium maintained at 48 C. The tubes were stoppered and rolled gently in cold water to obtain an even, thin film of blood-agar around the inside of the tube. (ii) Conventional anaerobic technique. Three blood-agar petri plates were inoculated in room atmosphere, and the sample was spread on the surface by means of a sterile glass rod. These plates were incubated in Brewer Jars containing 85% nitrogen, 10% hydrogen, and 5% carbon dioxide. In this procedure, residual oxygen in the presence of hydrogen is converted to water by means of a heat-activated platinum catalyst contained in the lid of the Brewer Jar. (iii) Conventional aerobic techniques. Three blood-agar plates treated as above except that they were incubated aerobically. All tubes and plates were incubated at 35 C for 5 days. Total microscopic counts were obtained by placing the dispersed material from the sonic oscillator in a Petroff-Hauser counting chamber and counting the bacteria by using a dark-field microscope. Clumps of bacteria were occasionally observed and were counted as a single microscopic unit. The statistical significance of the data was determined by the Student ttest. RESULTS All samples were collected, dispersed, and diluted under conditions of complete anaerobiosis, i.e., resazurin remained colorless. Samples of the final dilution were then used to inoculate roll tubes or were used to inoculate plates in the conventional manner. The results obtained when the OFG was 90% nitrogen and 10% hydrogen are shown in Table 1. The mean total microscopic count was 24.7 :1: 8.7 X 1010 per gram (wet weight) of crevicular debris. Under aerobic conditions, 14.8% of these organisms were cultured (Table 4). When conventional anaerobic incubation was employed, the recoveries improved to 22.2%. When oxygen was completely excluded, 41.3% of the bacteria proved to be viable. Carbon dioxide appears to be essential for the growth of certain oral anaerobes, i.e., Bacteroides melaninogenicus and Fusobacterium nucleatum (Loesche, unpublished results). These organisms, readily identified by distinct colonial appearance, were not observed in the role tube isolates. Thus, the omission of CO2 from the OFG would be expected to give lower recoveries than might otherwise be achieved by the inclusion of this gas. The experiment was repeated with the addition of 5 % CO2 in the OFG. Eight samples which contained an average of 26.9 i 11.7 x 1010 bacteria per gram (wet weight) were each treated as before. The aerobic count was 2.2 ± 1.5 X 1010/g (wet weight); the conventional anaerobic count was 5.4 ± 2.9 X 1010/g (wet weight); whereas the a Standard deviation. Differences between aerobic and anaerobic (Brewer Jar), P = 0.18; between aerobic and anaerobic (roll tubes), P = 0.03; and between anaerobic (Brewer Jar) and anaerobic (roll tubes), P = 0.06. aStandard deviation. Differences were significant between aerobic and anaerobic (Brewer Jar), P = 0.01; between aerobic and anaerobic (roll tubes), P = <0.01; and between anaerobic (Brewer Jar) and anaerobic (roll tubes), P = 0.01. roll tube count was 17. 9 7.1 X 10'0/g (wet weight; Table 2). All differences were highly significant, i.e., P < 0.01. The roll tube recovery was 70.4% of the microscopic count and included colonies of B. melaninogenicus which appeared in five of the samples. Viable counts of this organism alone averaged 5.4% of the microscopic count. Aerobic and conventional anaerobic counts represented 8.7 and 20.6%, respectively, of the total count High recoveries of bacteria from the rumen (5) and from the mouse cecum (10) were achieved with 100 % CO2 as the OFG. Accordingly, the experiment was repeated by using 100% CO2. Eight samples containing 15.5 + 3.9 X 10it bacteria per gram (wet weight) were dispersed, diluted, and plated as before. The recoveries obtained were as follows: aerobic, 2.0 4 0.4 x 1010/g (wet weight); conventional anaerobic, 3.7 i 0.9 X 101°/g (wet weight); roll tube, 9.8 i 2.8 X 1010/g (wet weight; Table 3). DISCUSSION Anaerobic incubation yielded significantly higher recoveries than aerobic incubation. Of the two anaerobic procedures tested, the roll tube method, when the incubating atmosphere contained CO2, permitted significantly higher recoveries than the anaerobic jar method ( Table 4). The only differences in sample manipulation between these methods in the present experiment had to do with the plating of the diluted sample. a Standard deviation. Differences were significant between aerobic and anaerobic (Brewer Jar), P = <0.01; between aerobic and anaerobic (roll tubes), P = < 0.01; and between anaerobic (Brewer Jar) and anaerobic (roll tubes), P = <0.01. a Per cent recovery = viable count divided by microscopic count X 100. b 4, standard deviation. Differences were significant between aerobic and Brewer Jar in an atmosphere of 100% CO2, P = <0.01; between aerobic and Brewer Jar in an atmosphere of 85% N2, 10% H2, and 5% CO2, P = <0.01; between Brewer Jar and roll tube in an atmosphere of 100% CO2, P = <0.01; between Brewer Jar and roll tube in an atmosphere of 85% N2, 10% H2, and 5% CO2, P = <0.01; and between atmospheres of 90% N2 and 10% H2 and 100% CO2 with roll tube, P = 0.02. In the roll tube method, the sample was added to agar medium maintained at 48 C in the absence of atmospheric oxygen. The colonies developed within and on the surface of the agar medium. In the anaerobic jar method, the diluted samples were exposed to atmospheric oxygen during the brief period in which they were spread over the agar surface. The inferior recovery of the anaerobic jar method would seem to be due to the exposure of the bacteria or medium, or both, to oxygen. In different experiments, the time between plating and removal of air from the anaerobic jar varied between 20 and 45 min. This brief exposure to air apparently is sufficient to kill many strict anaerobes (6). Similar findings were reported recently by McMinn and Crawford (7). They found that Propionibacterium propionicum would not survive beyond 30 min in aerobic transport broth. The higher counts obtained with the roll tube method raised the question as to whether these isolates are more oxygen-sensitive members of known oral species or whether they are to date undescribed species. Spears and Freter (10) reported that 10 roll tube isolates, on subculture, would not grow in anaerobic jars. Several rumen species, isolated and maintained in roll tubes, would not grow as surface colonies in the presence of oxygen levels greater than 0.7%. However, several oral species, isolated and maintained in Brewer Jars, grew reliably in the presence of 4% oxygen (6). It would seem that some anaerobic bacteria are uniquely sensitive to oxygen and are different from others that can survive short exposures to air and grow in the presence of low levels of oxygen. Thus, many of the roll tube isolates upon further investigation may prove to be species not presently known to reside in the oral cavity. The most difficult problem in maintaining strict anaerobiosis was during the sample collection. The subject was seated in the laboratory and the sample was transferred from crevice to an oxygenfree test tube in less than 5 sec. In previous count studies of human gingival debris (2,3,9), material was collected aerobically and subjected to atmospheric conditions for an unspecified time before anaerobic incubation. The present investigation in terms of total microscopic count and viable recoveries in anaerobic jars compared quite well with the results of these previous studies. However, the present recovery from roll tube was about three times higher. This would suggest that the failure to grow more gingival organisms in these earlier studies was due to a killing effect of atmospheric oxygen during the manipulation of the sample prior to anaerobic incubation (1). The roll tube procedure is a relatively simple technique for obtaining an anaerobic environment suitable for the isolation of strict anaerobes which colonize in large numbers the mucous membranes of mammals. The necessary equipment is inexpensive and requires little bench space. The technique itself is simple, and, given its marked superiority in isolating bacteria from a wide range of specimens, should be a routine procedure in various laboratories.

v3-fos

2020-12-10T09:04:10.941Z

1971-08-01T00:00:00.000Z

237232001

s2

Effect of Soy Proteins on the Growth of Clostridium perfringens Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an Eh of below −300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N2-10% CO2) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an Eh of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N2-10% CO2) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease. Clostridium perfringens foodborne disease continues to be a major concern to the food industry (2), especially that portion associated with cooked meat products which may be handled improperly and will support growth of C. perfringens. Increases in commercial production of synthetic meats and increases in protein supplementation of natural meat systems directed our interest into this area. There is little current literature to determine what significance this addition of proteins has on the potential growth of C. perfringens. Our objective has been to determine whether protein fractions from sources such as soy protein enhance, retard, or do not affect the growth of C. perfringens. Protein fractions that are used to fabricate imitation foods such as synthetic meats and to supplement standard meat formulations were evaluated. A preliminary report of these findings has been presented (F. F. Busta, D. J. Schroder, and M. W. Ewers, Bacteriol. Proc., p. 2,1970). MATERIALS AND METHODS C. perfringens strain S40 was obtained from H. E. Hall (National Center for Urban and Industrial Health, Cincinnati, Ohio). Stock cultures were maintained in Cooked Meat Medium (BBL) at room temperature. Inoculation cultures were grown at 45 C for 18 hr in Thioglycollate Medium without added dextrose (BBL). The culture inoculum was centrifuged at 4,080 X g for 10 min and decanted. The pellet was dispersed in 200 ml of sterile 6.25 X 1(4 M phosphate buffer (pH 7.0) and centrifuged. The pellet was dispersed in buffer, and the procedure was repeated twice. The final pellet was resuspended in 20 ml of phosphate buffer and serially diluted to obtain the proper inoculum. Microscopic examination indicated negligible clumping. All procedures were carried out under aseptic conditions. The generation time was calculated by the formula The stirring assembly and the inoculation needle were autoclaved in the medium at 121 C for 15 min. The platinum and glass electrodes were sterilized by immersion in a 0.05% sodium hypochlorite solution for 20 min followed by rinsing in sterile distilled water before inserting in the growth medium. Studies showing the effect of added protein to meat loaf were carried out by the methods previously outlined (D. J. Schroder and F. F. Busta, J. Milk Food Technol., in press). Growth media. Various test proteins were substituted for Trypticase (BBL) in the thioglycolate medium without added dextrose (Table 1) and autoclaved at 121 C for 15 min. The soy protein and sodium caseinate protein concentrations were adjusted to contain the same protein concentration (w/w) as the Trypticase control. Manufacturers' specifications were used for protein concentrations. This medium was selected as the growth medium because no sugar was present and the sole protein or peptide source was Trypticase. The meat loaf medium has been previously described (Schroder and Busta, J. Milk Food Technol., in press). One part protein additive was added to five parts meat unless stated otherwise. The growth media were steamed before inoculation to ensure a low oxidation-reduction potential. The pH of each medium was adjusted to 7.0 + 0.1. The growth vessels were placed in a constant-temperature water bath maintained at 45 C. The Eh and pH were monitored throughout the incubation. Samples were taken at appropriate times to determine the extent of the lag, the log, and final stationary growth phase. These were serially diluted in 0.1% peptone dilution blanks, and the viable cell populations were estimated by plate count in triplicate with freshly prepared Trypticase-sulfite-yeast-citrate-agar. This medium contained Trypticase (BBL), 15.0 g; yeast extract (BBL), 10.0 g; sodium sulfite (Allied Chemical). 0.5 g; iron citrate (Mallinckrodt), 0.5 g; agar (Difco), 13.9 g; water (deionized), 1,000.0 ml; final pH 7.0 + 0.1. This is similar to SPS agar (1) with the selective agents deleted. The agar plates were overlaid and incubated anaerobically at 35 C for 18 hr in an atmosphere of 90% N2 and 10% CO2. The t test was employed to test the significance of differences between growth in the presence of the soybean proteins, sodium caseinate, beef, turkey, or combinations and growth with the standard Trypticase or meat control (4). RESULTS The reproducibility in population, E1,, and pH in three trials with C. perfringens S40 in thioglycolate medium is demonstrated in Fig. 1. Initiating growth at several levels had little effect on the apparent growth rate, maximum population, or generation time. The relative pH and Eh values for each of the three trials are represented in the lower portion of the figure. The pH remained between 7.5 and 7.0, and the Eh generally registered below -300 mv down to -500 mv. The influence of eight soy protein supplements and one sodium caseinate protein supplement on the growth of C. perfringens was tested. Generation times of two trials are presented in Table 2. The two right columns of the table relate the ratio of generation time in the protein under test to that observed in Trypticase during the same trial. Isolated soy protein batch 1, whipping protein, and soy glycinin resulted in significantly shorter generation times in comparison to Trypticase (t test, 5% level). The soy flour brand B, soy protein concentrate, textured soy flour, and isolated soy protein batch 2 sources resulted in generation times similar to Trypticase. Sodium caseinate and soy flour brand D were significantly inhibitory to the growth of C. perfringens (5% level). Data presented in Fig. 2 show that, when isolated soy protein batch I was substituted for Trypticase, the generation time of C. perfringens S40 was reduced by more than one-third and the maximum extent of growth was increased 10-fold. Table 3 relates data obtained in eight trials comparing isolated soy protein batches 1 and 2 to Trypticase as the protein or peptide source in thioglycolate medium. In the case of batch 1, maximum populations of approximately 108 were obtained in each of the four trials. In batch 1, all four trials of the isolated soy protein resulted in a higher population. The ratios of generation times in isolated soy protein to Trypticase ranged from 0.61 to 0.85 in the four trials. With batch 2 of the isolated soy protein, the growth was essentially the same in either medium. Data in Table 4 show the influence of substitut-ing beef, soy beef, or combinations of beef plus soy beef or beef plus textured soy flour for the Trypticase protein source in liquid media. Data on growth in turkey, soy chicken, or combinations of turkey plus soy chicken or turkey plus textured soy flour in liquid media are also shown in Table 4. With Trypticase as the control, the use of beef or beef plus soy beef, soy beef alone, or beef plus textured soy flour as the sole source of protein produced significantly shorter generation times. The use of turkey, soy chicken, and combinations of turkey and soy chicken or turkey plus textured soy flour produced generation times that were not significantly different from those observed with Trypticase. Data used in Table 4 were reevaluated as shown in Table 5. These data show that soy beef or textured soy flour plus beef did not significantly influence growth of C. perfringens when compared to beef alone as the protein source. Generation times observed with soy chicken or textured soy flour plus turkey were not significantly different from those observed in turkey alone (Table 5) when these were used as sole protein sources in liquid media. Note that growth rates in beef or soy beef were more rapid than in turkey or soy chicken. Table 6 summarizes data on growth of C. perfringens under actual meat loaf conditions. The effects of the addition of soy beef, soy protein, and sodium caseinate to beef are shown as they relate to the beef control. Soy beef was used alone as a meat loaf but did not support growth to any extent. These data show no significant effect on growth by the additives to beef. The data also show that there was a significant difference between beef and turkey and between beef and turkey plus Trypticase, i.e., growth in turkey and turkey plus Trypticase was slower than growth in the beef control. The addition of sodium caseinate and soy glycinin to turkey increased the growth rate of C. perfringens to the level observed in beef. Under meat loaf conditions with turkey as the control, the addition of one part soy chicken to five parts turkey had no significant effect on the growth of C. perfringens (Table 6). The addition of a 1 :1 mixture of soy chicken and 1: 5 additions of isolated soy protein, sodium caseinate, Trypticase, or soy glycinin to turkey all significantly increased the growth rate in the turkey meat loaves. Soy chicken alone supported little or no growth. The Eh remained at +100 to +200 mv, and there was slow death or no growth until about the 6th hr of incubation. Some growth did take place when the Eh was reduced to approximately -30 to -50 mv. This is in contrast to the meat systems which quickly dropped the Eh to -300 mv and reached -500 mv after 7 hr of incubation. DISCUSSION Stimulation of the growth of C. perfringens was observed with some soybean protein products when compared to Trypticase. These include an isolated soy protein. A basic difference between the products that cause stimulation and those that do not is the amount of refinement by the removal of a major portion of the nonprotein components. Manufacturers' literature states that isolated soy proteins are over 92% protein and the whipping protein and soy glycinin are over 80% protein. The soy products that did not appear to stimulate growth ranged from 50 to 55% protein, with the exception of the soy protein concentrate which is approximately 70% protein. The processes involved in the concentration or isolation of the soy protein apparently improve it as a growth medium for C. perfringens. This could be due to the removal of some growth inhibitors or to some protein modification which could make the protein more available to the organism or to some change in availability of certain amino acids. The whipping protein has undergone some enzymatic modification which may effect some breakdown of the protein and facilitate easier uptake by the organism. Another factor may be the further concentration and isolation of the soy protein which in turn may affect the solubility of the soy protein and thus its availability to the organism. This study has shown that, in the case of the isolated soy protein, some batches may have stimulative activity whereas others may not. This suggests that inadvertent changes in the process may modify the end product and cause a change in solubility or in some modification of the soy protein which may affect the growth of C. perfringens. Another possibility is that some factor may be added to the product to increase its functional properties or to prevent some undesired reactions. Similarly, the converse may be applied to the soy flour brand D which caused some inhibition Generation time for beef appears in parentheses. b Generation time for turkey appears in parentheses. of the growth of C. perfringens. Sodium caseinate showed a major inhibition on the growth of the organism. Again protein availability and the presence of some inhibitory factors may play a part in its effects on growth. The rate of growth of C. perjringens in liquid medium in which beef or soy beef was the sole protein source was more rapid than in Trypticase. This may be due to readily available growth factors in beef. Under meat loaf conditions, the rate of growth was rapid in beef and the additions of various amounts of soy beef, soy glycinin, isolated soy protein, and sodium caseinate had little effect on the growth rate. This indicates that beef is an excellent medium for the growth of C. perfringens and added protein sources have little effect. Soy beef alone as a meat loaf supported little or no growth. Under meat loaf conditions, as in the liquid medium, the growth of C. perfringens was slower in turkey than it was in beef. The additions of soy chicken, isolated soy protein, sodium caseinate, Trypticase, and soy glycinin significantly stimulated the rate of growth of this organism in turkey meat loaves. The additions of sodium caseinate or soy glycinin to turkey brought the rate of growth to a level equivalent to that of beef. As with meat loaves containing soy chicken alone, soy beef alone did not support any substantial growth of C. perfringens. Eh values in these meat loaves remained high throughout the 6-hr incubation period. These observations with soy meat loaves may be due to the physical nature of the meat loaf (i.e., somewhat more porous than regular meat loaves) or due to some other inadequacy of the soy meats. In summary, these results show that certain soybean proteins were stimulative or inhibitory to growth of C. perfringens. Natural meat systems are highly stimulatory. Under actual meat loaf conditions, beef appears to be an excellent growth medium and the addition of soy meat, soybean, or other protein additives does not appear to affect its potential for fast growth of this organism. These observations are consistent with earlier findings (Schroder and Busta, J. Milk Food Technol., in press). This is not the case for turkey, in which protein additives did significantly enhance the growth of this organism. The stimulation may be due to the addition of certain factors which enhance the growth of C. perfringens, i.e., factors which are present in beef or soy protein and not in turkey. The presence of stimulative or inhibitory factors in some soybean products or other proteins could be of public health concern and warrant their identification. Food processors fabricating synthetic meats or supplementing natural meats with protein preparations should be aware of their potential influence on growth of C. perfringens.

v3-fos

2020-12-10T09:04:11.250Z

1971-05-01T00:00:00.000Z

237234464

s2

Detection of Sulfa Drugs and Antibiotics in Milk A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination of Dairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy. The Grade "A" Pasteurized Milk Ordinance of the Food and Drug Administration (4) requires that producers' milk or commingled milk be tested for antibiotics at least four times during any consecutive 6-month period. Although sulfa drugs are commonly used along with antibiotics to treat bovine mastitis, the test procedure used (1) is generally insensitive to sulfa drugs. Chemical methods are available for detecting sulfa drugs in milk; however, the analytical procedures involved are far more complex than the antibiotic test now in use for routine regulatory testing (3,5). We believed it would be desirable to develop a procedure for routine regulatory testing that would be useful in detecting sulfa drugs as well as the antibiotics commonly used to treat mastitis, provided that the test developed was not more complex than the Bacillus subtilis method described in Standard Methods for the Examination of Dairy Products (1). This paper reports the development of a testing procedure that meets this requirement. MATERIALS AND METHODS Preparation of assay plates. Three sporeforming and two nonsporeforming organisms were studied as candidate test organisms for sensitivity testing. These were B. subtilis ATCC 6633, B. cereus ATCC 11778, B. megaterium ATCC 9855, Sarcina lutea ATCC 9341, and Escherichia coli ATCC 11229. B. subtilis spores were obtained from Difco Laboratories, Inc. Spore suspensions of B. cereus and B. megaterium were prepared by growing the cells in AK Sporulation Medium No. 2 (BBL) in 6-oz prescription bottles incubated for 48 hr at 35 C. Suspensions of S. lutea and E. coli were prepared daily from slants of Mueller-Hinton agar that had been incubated for 18 to 20 hr at 32 C. Three successive transfers were made before the cells were harvested and used. Growth from sporeforming or vegetative cell cultures was washed from the agar with phosphate-buffered distilled water and centrifuged at a relative centrifugal force of 5,000 for 15 min at 3 C, and the centrifugation-washing process was repreated three times. Spore crops were stored in buffered dilution water at 4 C until used. All inocula (vegetative cells or spores) were adjusted in optical density to give a final concentration of about 5 X 104 per ml of agar. The inoculum was added to the agar at 50 C, the inoculated agar was mixed gently by swirling to avoid air-bubble formation, 4 ml of agar was pipetted into 90-mm inside diameter plastic petri dishes, and the agar was distributed over the dish by swirling the dish. Fresh agar was prepared for each day of testing, and the spores, when used, were not heat-shocked. Preparation and testing of milk samples. Farm-bulktank or dairy-storage-tank raw milk was used for this study. Discs containing a sulfa derivative or an antibiotic were prepared by weighing the inhibitor under test and suspending it in distilled water. From this, an appropriate amount was added to milk, and twofold serial dilutions were made of the inhibitor in milk. One-tenth mililiter of milk containing the inhibitor was added to 0.5-inch (12.7-mm) blank discs (Carl Schleicher and Schuell Co.), and the discs were placed on the inoculated solidified agar for inhibitory testing. Temperatures of 25, 32, 35, and 37 C were used for various incubation times. Zone measurements were made with a vernier caliper with the plates illuminated from the back by fluorescent light. Zones, 15 mm in diameter or larger, were recorded as positive (disc diameter is 12.7 mm). For routine testing, any zone should be recorded as positive providing the proper controls are run. The 15-mm criterion was used in this study because it gave a zone that could not be mistaken even by a relatively untrained analyst. All minimum sensitivities reported were obtained in at least two trials. RESULTS AND DISCUSSION Eight sulfa drugs and nine antibiotics were selected for study, and the inhibitor testing procedure listed in Standard Methods for the Ex- amination of Dairy Products was used to establish the sensitivity of the procedure now in general use for regulatory testing to the inhibitors selected (Table 1). We believe that the general insensitivity of the standard method to sulfa drugs was the result of the medium (Antibiotic Medium No. 1) containing p-aminobenzoic acid or folic acid or both. Since B. subtilis produces bacitracin, it is insensitive to it. Mueller-Hinton agar has been widely used for the detection of sulfa drugs, and this medium was selected to test the sensitivity of five species of bacteria to the 17 inhibitors under study ( Table 2). In all cases, incubation was at 37 C for a previously determined incubation time that gave optimal zones with the test organism. S. lutea and B. megaterium were more sensitive to the test inhibitors than were the other three organisms tested. B. megaterium was selected for further study inasmuch as a sporeformer is preferable to a nonsporeformer for routine testing because it can be purchased in readyto-use form and stored until used. This would make the test easier to standardize between laboratories. Heat shocking the spores of B. cereus, B. subtilis, and B. megaterium did not enhance sensitivity or significantly reduce assay time. Since 37 C incubation is not commonly used in milk testing laboratories, incubation temperatures of 35, 32, and 25 were studied to determine whether they might be as useful as 37 C VOL. 21, 1971 Incubation temp (C) 25 (12) ( Table 3). In general, the sensitivity of the procedure increased with increased incubation temperature, and 37 C was the temperature of choice. Standard Methods for the Examination of Dairy Products recommends that all milks be heated to 82 C for 2 to 5 min to avoid reporting false-positives resulting from natural inhibitory substances in raw milk. Since this is part of the testing procedure and would affect apparent sensitivity if any of the inhibitors were sensitive to heat, all 17 inhibitors under study were assayed in milk before and after heating when incubated at 25, 32, 35, and 37 C. In no case was the apparent sensitivity of the test changed by more than one step in a twofold dilution series after the milk containing the inhibitors had been heated. This variation is normal for this procedure when used for repetitive testing of milk containing an inhibitor. Unlike the results of Marth,Alexander,and Hussong (2) in studies of the effect of heating milk on apparent sensitivity of assay, the heating technique of 82 C for 3 min had no effect on penicillin. This may be due to the heating techniques used since Marth et al. steamed their test milks for 7 min before testing. With penicillin, penicillinase discs are used for identification in that the antibiotic is inactivated by the enzyme. Similarly, we tested 0.5inch (12.7-mm) discs impregnated with 50 ,ug of p-aminobenzoic acid for their usefulness in identifying an inhibitor as a sulfa drug. These discs were made by adding the acid in aqueous solution to the disc, followed by drying the disc at 40 to 44 C. These discs inactivated the inhibitory properties of all sulfa drugs studied in concentrations of at least 5 ,g of sulfa derivative per disc. Accordingly, we believe this technique is useful in identifying an unknown inhibitor as a sulfa drug. From the alternatives studied, we believe the following to be most useful for the detection of sulfa drugs and antibiotics in milk: (i) add B. megaterium ATCC 9855 spores to Mueller-Hinton agar at 50 C to give a final spore concentration of 5 X 104 per ml of agar; (ii) dispense 4 ml of inoculated agar into a flat-bottom petri dish of about 90-mm inside diameter and agitate the dish so that the agar will cover the dish surface; (iii) allow agar to solidify on a level surface; (iv) touch edge of 0.5-inch (12.7-mn) filter disc to milk sample to wet disc by capillary action, and place disc on surface of inoculated agar; (v) incubate plates at 37 C for 4 to 5 hr; (vi) examine for zones of inhibition; and (vii) identify zones using conventional techniques as described in Standard Methods for the Examination of Dairy Products (1) including heating the milk at 82 C for 2 to 5 min to detect natural inhibitors. Discs containing p-aminobenzoic acid may be used to identify inhibition from sulfa drugs. We believe that the substitution of the B. megaterium-Mueller-Hinton procedure for the one now in common use for testing milks offers the advantage of sensitivity to sulfa derivatives and to bacitracin. This is accomplished without significant change in sensitivity to the other antibiotics tested and without making the test procedure more difficult to perform. For these reasons, we feel that the procedure described merits consideration as a standard method for the regulatory testing of antitiotics and sulfa drugs in milk.

v3-fos

2020-12-10T09:04:10.767Z

1971-02-01T00:00:00.000Z

237229154

s2

Method for Studying Particle Size and Infective Potential of Infectious Bovine Rhinotracheitis Virus Aerosols A technique is described for estimating the number of potential respiratory infectious loci of aerosolized infectious bovine rhinotracheitis virus. A technique is described for estimating the number of potential respiratory infectious loci of aerosolized infectious bovine rhinotracheitis virus. The Anderson (1) aerodynamic particle sizing sampler was used in the past decade to collect and size bacterial particulates. Airborne particles are impacted directly on nutrient agar surfaces in six stages of aerodynamic sizes. Because of localized drying of the cell monolayer at the impaction site, it is implausable to collect virus particles directly on a cell monolayer. The usefulness of the Andersen sampler was extended to virus collection by Jensen (3), who washed the agar surface and assayed the washings for plaque-forming units (PFU). Guerin and Mitchell (2) and Wolfe et al. (4) proposed a gelatin collection surface which was melted and assayed. These procedures provide an estimate of PFU for each size category of the sampler but do not provide information as to the number of particulates involved. A single particle may contain many PFU but produce only one locus of infection. Alternatively, many particles may each contain few PFU, and thus a large number of loci could be produced from a small quantity of virus. These differences result not from variations in the virus but from dissimilarities in the manner in which it was aerosolized. A knowledge of these parameters is desirable in the careful study of dose response relationships of airborne infection. A technique capable of providing an estimate of the number of infectious particles and hence an estimate of the number of potentially infected loci in the lungs is presented here. The technique consists of collecting the aerosol by jet impaction on agar-solidified tissue culture media. After collection the agar is inverted on a monolayer of susceptible cells thus forming an overlay. Two strains in infectious bovine rhinotracheitis (IBR) virus were utilized in developing this sampling technique. Strain "V" is a vaccine strain originally obtained from Armour-Baldwin Laboratories. This particular strain was useful because it was a readily available attenuated virus and produced consistant plaque characteristics in testing the technique. The other strain, LY 985-3, was isolated from a field case of IBR at Utah State University. Specific viral suspensions were aerosolized into a test chamber of 2 ft3 with a deVilbis clinical atomizer and equilibrated for 1 min. The virus laden particulates were collected from the test chamber by using an aerodynamic sizer model 30 (AIR, Inc., Logan, Utah). Molded glass collection plates filled with 27 ml of tissue culture media solidified with 1.5% Noble Agar (Difco) were used. The agar surface may be conditioned by storage for several days or by inverting the opened dishes on a sterile rack for a few hours before use in the sampler. This conditioning removes the surface water which tends to distort the impaction pattern. Water removal also assists in applying the agar surface to the monolayer without slippage. Transfer of the agar from the collection plates to the cell monolayer is facilitated by cutting a ring in the agar }j inch from the collection plate wall. A no. 11 surgical blade fitted with a gauge may be used for this purpose. The peripheral ring of agar is pulled from the corner of the dish while inverted. The agar disc is then held vertical and pried away from the glass plate with a sterile spatula. About one-third of the disc can be separated from the glass without danger of the agar falling. The spatula is then carefully inserted under the agar as the plate is turned back to horizontal. The agar layer may then be transferred to the cell monolayer in a petri dish from which the liquid media have been aspirated. Any air trapped under the agar must be removed and can be done by stroking the agar with the spatula or a sterile swab. Plaques appear as necrotic areas in the cell monolayer when viewed with the low power microscope. The use of a petri dish clamp on a mechanical stage facilitates scanning the areas to be counted. The results of aerosols generated from two virus suspensions are shown in Table 1. The distribution of counts among the six stages reflects the particle size distribution of the aerosol. The LY 985-3 suspension had a virus titer approximately 10 times that of strain "V." Precautions against including areas of damaged cells in the plaque count should be observed. The immediate areas of the agar edge and bubble areas can be removed from consideration in the plaque count by use of counting masks made by punching holes of known size in self-adhesive paper stock. The masks can be placed on the petri dish over randomly selected areas that are not otherwise affected by the factors mentioned. For example, since the impaction areas of each stage of the sampler are approximately 7.07 square inches, a circular mask 1 inch in diameter represents 11.1 % of the collection surface. Therefore, the average count of 1-inch masks should be multiplied by 9.0 to obtain an estimate of the number of positive impact areas per stage containing at least one PFU. Characteristics of the model 30 aerodynamic sizer permit more than one particle to be collected on a specific impact area. This may result in a cluster of microplaques in one impact area. Such areas merge into single plaques with time and should be counted as one plaque. This estimate may then be transformed to a particle count estimate by use of a conversion table supplied with the instrument. This technique, therefore, estimates in each of six size categories the number of potentially infectious particles in the respirable air of experimental animals.

v3-fos

2020-12-10T09:04:17.490Z

1971-07-01T00:00:00.000Z

237234386

s2

Microtiter Tests for Detecting Antibody in Bovine Serum to Parainfluenza 3 Virus, Infectious Bovine Rhinotracheitis Virus, and Bovine Virus Diarrhea Virus Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera. This report is part of an investigation of the shipping fever complex of cattle in which parainfluenza 3 virus (PI 3), bovine virus diarrhea virus (BVD), and infectious bovine rhinotracheitis virus (IBR) have been incriminated. Since antibody to PI 3 is measured primarily by hemagglutination-inhibition (HI) tests whereas antibody to IBR and BVD is measured by the more timeconsuming serum-neutralization tests (SN), much more serological data have been obtained for PI 3 than for the latter two viruses. The miniaturization of the HI test by Takatsy (14) and Sever (12) and the development of suitable microtiter equipment have increased the ease with which the HI test can be performed. Recently the utility of the microtiter system has been further increased by its adaptation to SN tests (10,11,13). The purpose of this research was to develop micro-SN tests for measuring antibody to BVD and IBR and to investigate the micro-HI test for PI 3. Although nonspecific HI inhibitors to PI 3 have not been found in bovine serum (4,8), the significance of HI titers of 20 or less has been questioned (1). Our initial results with the micro-HI test also led us to question the significance of titers of 10 and 20. Since others have reported that treatment of bovine serum with kaolin, bentonite, trypsin, receptor-destroying enzyme (RDE), or periodate did not produce a reduction in the HI titer (4, 8), we investigated the effect 32 of treatment of serum with trypsin and periodate-We also compared the micro-SN with the micro-HI test for detecting antibodies to PI 3. Henceforth, HI and SN will refer to micro-HI and micro-SN, respectively, when used in reference to the work performed in our experiments. MATERIALS AND METHODS Cell cultures. The Madin-Darby bovine kidney (MDBK) cell line and bovine embryonic kidney (BEK) cells between the 20th and 40th passage were used. Cultures for SN tests were grown in a yeast extract-Eagle medium-lactalbumin hydrolysate-peptone (YELP) medium (9) with 10% fetal bovine serum, 200 units of penicillin per ml, 200 ,ug of streptomycin per ml, and 100,g of neomycin per ml. Viruses. The following viruses were used in HI and SN tests and were obtained from G. H. Frank of the National Animal Disease Laboratory, Ames, Iowa: (i) IBR, (ii) the SF4 strain of PI 3, and (iii) the NADL strain of BVD. Calves. All calves were between 6 and 10 months of age and were from Auburn University and two Auburn University Agricultural Experiment Substations. Immunization. Three New Zealand White rabbits were given 2 X 107 TCID15 of the SF-4 strain of PI 3 intranasally four times biweekly. Three calves (no. 46-48) were given 8 X 107 TCID50 of the SF-4 strain of PI 3 intranasally followed by an intramuscular injection with the same amount of virus 2 weeks later. The calves were kept in isolation after immunization Vol. 22, No. 1 was started. Three calves (no. 49, 50, and 109) from the same herd were kept as controls in another isolation room. Thirty calves from a single herd were divided so that 15 calves were given a combined live BVD-IBR-PI 3 commercial vaccine (Pitman-Moore, Indianapolis, Ind.) intramuscularly twice biweekly; 15 calves were kept as contact controls. This sera were tested for neutralizing antibody to BVD and IBR. Treatment of sera used in HI test. All sera, unless otherwise indicated, were treated with trypsin and periodate as described by Davenport and Minuse (3), which includes heating at 56 C for 30 min, and subsequently adsorbed with guinea pig red blood cells (RBC). Untreated sera were heated at 56 C for 30 min and adsorbed with guinea pig RBC. Sera used for comparing HI and SN titers to PI 3. Two hundred sixty-seven serum samples were obtained from 61 unimmunized calves over a period of 2 months. Some animals had clinical signs of respiratory infection for 1 or 2 weeks, whereas others appeared healthy during the interval sera were collected. test. The HI test described by Sever (12) was performed as follows. Phosphate-buffered saline (pH 7.2) was used as diluent. Dropper pipettes and dilution loops calibrated to deliver 0.025 ml were used with "U" bottomed Lucite plates (Cooke Engineering Co., Alexandria, Va.). Duplicate wells were used for each dilution of serum. A drop of serial twofold dilutions of serum was incubated with a drop of the SF-4 strain of PI 3 containing 4 to 8 units of hemagglutinin at ambient temperature for 1 hr. Two drops of guinea pig RBC, standardized to contain 5 X 107 cells/ml as recommended by Hierholzer and Suggs (6), were added, and the plates were held at 5 C overnight. The HI titer was considered the reciprocal of the highest dilution of serum in which complete agglutination did not occur. The lowest dilution of serum tested was 1::0. SN test. The SN test was performed with the same equipment used in the HI test except MicroTest II plates and lids (Falcon Plastics, Los Angeles, Calif.) were used instead of those used in the HI test. Fetal bovine serum was pretested for antibodies to BVD, IBR, and PI 3 before use in growth medium. The general procedure for the SN test has been described (10,11,13). Duplicate wells were used for each dilution of serum. A drop of serial twofold dilutions of serum was incubated with a drop containing 50 to 100 TCIDw of IBR, PI 3, or BVD at 37 C for 1 hr. A drop containing 200,000 cells per ml was added to each well, and the cultures were incubated at 37 C. MDBK cells were used for PI 3 tests, and BEK cells were used for IBR and BVD tests. Titers were determined for PI 3 on the 6th day, for IBR on the 4th day, and for BVD on the 7th day. Titers were taken as the reciprocal of the highest dilution of serum to protect completely cells in both wells. The lowest dilution of serum tested was 1:4. With continuous use, the microtiter dilution loops became toxic and prevented growth of cells. Considerable care in washing and flaming loops was necessary to prevent toxicity from developing. RESULTS Effect of trypsin-periodate treatment on HI titer to PI 3. Two hundred forty-one serum samples obtained from 153 calves were tested both untreated and trypsin-periodate-treated for antibody to PI 3. Thirty-seven sera had titers of less than 10 in both the untreated and treated samples. One hundred fifteen sera had their titer reduced after treatment, whereas 89 sera had the same titer whether treated or untreated (Table 1). No instance was found in which a treated sample had a higher titer than an untreated sample. No bovine serum sample had its titer reduced more than a twofold dilution. Sera obtained from calves at different times did not respond consistently to trypsin-periodate treatment. Correlation between HI and SN titers to PI 3. HI titers of trypsin-periodate-treated sera were compared with SN titers of the same sera. Of the sera with HI titers of either less than 10 or 10, 74 and 14%, respectively, had SN titers of less than 4. When HI titers were at least 10, SN titers averaged higher than corresponding HI titers ( Table 2). Development of antibody to PI 3 after immunization. A comparison between the HI and SN tests for measuring the antibody response to PI 3 was made. The response of calves to immunization with PI 3 was not noticeable with the HI test, whereas the response was very obvious with the SN test (Table 3). Rabbits responded with considerably higher titers than calves, and their response was measurable by both HI and SN. As with bovine serum, rabbit serum yielded higher titers by SN than HI ( vaccine, none had prevaccination titers to IBR' Antibody was first detected in the serum of 8 of the 15 calves by 2 weeks. Maximum titers were detected 6 to 8 weeks after starting immunization and then declined. All vaccinated calves developed antibody to IBR. The distribution of the SN titers is shown in Table 5. Titration end points were usually sharp so that the transition from complete neutralization to no neutralization occurred within two or three dilutions. None of the control animals had antibody to IBR. SN test for detecting antibody to BVD. Of the 15 calves given the combined BVD-IBR-PI 3 vaccine, none had SN antibody for BVD in their prevaccination sera or in sera collected 2 weeks after starting vaccination. One month after the first injection, SN antibody was present in all vaccinated calves. Antibody persisted throughout the duration of the experiment and gradually increased. Distribution of the antibody titers is shown in Table 6. Titration end points with BVD were not always sharp as with PI (4) obtained similar results. He found a good corresponce between HI and SN titers. However, only 7 sera with HI titers of 32 or less and 13 sera with HI titers greater than 32 were tested for neutralizing antibody. He also found that treatment of sera with either RDE, trypsin, periodate, kaolin, or bentonite failed to reduce HI titers from those of the corresponding untreated sera. We found that treatment of bovine serum with trypsin and periodate frequently reduced the titer of a serum by a twofold dilution. However, there was no correlation between the titer of a serum sample and its titer after treatment. Lowtitered sera as well as high-titered sera were equally affected by trypsin-periodate treatment. Furthermore, biweekly serum samples from individual calves varied in their response to trypsinperiodate treatment. Our data do not provide sufficient evidence to contradict the prevalent opinion that nonspecific HI inhibitors to PI 3 are either absent or infrequent in bovine serum. The significance of low HI titers is independent of the existence of HI inhibitors. Low HI titers may merely reflect an artifact of the HI test. Abinanti et al. (1) examined the sera of 788 cattle and concluded that HI titers of 20 or greater could be considered positive for antibody. This agrees with our results. Although several investigaors who have used tube tests have found the HI test to be more sensitive than the SN test for detecting antibody to PI 3 (1, 5, 7), our results with microtiter tests have been the opposite. We found the SN test was more sensitive than the HI test as judged by (i) its ability to detect antibody production in immunized animals (Tables 3 and 4), (ii) its ability to detect seroconversion more frequently than by use of the HI test, and (iii) by its ability to attain titers higher than those attained in the HI test (Table 2), except for instances at low dilutions in which no antibody could be found by SN when a positive reaction was obtained by HI. We found the SN test more specific than the HI test as judged by the fact that it demonstrated neutralizing antibody in all sera with HI titers above 20, but it did not demonstrate comparable neutralizing antibody in every serum which had an HI titer below 20. Considering the SN test was found to be more sensitive than the HI test and that HI and SN antibodies to parainfluenza viruses are probably directed against the same antigen(s), since they have the same specificities (2), it is likely that the presence of low HI titers without corresponding SN titers cannot be attributed to reactions against different antigens. SN tests for PI 3 and IBR both gave sharp titration end points. BVD end points often extended over more than three dilutions. Improvement in the test for BVD might be achieved by using fewer cells since the BEK cells used were more susceptible to BVD during stages of active growth than when confluent, especially when infected with small amounts of virus. In these experiments, SN end points were calculated as the last dilution of serum which gave complete protection of cells. Since BVD end points often extended over several dilutions, when the SN test is used for diagnostic purposes, it would be advisable to use more replicates per serumdilution and one of the standard methods of calculating 50% SN end points to increase the accuracy of the test.

v3-fos

2018-04-03T04:07:38.576Z

1971-03-01T00:00:00.000Z

24642545

s2

Microorganisms of the San Francisco Sour Dough Bread Process Two hundred isolates from San Francisco sour dough French bread fermentations (40 from each of five different bakeries) were screened by fermentation tests and for their ability to grow in the presence of cycloheximide (Actidione). All of the isolates from four of the bakeries and 70% of those from the fifth were unable to utilize maltose but grew well on other sugars, even in the presence of cycloheximide. The remaining few isolates from the fifth bakery utilized maltose but not galactose and were inhibited by cycloheximide. No bakers' yeast types were found. Sixteen of the maltose-negative and five of the galactose-negative isolates were subjected to more rigorous taxonomic procedures. All of the maltose-negative isolates were identified as asporogenous strains of Saccharomyces exiguus (Torulopsis holmii) and the galactose-negative ones, as S. inusitatus. The predominance of S. exiguus, its vigor in the particular acidic environment of the sour dough, and the correlation of its numbers with the leavening function constitute strong evidence on the role of this organism in the sour dough system. This and the subsequent study on the isolation and characterization of the microorganisms involved in the San Francisco sour dough bread process (3) were prompted by the absence, to our knowledge, of any prior reports on this subject although the process has been carried out in the San Francisco bay area for over 100 years. In the bakery, the microbiological activities responsible for the leavening (rising) and souring actions in the dough are perpetuated by rebuilding a special piece of dough known in the trade as the "starter" or "mother sponge" approximately every 8 hr with fresh flour and water. This starter sponge, when fully developed, serves as the "inoculum" for each batch of bread dough. In recent reports (4,7), some of us have described the mechanics of the sour dough process and the nature of the acidity developed (2) and have presented preliminary information on the two principal types of microorganisms involved. One, a readily isolatable yeast, is responsible for the leavening function. The other, an unidentified bacterial type, isolated with some difficulty and apparently for the first time, has been correlated with the souring activity. The present report supplies detailed information on the isolation, occurrence, and character-ization of the sour dough yeast isolates. Characterization of the sour dough bacteria is presented in a subsequent publication (3). MATERIALS AND METHODS Isolation of yeasts. Sour dough starter sponges were obtained from five different sources (bakeries) in the San Francisco bay area and maintained as needed by rebuilding with flour and water as described elsewhere (4). For isolation and enumeration of the yeasts, 11 g of sour dough was aseptically blended for 90 sec at reduced speed with 99 ml of sterile 0.1% aqueous peptone and a few drops of antifoam (tributyl citrate) in a sterilized 8 oz (ca. 226.8 g) Osterizer jar. After further serial dilution, also in the sterile 0.1% peptone, samples were plated out on selected agar media by either pour or spread plate techniques and incubated 2 days at 30 C. Total yeast counts were determined by using either Mycophil agar (BBL), adjusted to pH 4 after autoclaving with 20% lactic acid, as the plating medium or by using yeast extract (0.5%)-glucose (1.0%)-Trypticase (1.0%) agar. APT (BBL) agar, containing 100.,g of cycloheximide (Actidione) per g added before autoclaving, was used to test the ability of yeasts to grow in the presence of cycloheximide (5,8). Studies showed that cycloheximide added in this manner is effective in inhibiting the growth of those yeasts susceptible to it if autoclaving is kept to a minimum (15 min at 120 C) and Sourdough type I + + -+ -+ + Sour dough type 2 + + --+ + -Bakers' + + -+ ++ -aLiquid media were prepared by the method of Wickerham (9). Two hundred isolates were tested (40 from each of 5 sources). b +, Growth, acid and gas; -, no growth. the media are used within 2 months of preparation. The sour dough bacteria described in a subsequent publication (3) did not grow on any of the above media and hence did not pose a problem in the yeast isolation and enumeration. Identification of yeast isolates. Approximately 40 colonies for each of the five sources were picked from the Mycophil plates, transferred to Mycophil (pH 4) slants, and incubated 24 to 48 hr at 30 C. A loopful was then transferred to wort broth (BBL) which, after being incubated 18 to 24 hr at 30 C, was used as the inoculum in preliminary screening tests for carbohydrate assimilation or fermentation, or both, according to the procedure of Wickerham (9). Approximately 200 isolates were tested in this manner on glucose, sucrose, galactose, lactose, maltose, and raffinose. As will be shown, the yeast isolates appeared to fall into two types. Sixteen of the predominant type from four different sources and five of the other type, all from a single source, were then subjected to the more rigorous identification procedures described by Lodder and Kreger-van Rij (6) and the supplementary procedures of Lodder (5). Ability to assimilate various carbon compounds in these tests was measured by inoculating the yeasts on solid carbon assimilation medium composed of 0.7% yeast nitrogen base (Difco), 0.5% of the carbon compound to be tested, and 2% agar. Inoculation was done with a multipoint inoculating device as modified from Beech et al. (1). Where necessary, the liquid medium technique of Wickerham (9) was used to confirm assimilations obtained on the solid medium. Sporulation was tested by incubating 14 days on Kleyn's acetate agar and V-8 agar. RESULTS The 200 yeast isolates from five sources were found to fit into one of two types ( Table 1). The predominant type (sour dough no. 1) was unable to assimilate maltose but utilized other sugars, even in the presence of cycloheximide. The other type (sour dough no. 2) was unable to utilize galactose, and its growth on other sugars was inhibited by cycloheximide. A strain of bakers' yeast, isolated from commercial compressed yeast, was also tested for comparative purposes and, as expected, was able to utilize both maltose and galactose, thus being readily distinguished from the sour dough yeasts. The distribution of the two types of yeast isolates recovered from the five sources was determined in two ways. First, the 40 isolates from each source were grouped by their fermentation characteristics and cycloheximide-resistance (Table 2). The maltose-negative type which grew well in the presence of cycloheximide was found to be the only yeast present in four of the five sources b Symbols: F, fermentation and assimilation positive; +, positive assimilation or growth reaction; W, positive but weak; -, no assimilation or growth; and *, ferments only half of the disaccharide. 457 VOL. 21,y 1971 and the predominant type in the fifth. Differential counts in the sour doughs were also run on various agars as follows. The maltose-negative type was specifically isolated and enumerated on the APT agar (BBL) containing 100 jig of cycloheximide per g. The galactose-negative type was determined either by difference between the counts on Mycophil and APT-cycloheximide agars or specifically on a medium containing yeast extract (0.25%), Trypticase (0.5%), and maltose (0.25%). The level of maltose was kept low in this medium so that its impurities would not promote the growth of the other yeast type. These differential counts over a period of time confirmed the distribution of the two types of yeast in the five sources as established by the fermentation tests. Thus, for the four sources containing only the maltose-negative cycloheximide-resistant yeast, counts determined on the APT-cyclohexirride agar were always as high as or higher than those determined on Mycophil agar. The proportions of the galactose-negative strain in the fifth source averaged out about 30% as determined either by the difference between the counts on Mycophil and APT-cycloheximide agars or specifically on the yeast extract-Trypticase-maltose agar. Sixteen of the maltose-negative isolates selected from four difterent sources and five of the galactcse-negative isolates from the one source were subjected to the more rigorous taxonomic testing illustrated in Table 3. Based on these characteristics, the maltose-negative isolates were all identified as the imperfect form of Saccharomyces exiguus (Torulopsis holmii). Ascospore formation was not observed. The galactose-negative isolates were all identified as Saccharomyces inusitatus. Representative cultures of each type have been deposited in the culture collection at the USDA Northern Regional Research Laboratory, Peoria, Ill. DISCUSSION The presence of S. exiguus in sour doughs to the virtual exclusion of other yeasts and the correlation of its presence and numbers with the leavening function in San Francisco sour dough as de-scribed in an earlier report (7) appear to establish its role in this system. Its presence after so many years of being subcultured in the sour dough can probably be attributed, in part, to its vigor in this particular acidic environment which contains a substantial proportion of acetic acid in the pH range of 3.8 to 4.5. However, in view of the extremely unusual ability of this sour dough system to resist invasion by other microorganisms, the possibility is also suggested that S. exiguus may coexist with the sour dough bacteria because of its resistance to an antibiotic substance produced by the latter. Also contributing to the successful coexistence of S. exiguus with the sour dough bacteria is the observation that the latter require or greatly prefer maltose which S. exiguus does not utilize. Thus, they are not competitive for the same carbohydrate source in the dough. No carbohydrates are added in the formulation of this sour dough and the maltose, utilized by the bacteria, is formed in the dough by amylase action on free starch. The S. exiguus utilizes the approximately 2% of nonmaltose carbohydrates contained in the flour.

v3-fos

2020-12-10T09:04:11.645Z

1971-05-01T00:00:00.000Z

237229849

s2

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen. formnis W has been used to assess relative nutritional quality, growth being measured by microscopic counting of the cells, and is described in recent reviews by Celliers (9), Reynolds (21), and Evans and Bandemer (12). Microscopic cell counting to assess the growth response is very tedious, time-consuming, and subject to error particularly in the range of relatively high concentrations of the cells, food, and other particles. Several investigators have stressed the need of a quantitative method of separating the cells to allow automatic counting and to allow the criteria for protein quality to be based on parameters such as weight gain or nitrogen balance (W. R. Fernell and G. D. Rosen, Abstr. Proc. Nutr. Soc., vol. 13, p. xviii, 1954) measured in terms of protein synthesis (13, 1; W. R. Fernell and G. D. Rosen, Abstr. Proc. Nutr. Soc., vol. 13, p. xviii, 1954), dry weight (24), or total nitrogen (24) in addition to cell yield. This is a report of a quantitative method of separating preserved T. pyriformis W cells without microscopically visible damage from particulate substrates, especially peanut and cottonseed meals, and the development of a method for electronically counting the cells of various sizes in each population using a Coulter counter. MATERIALS AND METHODS Assay. The procedure was essentially that previously described (28), with some exceptions. The peanut and cottonseed meals were fed to the test organism in the same form as they were fed to animals in feeding tests and were ground to pass a 40-mesh screen. The residual oil and any fatty acids left after processing were not extracted before microbiological assay, although it is known that some free fatty acids inhibit the growth of this test organism (16). The aqueous suspensions of the test substances were prepared to contain approximately 5 mg of nitrogen per ml after adjustment of the pH to 8.2 and were then analyzed for total nitrogen (3). The analyses of homogeneous test substances, e.g., casein, were made on duplicate subsamples of 3 ml each. The analyses of heterogeneous substances, e.g., the oilseed meals, were made on duplicate composites each prepared by transferring 1.0, 0.75, 0.50, 0.40, 0.25, and 0.1 ml of suspension with a bacteriological pipette into a macro-Kjeldahl flask. Each composite then consisted of 3 ml estimated to contain ca. 15 mg of total nitrogen. The composites were made in this manner to include sampling error similar to that possible in the assays. The components of the basal medium were not lyophilized. The various stock solutions for this basal medium were prepared separately by using the methods of Baum and Haenel (5) For each assay, 4 ml of solution or suspension of test substrate of known total nitrogen concentration, 1 ml of the mixed vitamins, 1 ml of 20% soluble starch (Difco), and 4 ml of mixed solution of the salts, purines, and pyrimidines were autoclaved in a 2-oz bottle with a screw-cap at 120 C for 15 min. After cooling, each bottle was inoculated with two drops (approximately 0.03 ml each) of actively motile cells of T. pyriformis W from a composite of two 3-day 10-ml proteose peptone broth cultures. The bottles with loose caps were placed in special racks at a slope of 150 to the horizontal to insure a high surface to volume relationship of the culture medium. The inoculated cultures were incubated for 4 days to ensure that comparisons were made on cultures of motile organisms of near maximal population (14), at 24.9 +: 0.5 C at 60 to 90% relative humidity. Sampling for counts was made from single cultures. Elutriation of the cells from the particulate substrates. A suitable subsample (usually 1 ml) of each culture of known substrate nitrogen content mixed on a shaker was transferred to 4 ml of potassium phosphate preservative (28) in a 12or 15-ml graduated, conical centrifuge tube with a screw-cap or a tightly fitting plastic cap. The preserved culture is later referred to as a subsample. The tube was shaken during addition of the culture and for several minutes afterwards to avoid clumping of the cells. The elutriation apparatus is shown in Fig. 1. A stirring magnet (3 by 10 mm, Teflon-covered) was inserted in the tube of preserved subsample, saline (0.9% NaCl plus 0.1% formalde-hyde) was added to bring the volume to ca. 10 ml, and the suspension was mixed with a magnetic stirrer. The suspension was viewed with a hand lens (Aspheric Cataract Reader, 50 mm in diameter, 20 diopters, Combined Optical Industries Ltd., Bath Road, Slough, England). After extraneous particles settled from the upper part of the suspension and the cells looked clean, part of the suspension was drawn off through the plastic tube with the syringe in the continuous pipetting outfit (Becton, Dickinson, and Co., Rutherford, N.J.) and transferred through a 100-mesh and then a 200-mesh (sieve opening, ca. 0.074 mm) stainless-steel screen, each held in a stainless-steel membrane-holder (Millipore Corp., Bedford, Mass.) into a volumetric flask of suitable size for the final dilution desired for counting. The dilution was usually 1: 200. The elutriation process was repeated until the extraneous particles left in the centrifuge tube were washed free from cells, a clean tube was substituted, and the apparatus was washed well with saline-preservative. This saline was also transferred into the volumetric flask. Coulter counting of the cells. For Coulter counting, the cells are suspended in an electrolyte. The suspension of cells is drawn under vacuum through an aperture of known size in a tube which has an immersed electrode within it and another outside of it. These electrodes cause an electric current to flow through the aperture. Each cell passing through the aperture displaces an equal volume of electrolyte causing a modulation (or resistance) in the electric current. This modulation is detected by the instrument as a signal or pulse. The pulses are electronically amplified and automatically counted. Threshold settings of the in- VOL. 21,1971 strument must be determined for cells of particular volumes. An upper threshold setting is chosen to screen out counts of particles above a predetermined volume, and a lower threshold is selected to screen out counts of particles below a selected volume. Therefore, the dimensions of cells of various sizes and shapes grown in several substrates, including casein, were measured microscopically with a calibrated micrometer. The volumes of the cells were then calculated as prolate spheroids, since the predominant type was pyriform, or as spheres, even though the round types are not quite spheres. Cell dimensions and counter settings. Of numerous cells measured, the smallest were almost round, approximately 15 to 20 ,m in diameter. The largest cells, pyriform, were 30 by 70, 28 The ranges of the calculated volumes of the cells, assumed to be primarily prolate spheroids and secondarily spheres, are given in Table 1 with the settings of the Coulter counter (model B, aperture 140 Mm) required for counting these volumes. The particles are counted according to volume and not as to shape. Even if the cells were rectangular, the volume of the largest measured would be included for counting. Each time the instrument was repaired, it was recalibrated, and the "zero" of the threshold settings was checked frequently. A model B Coulter counter, described by Barnes et al. (4), was used with a tube with an aperture of 140 M4m and without the size distribution plotter. It was calibrated with ragweed pollen, diameter predominantly 20.3 MAm, to obtain a constant for determining the volumes of particles that would be counted at stated thresholds and settings of the instrument. The method of calibration is described in the manual supplied by the manufacturer of the instrument. The settings were chosen to include the range of the volumes of the smallest, the predominant size, and the largest cells measured. Two-milliliter subsamples of the diluted cell suspension were counted. At least three readings were made and averaged for each of the three ranges of cell volumes. The recorded total cell count is the sum of these means. The electrolyte used in the counting assembly was 0.9% NaCl plus 0.1%,O formaldehyde, filtered through a membrane (Millipore Corp.) of 4-,Mm porosity and counted to be sure it was substantially free from particles the same size as the particles to be counted. The functioning of the instrument was checked periodically by testing the zero settings of the thresholds, by recalibrations with the ragweek pollen, and by counting the particles in weighed samples of pecan pollen (Hugh Graham Laboratories Division, Hollister-Stier Laboratories, Dallas, Tex.). The pollens were suspended in the electrolyte at known volume and counted at the same settings used for the T. pyriformis cells. The diameter of the pecan pollen was reported to be predominantly 45 to 50 ,m (48,032 to 65,888 Mm3), and the particles were considered to be "essentiallv mono-sized." It was expected that these particles could be counted at the same instrument settings that were selected for counting the cells of the test organism. As shown in Table 2, this was true. If blocking of the aperture occurred too frequently for one subsample, another elutriated subsample was poured through a 200-mesh stainless-steel funnel from the volumetric flask into the beaker for counting. If another subsample was not available, the one partly used for counting was filtered in the same manner into another beaker, stirred well, and counted. No corrections were made for coincidence counting. Growth response. The growth response was evaluated by computing the linear regression coefficient for the regression of numbers of organisms per unit of medium, without the restriction that the line of response must pass through the origin. RESULTS Elutriation of the cells. During development of the elutriation method, its adequacy for quantitatively transferring the cells from the particulate substrates to another medium for counting was checked by microscopic examination of rinsings of the stainless-steel screen after elutriation of numerous samples. Few, if any, cells were found in the rinsings. The adequacy of the method was also checked to determine if extraneous particles of sizes similar to the cells were being transferred into the suspensions to be counted. Microscopic examinations of numerous cell suspensions diluted for counting showed that they were virtually free from extraneous particles that might be counted as cells. The number of cells lost during elutriation was negligible. This is shown by examples of counts of cells not elutriated and elutriated from proteose peptone broth in Table 3 and from casein in Table 4. Six subsamples were elutriated in about 1 hr, and usually two sets of six subsamples could be partitioned in 1 day. This included setting up parts of the equipment, washing the necessary parts at the end of the runs, and diluting the elutriated cells for counting. The time for the 21,1971 procedure could be reduced by making the apparatus automatic with solenoid valves and timing devices, by the addition of saline under pressure, and by the removal of the cell suspension under vacuum. The volumetric flask was chosen so that the cells were diluted properly for counting without further transferring. By trial, a 1:200 dilution of the original sample was usually suitable. Cells were stable in cultures preserved in phosphate buffer with 6% formaldehyde (28) for 2 years before elutriation. Growth response evaluated by visual and Coulter counts. Results of visual and Coulter counts of the test organism grown in several substrates and results of replicated automatic counts are shown in Table 3. The automatic counts tend to be lower than the visual ones at the higher test substrate nitrogen concentrations assayed but check fairly well at different dilutions and volumes of subsamples. Previous studies of assays of various protein concentrations (28) and of numerous cottonseed meals (unpublished data) showed that their relative nutritive value could be determined by comparing the growth response of the organism, determined by visual counts, at various levels of nitrogen of the test substance up to about 1 mg per ml of final medium, the range in which the response was linear. This is illustrated in Fig. 2 with results obtained with a very good quality meal and a moderately good one, before and after heat damage. In developing the elutriation and Coulter counting methods, a cottonseed meal and a peanut meal were assayed at various nitrogen concentrations up to ca. 2 mg per ml of final medium, and the growth response was determined by both types of counts. The results (Fig. 3) were linear up to at least 1 mg of nitrogen per ml. The visual counts of cells in diluted substrate were made within a few days of sampling to avoid possible evaporation of the liquid, since it was not feasible to make up the volume just before counting. For these two meals, preserved subsamples for Coulter counts were stored diluted with the phosphate buffer preservative in 25-ml volumetric flasks. Just before elutriation, each suspension was quantitatively transferred to a centrifuge tube. Therefore, for these two meals, one more transfer was required than for subsequent assays. For the regression lines shown in Fig. 3, the slope b for the cottonseed meal is 0.5769 for the visual counts and 0.4608 for the Coulter counts. For the peanut meal in this same range, b is 0.6947 for visual counts and 0.5193 for the Coulter counts. The statistics for all of the data for each of these two meals are shown in Table 5. The probability for the correlation between the nitrogen levels and the growth response for each type of counts for both meals is > 99.9. Although for each meal b is somewhat greater for the visual than for the Coulter counts, it seems feasible that the latter could also be used in determining the nutritional index. Two more peanut meals were assayed with the Coulter counts spot-checked by visual counts (Fig. 3). The efficacy of the elutriation and automatic counting methods was tried on five more peanut meals before using the methods to determine the Oval svmbols, xy. relative nutritive value of a series of treated and untreated meals with the growth response determined by Coulter counting only. As shown in Fig. 4, the response was again linear in a meal nitrogen range of as high as ca. 1 mg per ml of final medium. These results indicate that the growth response of T. pyriformis W to peanut and cottonseed meals at various meal nitrogen contents of at least 1 mg per ml of final medium, evaluated by Coulter counting of cells elutriated by the described method, can be used to demonstrate differences between meals. A study is to be reported on the successful use of this assay by the described procedures to predict for higher 884 TEUNISSON APPL. MICROBIOL. Table 4. Fernell and Rosen (13) reported that they were nitrogen levels. Solid symbols, xy~. unable to separate Tetrahymena cells from pro-tein media by differential centrifugation in sucrose solutions or by electro-migration techniques. Waithe (30) reviewed reports of other investigators who tried to obtain undamaged, washed suspensions of protozoa from nonparticulate media for physiological studies, but no report is known of an adequate method of separating Tetrahymena or other protozoa from a particulate medium. In addition, no report was found giving details of Coulter counting of a population of Tetrahymena of various sizes. Elutriation of the cells from particulate substrates. Attempts were first made to partition the cells from other particles in the media by methods similar to those suggested by Albertsson (1). Two water-soluble polymers, arabinogalactan (kindly supplied by Stein, Hall and Co., New York, N.Y.) and sodium carboxymethyl cellulose, were tried at various concentrations. Both were satisfactory in removing the relatively large meal particles, but the lightest-weight extraneous particles of various sizes remained in the phase with the cells. The most dilute concentrations of the polymers used were the best for partitioning the cells, so the potassium buffer of the basal medium and then 0.9% aqueous sodium chloride were tried. Both were satisfactory for the purpose. The latter plus 0.1 % formaldehyde is suitable for use in a Coulter counter. In the buffer and in the saline, the meal particles in a mixed suspension settle faster than living or preserved cells in separatory pyriform funnels and conical centrifuge tubes but not in straight-sided vessels, such as cylinders or ordinary test tubes. The funnels of various sizes, especially 125 ml, are satisfactory for large volumes of culture. Volumes of the cells. The volumes given are within the ranges of those reported for T. pyriformis W calculated as prolate spheroids by Cashland and Johnson (8) and Reynolds and Wragg (22). These volumes are also within the ranges of other strains whose volumes were calculated by other methods by Thompson (Ph.D. Thesis, Univ. of Alabama, 1960), Thormar (29), and James and Read (14), and for an unidentified strain during different phases of growth but with the method of volume calculation not reported by Summers, Bernstein, and James (26). The dimensions of width and length are within those given for strain W by R0lle (23) and for 16 strains of T. pyriformis, including W, by Loefer (19). Method of Coulter counting. The method of using the instrument had to be devised because of the scant information found in the literature. There are a few reports of Coulter counting of Tetrahymena in nonparticulate media but few details of the method used are given. Using a model A, Leboy, Cline, and Conner (18) used a tube with a 200-.sm orifice. The model A is similar to the model B without the automatic recording device, except that the model B has the advantage of more refined electronics, according to Barnes,Parker,and Bradley (4). Using the model B, Kovatcs et al. (17) counted unpreserved T. pyriformis G, precooled to 0 C, with "a capillary tubesize 100 ,um," Byfield and Scherbaum (6, 7) counted T. pyriformis GL and WH-14, and Wille and Ehret (32) and Szyszko et al. (27) reported counts of T. pyriformis using a model B but did not give details of the method used. Coincidence counting. The results were not corrected for coincidence counting. For the assays of peanut and cottonseed meals, this apparently was not necessary under the conditions of this study. It is not feasible to determine the coincidence error per se for every type of sample counted at various cell-volume ranges and substrate nitrogen levels because of the amount of time required to assay, preserve, and elutriate enough subsamples and then make dilutions and counts to calculate the correction required. The method of averaging at least triplicate counts for each of the three cell-volume ranges and using the sum of the means for the final count should reduce this error to a minimum. Each of these nine counts of the cells grown at a particular nitrogen concentration is made on a different sample of the cell suspension of high dilution. Actually, determining the final counts at various substrate nitrogen concentrations is in effect determining the counts at various dilutions of the original sample. The results of final Coulter counts of various dilutions and sizes of subsamples of the organism grown in proteose peptone broth, peanut meal, and cottonseed meal (Table 3) and the good checks of final counts of replicate subsamples of the organism grown in the higher levels of casein during several years (Table 4) indicated that corrections for coincidence were not required. Only a few reports were found on correction of Coulter counts of Tetrahymena for coincidence, and the correction was very small or negligible when the organism was grown in nonparticulate media. Macdonald (20) found that coincidence effects were negligible for T. pyriformis W. Byfield and Scherbaum (7) reported a coincidence of less than 1 % for cells of T. pyriformis GL and WH-14 fixed in 4% Formalin. Wille and Ehret VOL. 21,y 1971 (31) made counts of T. pyriformis W in duplicate with a model B instrument without the distribution plotter and found they usually agreed within 0.5% when the average counts were corrected for coincidence. These investigators did not describe the method they used for counting or for the coincidence correction. Discrepancies between the two types of counts are probably not due to changes in temperature at the aperture (4), since the model B has built-in automatic compensation for such changes (Coulter Manual for the Model B Coulter Counter, 5th ed. 4-67, 420100201, Coulter Electronics, Inc.), or of changes in the temperature of the cell suspensions which were counted at room temperature regulated by air conditioning. Degkwitz and Selle (10) found discrepancies due to variations in ambient temperatures in a laboratory without air conditioning. Anderson, Petersen, and Tobey (2) observed anomalies in the determination of cell number by Coulter counting of Chinese hamster ovary cells due to the failure of daughter cells to separate after mitosis. However, this is not true for the T. pyriformis W assays incubated for 4 days. Only an occasional pair of dividing cells was found during visual counting of each assay of more than 50 substrates, including casein (Teunisson, unpublished data), each assayed at six or more nitrogen levels. Discrepancies would more probably be due to sedimentation of cells in the more dense suspensions during counting, but more study is required on this aspect of the problem. The method has not yet been tried for separating living cells, but it seems reasonable to assume that it could be used for this purpose. Since T. pyriformis W is negatively geotrophic and grows best with aeration, the actively motile cells swim near the surface of stationary medium, and the less motile cells can be easily suspended by gentle shaking of the container and could also be removed from the upper portion of the diluted suspension. A method of harvesting Tetrahymena cells without the deleterious effect of centrifuging is desirable. This is especially so for cells grown in Tween (15), for cell extraction procedures, and for enzyme and cytological studies (11). The procedure might also be used to separate other organisms from particulate substrates for similar purposes; in cell culture studies, e.g., to separate trypsinized cells from fibrous tissues; or to determine the microbial populations in food slurries, soil suspensions, washed fibers, etc., without adding nutrients or inhibitory substances from the test substrates. Other possible uses for the elutriation procedure. In addition to cell yield, other parameters of growth response can be determined on cells elutriated from particulate substrates. For the assay of nutritional quality of oilseed meals or of other proteinaceous substances with T. pyriformis W, measurement of cell nitrogen or dry weight produced would be desirable.

v3-fos

2014-10-01T00:00:00.000Z

1971-03-01T00:00:00.000Z

4670969

s2

A focus of rumenal cancer in Kenyan cattle. A minimal incidence rate of 2·5% of rumenal cancer of cattle in the Nasampolai valley of Kenya Masailand has been established. Carcinoma of the oesophagus and oesophageal region of the stomach in two free-living giant forest hogs from the same area is reported. The high incidence of the bovine disease is thought to be associated with the abnormal forest grazing of the cattle. The possible aetiology of the disease is discussed. IN 1955, Plowright reported that the residents of a valley in the Sakutiek area of the Narok district of Kenya Masailand recognized that a number of their cattle died from a disease which they claimed to diagnose clinically 6 to 36 months before death or slaughter. The disease was sufficiently common to cause grave economic loss and the annual incidence was said to be as high as 10% of the cattle population, causing some owners with grazing lands elsewhere to move their cattle from the valley. The character of the disease was established by finding squamous cell carcinoma of the rumen and oesophagus in three cases following field autopsies. No further investigation took place until September 1968, when during a preliminary visit, four clinical cases were presented and retrospective enquiry indicated that the incidence had not greatly changed. The immediate slaughter of one of these cases and subsequent post-mortem examination again showed a carcinoma of the rumen with secondary deposits in the regional lymph nodes. Since February 1969, a Masai field worker has been employed to gather data on tlle cattle population and grazing areas. He has also made periodic checks on the appearance and development of new cases, collecting tissues for histological examination when it was not possible for autopsy to be carried out by one of'us. The Valley of Nasampolai This somewhat remote valley is on the south-western slopes of the Mau Escarpment at approximately 36' 07' E., O' 50' S. The valley lies at altitudes between 9000-10,000 feet and runs into the forested Mau hills. The valley floor is approximately 5 square miles in area and a small stream runs through it. The sides of the valley are steep and dense bamboo forest is present only 200 feet above the valley floor in some places. The animal population of Nasampolai The human population of Nasampolai in February 1969, was 384 distributed between 1 1 " bomas " , each consisting of the living quarters of family groups and fenced compounds for confining animals at night to prevent losses from predators. The total number of cattle at risk in February 1969, was 961, of which 252 were classed as " calves "-i.e. animals up to 6-7 months old, whilst 132 were " heifers " -i.e. female animals not more than 3 years old which have never calved. Part of the total of 99 " bullocks " (castrated males) was also probably less than 2 years old and hence about half of the total cattle population was immature. Also occupying the same area were 1792 sheep, 304 goats and 209 donkeys. A further animal census in November 1969, did not show any significant changes in this cattle population or herd structure. The forested areas on the ridges towards the periphery of the valley harbour a number of large wild ungulates and carnivores, including buffalo (Syncerus caffer, Sparrman), giant forest hog (Hylochoerus meinertzhageni, Thomas), bush-pig (Potamochoerus porcus, Linnaeus), hyena (Hyaena hyaena, Meyer), and leopard (Panthera pardus, Linnaeus). Animal husbandry in Nasampolai The domestic animals are taken from the night " bomas " to their grazing every morning and returned each evening, calves being retained near the " boma " for the first 6 months or so and then accompanyiDgtheherdstomoredistantgrazing. In this district there is no allocation of grazing rights to individual families, as is usual elsewhere in Masailand, and all grazing is communal and essentially over-stocked. Because the population of goats and sheep is so large and they graze, of necessity, on the valley floor, pasturing of cattle in partially cleared forest is often necessary although it is recognized as an unsatisfactory source of food. Whilst grazing is communal, the owners of some " bomas ", by reason of their marginal po4ition in the valley, are forced to use high forest clearings to a greater degree than their more fortunate neighbours lower in the valley. Supplementary feeding is not practised with the exception that maize stalk residues are fed in small quantities and that earth from a natural " lick " is periodically provided to prevent a form of marasmus, possibly due to phosphorus and/or cobalt-copper deficiency. Osteophagia by cattle is also reported by the owners. Clinical course of the disease It must be stressed that the first clinical diagnosis of all cases has been made by the Masai themselves. Cattle are their main source of wealth and, indeed, subsistence, and great interest is taken in all matters pertaining to animal husbandry. They are familiar with a wide range of animal diseases and it is only due to their continued co-operation that this study has been possible. It is their custom to examine carefully the viscera of all animals slaughtered and illness in their cattle is the subject of communal consultation. This disease at Nasampolai is sufficiently common to have acquired a vernacular name-" Embonget "-which simply refers to the rumenal tympany described below. In every case made available to us so far, the chnical diagnosis of the Masai has been supported bv autopsy and subsequent histological examination. The clinical signs which characterize the disease are: (i) Apparent pain and difficulty in swallowing or regurgitation of food for rumination. This may be accompanied by arching of the neck. Some animals regurgitate watery rumenal contents which dribble from the mouth and nostrils as if vomiting. (ii) Recurrent rumenal tympany, easily visible as a distention in the left flank region. (iii) Abdominal pain evidence by grunting, trismus, arched back and slow, stiff movements. Partial anorexia and slow eating are noted occasionally. (iv) Loss of condition in advanced cases, with the hair-coat harsh and dry and the skin tightly adherent to underlvina structures. (v) Excessive thirst is sometimes recorded. We were at first informed that death usually occurred 6 to 9 months after the first clinical signs, the terminal stages being marked by a progressive weakness and eachexia. Some cases were said to run a longer clinical course of 2 to 3 years with periods of remission but this is difficult to confirm. More recently, constant surveillance has shown that the overt disease in some anima-Is develops rapidly and slaughter may become imperative in a month or even less. Pathological findings A summary of the autopsy findings is found in Table 1, and a detailed description of the histopathology and cytology of the lesions will be published at a later date. Thirteen of the 20 autopsies were carried out by the field assistant who was instructed to fix a wide range of tissues whether apparently normal or not. The mouth cavity showed no abnormalities but small papillomata occurred on the mucosae of the pharynx or soft palate in a few cases. The oesophageal mucosa TABLE I Sex in half the bovine cases exhibited papillomata, usually pedunculated and found especially in the intrathoracic region within about 20 cm. of the cardiac opening. In three cases there were elongated or circular foci of brownish erosion which represented areas of undoubted malignant change; gross thickening of the oesophageal wall, as observed and illustrated earlier (Plowright, 1955) was not seen, however, in the present series. In several cases, brownish, roughened lesions showed hyperplastic epithelium with marked activity and downgrowth of the basal cell layers and sub-epithelial mononuclear-cell reaction. Although not frankly malignant, these changes could be interpreted as " carcinoma in situ " or " precancerous Histological examination revealed that squamous cell carcinoma of the rumen was present in every case, an invariable site being the anterior wall of the dorsal sac of the rumen, an area which is characterized by having no distinct mucosal papillae (the atrium). Lesions were also sometimes present at the cardiac opening and on the oesophageal groove; in a few cases, the pillars of the rumen were the site of both carcinomata and papillomata. Macroscopically, the tumours were of both the ulcerative and fungating types and appeared to be multicentric in origin. Wide infiltration of the rumenal wall was common, in some cases extending through the muscle layers to the sub-serous connective tissue. In each case, many lymph nodes on and near the rumen and oesophagus were examined for metastases, both macroscopically and histologically. Secondary carcinomata were found in only 4 animals, the glands involved being particularly those of the atrial and posterior mediastinal groups (Sisson and Grossmann, 1940). Large and numerous metastases were also found in the livers of 2 animals and the lung tissue of 1 case. Two animals showed papillomata of the bladder mucosa but these did not exceed a few millimeters in diameter and were not apparently associated with gross haematuria. In case No. 13 there were haemorrhagic, eroded areas in the bladder which reached I x 2 cm. in size and were associated with gross, oedematous thickening of the wall; this animal must have exhibited clinical signs of haematuria. The incidence of rumenal cancer in Na-sampolai The cattle owners claim that the disease has been recognized since 1935 and that the incidence has increased over the years, particularly since 1942 (Plowright, 1955). The name of the valley, Nasampolai, in the Masai dialect, is associated with excessive salivation. This and the fact that rumenal cancer has a vernacular name, " Embonget ", indicates that this is a long-standing disease well understood by the Masai. In 1955, it was estimated that the disease affected 10% of the total cattle annually but it is well known that animal holdings are not usually fully declared. The cattle population in the same year was reported to be 250 and in September 1968, to be between 500 and 600, whereas a physical check in February 1969, revealed 961. A later animal census in November 1969, gave a total of 982 and indicated some movement of cattle to and from the valley. In the first 15 months of the field survey, 18 cases have been proven histologically and of the additional clinical cases reported, at least 12 were traced and the diagnostic criteria checked by the field worker. Therefore, in Nasampolai, we have good evidence for a minimum annual loss due to rumenal cancer of 2-5% of the total cattle population or 5% of the adult animals. Cattle owners in several neighbouring valleys were interrogated and although a few cases of a similar disease were reported during 1969, the animals were stated to have been pastured in Nasampolai earlier in life and developed the disease within 6 months of translocation. In the next valley, Nosupukia, the residents recognize the disease clinically and at slaughter; it was said to have occurred there first in 1955 but they have only lost 8 animals from this cause since then. Healthy animals brought into Nasampolai are said to acquire the disease within as little as a year after transfer. Cattle which develop " Embonget " subsequent to transfer from the valley do not seem to introduce the disease to cattle in an unaffected area. There is no seasonal predominance of new cases but the Masai claim that the incidence is higher as the result of prolonged periods of drought and when forest grazing is used more frequently. An estimate of the age of affected animals was obtained in 10 cases (Table I) and whereas previously the disease was seen predominantly in females 7-8 years old, in the current series it was not uncommon in cows suckling a second calf (i.e. about 4=5 years old). Both male and female cattle are susceptible; the predominance of females (Table 1) can probably be attributed to the four-fold greater number of female animals retained in the herds. The Masai insist that the disease is not seen in goats and sheep, these animals grazing, as already noted, on the grasses of the valley floor but never in the forest area. The over-grazing of the valley floor bv goats and sheep leaves only a tough, dense, tussock grass which is generally unpalatable to cattle. Cases of rumenal cancer in cattle are not, to our knowledge, reported from other areas in Kenya or East Africa and, as far as can be ascertained, oesophageal and stomach cancer are not recognized in the human population of Nasampolai. Similarly, no reports have been received of a disease resembling " Embonget " in the numerous buffaloes which inhabit the higher forested areas, or in bushpigs which are fairly common. The occurrence of stomach cancer in Giant Forest Hogs in Nasampolai In -September 1968, some of the Masai elders informed us that they associated the disease in cattle with the presence in the area of giant forest hogs. One man clearly remembered having seen a case in this species confirmed at post-mortem examination about 15 vears previously. During the period under review, fixed tissues were obtained f.oni 2 giant forest hogs which were speared after they had been observed to show signs of illness, including abdominal tympany in I animal. In each case, there were large, ulcerated areas in the mucosae of the oesophagus and in the oesophageal region of the stomach. In these areas, which attained several cm. in diameter, there was often gross thickening of the wall and keratinized papillomata. In both oesophageal and stomach lesions some epitheliomatous cell cords were seen in direct continuity with the hyperplastic or papillomatous epithelium. Small secondary deposits were found in the lung of the first animal and malignant cell islets infiltrated the thyroid gland tissue in the second (Table I). In addition, the first case exhibited an irregular enlargement of the liver which histologically was shown to be due to haemorrhagic necrosis and regenerative changes, typical of a sub-acute toxic hepatitis. DISCUSSION Few cancer surveys of large cattle populations have been related to the numbers at risk and it is not possible to establish absolute incidence rates except in the series reported by Monlux et al. (1966) in Colorado, Misdorp (1967) in Holland, and recently, Anderson et al. (1969) in Britain. The crude incidence rates from these surveys vary between 23 and 60 per 100,000 animals. It is usually considered that the economic slaughter of ageing animals changes the structure of the population so radically that the number of animals of susceptible age at risk is effectively reduced. However, of more than one thousand million adult animals slaughtered for food in the United States during the period 1955-62, 80"' were slaughtered under the supervision of the Department of Agriculture and 227 cattle per 100,000 were condemned with a diagnosis of neoplasia (Brandly and Migaki, 1963). This crude incidence is much higher than in the animal series quoted earlier and, indeed, higher than that reported in man, particular if adjusted for comparable physiological age (Steele, 1963). The frequency ratios in these various series are very different, cancer of the endometrium being very common in the United States, as indeed is squamous cell carcinoma of the conjunctiva and eyelid. The importance of lymphosarcoma and its geographical pattern is well recognized. However, so far as rumenal cancer is concerned, this appears to be extremely rare in cattle. Misdorp (1967) found one case in 208 bovine tumours from an estimated cattle population of 340,000 and Anderson et al. (1969) record one case in 302tumoursfromasurveyofl-3millioneattle. SmithandJones(1966),reporting on a series of 1371 bovine tumours from a number of sources, also recorded only one case and comment that such neoplasms are almost non-existent. An analysis of 1000 consecutive cattle tumours from the U.S. study of Brandly and AEgaki (1963) did not reveal a single squamous cell carcinoma of the rumen or oesophagus. Moulton (1961) considers cancer of the stomach rare in all domestic animals but states that cancer of the oesophagus and, in particular, papillomata, are occasionally seen in cattle. Cancer of the oesophagus is well recognized in elderly cats, Cotchin (1962) reporting 13 cases in a series of 66 feline tumours of the gastrointestinal tract. The minimal incidence of rumenal cancer of cattle in Nasampolai-2500 per 100,000-is, therefore, exceptional. The incidence of cancer in wild animals is even less well documented and we must rely on data acquired under the artificial conditions of zoos. Ratcliffe (1963) states that cancer has been encountered in many captive animals and analysing material from the Philadelphia Zoo, Snyder and Ratcliffe (1963) found 100 cancers in 1702 autopsies, 284 of which were of Bovidae. No gastro-intestinal cancer in Bovidae wag seen, although 2 cases were noted in the Felidae and one each in the families Mustelidae and Viverridae. Heuschele and Herrick (1962) have reported a case of cancer of the oesophagus in an antelope (Antilope cervicapra) in a zoo. The present report of 2 cases of squamous cell carcinoma of the oesophagus and stomach in free-living giant forest hogs from Nasampolai in one year must, we consider, be unique. It is of interest that both ruminants and monogastric animals at risk have developed this carcinoma at a similar anatomical site within this small geographical area. Dobereiner et al. (1967) reported numerous cases of carcinoma, primarily of the pharynx, in cattle in Brazil associated with papillomata of the bladder and enzootic haematuria. They associated these bladder lesions with the ingestion of bracken feril and there is experimental evidence to support this suggested aetiology as gastro-intestinal cancer was recorded in long-term feeding experiments with bracken (Evans, 1968). The incidence of enzootic haematuria is world-wide (Pamukcu, 1963) whereas gastro-intestinal cancers have not been reported from areas where it is common and where animal inspection is of a high standard. Enzootic haematuria is not infrequent in bigh altitude areas of Kenya (Mugera and Nderito, 1969) and bracken fern is found in the Nasampolai valley but it is unlikely to cause " Embonget " as it does not form an item of cattle forage and clinical enzootic haematuria has not been reported by the Masai. Oesophageal tumours in sheep have been reported from South Africa, associated with a nicotine-copper sulphate drench and grazing at an altitude of about 4000 feet (Schutte, 1968): no histological identification of the tumours was obtained, however. In Nasampolai, the Masai are adamant that neither sheep nor goats contract the disease and they connect this with the fact that they are never herded in the forest. It is impossible to obtain any idea of the incidence of cancer in the giant forest hog which confines its feeding mainly to the forest flora but our findings of two cases in such short time supports the hypothesis that it is relatively common and associated with the ingestion of the same plants that cause rumenal cancer in cattle. The Masai identify by name about 30 broad-leaved plants and grasses which cattle eat and a botanical survey of this forest flora is in progress. As scientific identification requires specimens of the plants at all stages of growth, the survey is not yet complete and the final results will be reported elsewhere. We tentatively put forward the hypothesis that a potent carcinogen is either ingested with the forest plants at Nasampolai or rapidly produced in the stomach from precursors. In this connection the discovery of the carcinogenic properties of dimethyl-nitrosamine in rats by Magee and Barnes (1956) has stimulated an expanding literature on a range of nitroso-compounds recently reviewed by Magee and Barnes (1967) and Druckrey et al. (1967). The rate of administration, dosage regime, type of nitrosamine and host animal, can all affect the site of the primary lesion. Oesophageal tumours have been produced, mainly in rats, by 13 different N-nitroso-compounds out of the 29 nitroso-and chemically related azoxycompounds listed by Magee and Barnes (1967) and Druckrey et al. (1967) report oesophageal tumours in rats after treatment with more than 25 nitroso-compounds. Many of the nitroso-compounds that have evoked oesophageal tumours could be expected to produce a lesion at the site of administration because of their potentially high reactivity but Druckrey et al. (1963a) have pointed out that lesions in the mouth and pharynx are uncommon with animals on a per os regime of various nitrosamines. Oesophageal tumours have also been produced in rats by three different routes of administration of N-nitroso-piperidine (Druckrey et al., 1967). The possible dangers of nitrosamine compounds in the environment were first pointed out by Druckrey et al. (1963b) but apart from the potential hazard in the chemical industry and to laboratory workers, these compounds were mainly of interest as laboratory carcinogens until the report by Ender et al. (1964) of an outbreak of acute toxic hepatitis in sheep. This outbreak was associated with the feeding of nitrite-preserved fishmeal which was subsequently demonstrated to contain dimethyl-nitrosamine (Sakshaug et al., 1965). Burrell et al. (1966) have correlated high nitrate accum-ulation in molybdenum-deficient plants with the high incidence of human oesophageal cancer in locahzed areas of the Transkei region of South Africa and, more recently, Du Plessis et al. (1969) have demonstrated the presence of dimethyl-nitrosamine in the ripe fruit of Solanum incanum grown on such molybdenum-deficient soils. These fruits are said to be used to sour milk and human dietary exposure to nitrosamines could, therefore, have arisen. Before this the natural occurrence of compounds of this type in plant material likely to be involved in human and animal diets was, apparently, limited to cycasin (reviewed by Whiting, 1963). Excessive nitrate accumulation can occur in Plants and this could give rise to nitrite by reduction during rumenal metabolism. Acute nitrate poisoning leading to methaemoglobinaemia in cattle has been reported and in some cases the formation of nitrosohaemoglobin has been demonstrated (Case, 1957). Sodium nitrite itself is not carcinogenic on prolonged feeding to rats (Druckrey et al., 1963b). Recent reviews bv Ma-aee and Barnes (1967), Lancet (1968), and Lijinsky and Epstein (1970) have underlined the possible dangers of nitrosamine compounds in the environment. Lijinsky and Epstein (1970) have stressed that the presence of nitrosatable secondary amines is possibly more limiting than nitrate or nitrite for the in vitro production of nitrosamines during cooking or for their in vivo production under physiological conditions in the mammalian stomach. Sander (1967) (1969) demonstrated that nitrate can be reduced to nitrite in human stomachs by bacterial action and that the conditions were then optimal for the formation of nitrosamines from secondary amines. Sen et al. (1969) bave demonstrated the in vitro formation of diethyl-nitrosamine by the incubation of nitrite and diethylamine with the gastric juices from rats, rabbits, cats, dogs and man, and nitrosation was also shown to occur in vivo in cats and rabbits. Mirvish (I 970) has studied the kinetics of dimethylamine nitrosation and estimated the likely amounts of dimethyl-nitrosamine formation in foods during storage and in the human stomach from the consumption of foods conta'ming nitrite and dimethylamine. Oesophageal tumours have been produced in rats by the combined feeding of nitrite and a secondary amine, methylbenzylamine (Sander, 1968) and liver tumours have been evoked by a regime of nitrite plus morpholine (Sander, 1969). This author has also induced neurogenic tumours in rats fed sodium nitrite and NN' dimethylurea (Sander, 1970). Magee and Barnes (1967), commenting on Plowright's original observation in 1955, speculated that a nitroso-compound could be present in one of the plants consumed by the cattle. Botanically, the Valley shows no immediately obvious differences from other nearby valleys and we consider that the socio-economic situation in Nasampolai whereby the Masai use forest grazing atypical of normal cattle grazing, is the probable reason for the high incidence of this cancer within such a small community. If iiitroso-compounds are involved in the aetiology of this focus of rumenal cancer in the cattle of Nasampolai, then they could either be present in the plants as eaten or be formed in vivo during rumenal digestion. These possibilities are being investigated. We thank Dr. Maloiy of the East African Veterinary Research Organization for his considerable help and advice, Dr. Greenway of the East African Agriculture and Forestry Research Organization for extensive botariical investigations, and Joseph ole Swakei, our field worker, wbose tact and enthusiasm have made this study possible. The work has been financed largely from funds made available by the Nairobi Regional CeD.tre of the International Agency for Research on Cancer.

v3-fos

2016-04-23T08:45:58.166Z

2020-12-07T00:00:00.000Z

240842465

s2

Sucrose Experimental Single crystals of C12H22O11 [Sucrose] were supplied. A suitable crystal was selected and mounted in inert oil and transferred to the cold gas stream of a Moly diffractometer. The crystal was kept at 100 K during data collection. Using Olex2 [1], the structure was solved with the ShelXS [2] structure solution program using Direct Methods and refined with the XL [3] refinement package using Least Squares minimization. Introduction To recognize glucose look for the horizontal projection of the -OH on carbon #4. The alpha acetal is is really part of a double acetal, since the two monosaccharides are joined at the hemiacetal of glucose and the hemiketal of the fructose. There are no hemiacetals remaining in the sucrose and therefore sucrose is a non-reducing sugar. Sugar Processing Sugar or more specifically sucrose is a carbohydrate that occurs naturally in every fruit and vegetable. It is the major product of photosynthesis, the process by which plants transform the sun's energy into food. Sugar occurs in greatest quantities in sugar cane and sugar beets from which it is separated for commercial use. In the first stage of processing the natural sugar stored in the cane stalk or beet root is separated from the rest of the plant material by physical methods. For sugar cane, this is accomplished by: 1. pressing the cane to extract the juice containing the sugar 2. boiling the juice until it begins to thicken and sugar begins to crystallize 3. spinning the sugar crystals in a centrifuge to remove the syrup, producing raw sugar; the raw sugar still contains many impurities 4. shipping the raw sugar to a refinery where it is washed and filtered to remove remaining non-sugar ingredients and color 1 5. crystallizing, drying and packaging the refined sugar. Beet sugar processing is similar, but it is done in one continuous process without the raw sugar stage. The sugar beets are washed, sliced and soaked in hot water to separate the sugar-containing juice from the beet fiber. The sugar-laden juice is purified, filtered, concentrated and dried in a series of steps similar to cane sugar processing. Acetal Functional Group Carbon # 1 (red on left) is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Alpha position is defined as the ether oxygen being on the opposite side of the ring as the C # 6. In the chair structure this results in a down projection. This is the same definition as the -OH in a hemiacetal. A second acetal grouping is defined by the green atoms. This result because the the formation reaction of the disaccharide is between the hemiacetal of glucose and the hemiketal of the fructose. Invert Sugar When sucrose is hydrolyzed it forms a 1:1 mixture of glucose and fructose. This mixture is the main ingredient in honey. It is called invert sugar because the angle of the specific rotation of the plain polarized light changes from a positive to a negative value due to the presence of the optical isomers of the mixture of glucose and fructose sugars. 2 Hydrolysis of Sucrose In the hydrolysis of any di-or poly saccharide, a water molecule helps to break the acetal bond as shown in red. The acetal bond is broken, the H from the water is added to the oxygen on the glucose. The -OH is then added to the carbon on the fructose. Answer The -OH on carbon # 1 is projected down therefore, alpha. Answer The -OH on carbon # 1 is projected downand is on the same side of the ring as C#6, extreme right on fructose therefore, beta.

v3-fos

2020-12-10T09:04:16.633Z

1971-06-01T00:00:00.000Z

237235475

s2

Effects of Citrate on the Composition and Metabolism of Lactobacillus casei Lactobacillus casei ATCC 393 converted small amounts of citrate to diacetyl, other volatile compounds, and lipids. Citrate was accumulated passively by the organism. The presence of citrate in the growth medium decreased the uptake of acetate and its conversion to cellular lipids. Cells grown in citrate media contained more protein per cell than did controls. This increased protein content was reflected mainly in the soluble fraction when cells were subjected to sonic lysis. Soluble fractions from cells cultured in the presence of citrate contained more total protein as well as more individual proteins than these fractions from control cells. The presence of citrate caused extensive flocculation and increased the susceptibility of cells to lysis. Lactobacillus casei ATCC 393 converted small amounts of citrate to diacetyl, other volatile compounds, and lipids. Citrate was accumulated passively by the organism. The presence of citrate in the growth medium decreased the uptake of acetate and its conversion to cellular lipids. Cells grown in citrate media contained more protein per cell than did controls. This increased protein content was reflected mainly in the soluble fraction when cells were subjected to sonic lysis. Soluble fractions from cells cultured in the presence of citrate contained more total protein as well as more individual proteins than these fractions from control cells. The presence of citrate caused extensive flocculation and increased the susceptibility of cells to lysis. Microbial metabolism of citric acid and its salts has been investigated extensively. With lactic acid bacteria, citrate is known to stimulate both growth rate and diacetyl production (5,9). The metabolism of citrate has been studied more extensively with Streptococcus diacetilactis than with any other lactic acid bacteria. Although this organism possesses the necessary permease system, it does not catabolize citrate by the ordinary citric acid cycle (6,11). Instead, citrate is cleaved by citrate lyase to acetate and oxalacetate; the oxalacetate formed is subsequently decarboxylated to pyruvate, which can then be reduced to lactate or converted to acetoin and diacetyl (9,11,20). Campbell and Gunsalus (4) found that citrate could be utilized by Lactobacillus casei in the absence of fermentable carbohydrate, and it was thus conceivable that citrate could stimulate L. casei by acting as an additional energy source. Citrate is known to influence the metabolism of acetate (24), a compound which also stimulates growth of L. casei (22). In conjunction with studies on diacetyl metabolsm by L. casei, we observed that addition of citrate to the fermentation medium markedly enhanced the growth of this organism (3). Addition of citrate increased both the rate of diacetyl synthesis (4) and the level of diacetyl reductase (2) in L. casei cultures. Citrate also profoundly influenced other aspects of the metabolism as well as the composition of L. casei. The present communication details some of these effects. MATERIALS AND METHODS Organism and culturing conditions. L. casei 393, obtained from the American Type Culture Collection, was propagated routinely in Elliker broth (Difco) with or without the addition of 14 ,umoles of sodium citrate per ml. Growth rates were determined by relating absorbance at 660 nm to dry weight of cells by using standard curves prepared under the experimental conditions. Determination of cellular composition. Two-liter portions of the citrate-containing and control sterile broths were inoculated and incubated for 48 hr at 30 C. Cells were harvested by centrifugation at 10,000 X g for 20 min at 4 C, washed once with distilled water, and resuspended in distilled water. Lipids were recovered by extraction of cell suspensions three times with 6 to 8 volumes of chloroform-methanol (2:1, v/v). The chloroform-rich phases were combined, washed (8), and evaporated to dryness under reduced pressure at room temperature. Lipid residues were redissolved in accurately measured volumes of chloroform, and the quantities of lipid obtained were determined by the dichromate method of Bragdon (1). Protein (15) and carbohydrate (19) contents of cells and lipid-depleted residues were determined with crystalline bovine serum albumin and glucose, respectively, as reference standards. Nucleic acids were extracted from the lipid-depleted residues with trichloroacetic acid, and the amounts of deoxyribonucleic acid (DNA; reference 5) and ribonucleic acid (RNA; reference 23) recovered were determined. Cell-free extracts were prepared by sonic oscillation of cells suspended in 20 ml of 0.1 M potassium phosphate buffer, pH 7.0, for 10 min in a Branson B110 Sonicator operated at maximum output. During this treatment, the suspension was cooled in an ice-water bath. Cellular debris was removed by centrifugation at 10,000 X g for 20 min at 5 C and was resuspended 993 in distilled water. Protein contents of debris and soluble fractions were determined. Portions of the soluble fractions were applied to 4 by 50 cm columns of Sephadex G-200. Elution was accomplished in a 5 C cold room with a 0.1 M potassium phosphate buffer (pH 7.0); 5-ml fractions were collected, and the absorbance at 280 nm was determined. Portions of soluble fractions were subjected to disc electrophoresis in both largeand small-pore polyacrylamide gels by the method of Davis (7). Accumulation of citrate and acetate. The ability of cells harvested from control and citrate-containing media to accumulate citrate and acetate was evaluated by the method described by Harvey and Collins (10) or by this method modified to permit the use of radioactive substrates. Sodium citrate-1, 5-14C or sodium acetate-1-14C was added to sufficient amounts of the respective cold salt in 0.1 M potassium phosphate buffer, final pH 4.5, to yield a concentration of 2.5 mmoles in 20 ml. To this was added 0.5 ml of cell suspension in 0.1 M potassium phosphate buffer and 2.5 mmoles of magnesium sulfate. These mixtures were incubated at 30 C with constant agitation, and 2-ml samples were taken from each at intervals throughout the incubation period. Cells were removed from these samples by membrane filtration (0.45 ,um pore size, Millipore Corp., Bedford Mass.) and were washed with 1.0 ml of distilled water. Filters containing the cells, and also the supernatant fluids and washings, were dissolved separately in 20-ml quantities of xylene-dioxane-cellosolve (1:3:3 by volume) containing 1' 2,5-diphenyloxazole (PPO), 0.05c%;c 1,4bis-2-(5-phenyloxazolyl)-benzene (POPOP), and 8% naphthalene, and counted in a Packard Tri-Carb liquid-scintillation-spectromoter. Utilization of citrate and acetate. To determine utilization of citrate and acetate, 1 ,Ci of sodium citrate-1,5-14C or sodium acetate 1-'4C was added to 50 ml of sterile Elliker broth with or without 14 /Amoles of sodium citrate per ml. These broths were inoculated with 1 %by volume of an actively growing L. casei culture and were incubated for 72 hr at 30 C. Lipids were extracted from 25-g portions of the well mixed cultures and were washed as described. Total neutral lipid and polar lipid fractions were obtained by silicic acid column chromatography (13). Polar lipid was defined as that fraction retained during elution with diethyl ether containing 0.5%O formic acid, but eluted with methanol. Free fatty acids were separated from the neutral lipid fraction by the method of McCarthy and Duthie (16). Known portions of lipid fractions were counted in a liquid scintillation spectrometer in toluene fluid (5 g of PPO and 100 mg of POPOP per liter). Diacetyl and other volatile compounds were purged from other portions of the 72-hr broth cultures by using a modification of the method of Pack et al. (18). The apparatus was the same, except that a second graduated centrifuge tube containing 1 ml of 10%o aqueous KOH was connected to the tube containing hydroxylamine by means of a glass U-tube. This centrifuge tube was cooled in ice water. The culture was held at 65 C for 2 hr, and nitrogen was bubbled through at the recommended flow rate (18). After purging, accurately measured volumes of the hydroxylamine and KOH solutions were added to XDC scintillation fluid and counted. Portions of the purged broth from which lipids had previously been extracted were also taken for counting. The utilization of citrate was followed by determining citrate content of cultures at intervals during incubation by using the method of Marier and Boulet (17). Microscopic examination. A few drops of culture or cell suspension were spotted on slides and examined with a phase-contrast microscope. Storage stability of cells. To determine the effect of citrate on the storage stability of cells, cells were isolated from both types of media, washed, and resuspended in 0.1 M potassium phosphate buffer, at pH 4.5 or 7.0, to a concentration of cells equal to that in the original culture. Suspensions thus obtained were dispensed into tubes and held at 4 C. At different intervals, duplicate tubes were removed and small portions were used to inoculate sterile 10%70 nonfat milk, which was then incubated for 24 hr at 30 C. Acid production was measured by titration of milk cultures to the phenolphthalein end point with 0.1 N NaOH. Cells were assayed for their ability to produce diacetyl plus acetoin from pyruvate by recovering cells from suspensions by centrifugation, resuspending them in 3 ml of 0.1 M phosphate buffer (pH 4.5), adding 100 Amoles of sodium pyruvate, and incubating the mixture at 30 C for 2 hr. Diacetyl plus acetoin was quantified by the method of Hill et al. (12). Protein content of the cell-free, spent buffer was determined (15). Chemicals. All solvents used were of reagent grade quality, and those used for lipid extraction and fractionation were redistilled in glass. DNA and RNA were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. Bovine serum albumin was from Sigma Chemical Co., St. Louis, Mo. Sodium citrate-1 ,5-14C (1.9 mCi/rmmole) was from Tracerlab, Waltham, Mass., and sodium acetate-l-14C (52.9 mCi/mmole) was from Amersham/Searle, Des Plaines, Ill. RESULTS Only small amounts of labeled citrate were incorporated into diacetyl, other volatile compounds, and lipids (Table 1). By far the greatest amount of radioactivity was present in the lipiddepleted, purged broth. Analysis revealed that 20 to 30% of the original citrate disappeared from the medium during incubation. Acetate was incorporated primarily into lipids; small amounts of label were recovered also in diacetyl and other volatile compounds ( Table 1). The presence of citrate in the medium reduced significantly the amount of labeled acetate which was incorporated into lipids and volatile compounds. Optimum uptake of citrate occurred at pH 4.5, as determined by the method of Harvey and Collins (10). By using labeled citrate, low levels of uptake were observed over 60 min with cells cultured in the presence of citrate (Table 2). Con- trol cells did not accumulate citrate although some citrate was associated with cells. The low pH necessary for optimum uptake, together with the observation that only small amounts of citrate were accumulated, indicated passive uptake of citrate by this organism. Since the presence of citrate decreased utilization of exogenous acetate, the uptake of acetate was studied also. When labeled acetate was added, an almost immediate association of acetate with L. casei cells was found, and the quantity of bound acetate increased during 60 min of incubation. There was little difference between citrate-grown and control cells ( Table 2). Addition of citrate to the assay mixture reduced the initial uptake of acetate by 10 to 20% with both control and citrate-grown cells. Based on DNA content, cells cultured in the presence of citrate contained more protein than did control cells (Table 3). When tested by the new multiple-range test of Duncan, this increase in protein was significant at the 0.01 level of probability (21). Although not statistically significant, citrate-grown cells had smaller quantities of RNA, carbohydrate, and lipid than did control cells. On a dry weight basis, cells cultured in the presence of citrate had higher amounts of protein than did control cells throughout 48 hr of incubation (Table 4). Fractionation revealed that this increased protein content was reflected in the soluble fraction; cellular debris from citrate grown cells generally contained smaller quantities of protein than did that from control cells on a dry weight basis (Table 4). This increase in protein released by sonic treatment was noted first after 24 hr of incubation and was maintained throughout the 48-hr period. By 24 hr, growth stimulation by citrate was also evident (2), indicating that this stimulation is paralleled by an increase in protein which can be released by sonic treatment. Comparison of protein elution profiles of soluble fractions derived from cells after 48 hr of incubation showed some pronounced differences between cells grown under the two sets of conditions (Fig. 1). Soluble fractions from cells cultured in the presence of citrate showed a much higher protein peak IV and slightly lower peaks II and III than those from control cells. Peak I was found to be very similar when the two cells types were compared. Elution profiles of soluble fractions from 24-hr cultures were essentially identical to those of 48-hr cultures (Fig. 1), indicating that the increase in peak IV with citrate-grown cells may be due to increased susceptibility of these cells to sonic treatment. Care was taken to subject cells to sonic oscillation in exactly the same manner, and these observations ( Table 4, Fig. 1) were replicated in several trials. Under the conditions of sonic treatment, virtually all cells were disrupted. Gel electrophoresis of the soluble fractions confirmed the differences between the two cell types (Fig. 2). On both large and small-pore polyacrylamide gels, soluble fractions from cells cultured in the presence of citrate showed more protein bands than did these fractions from control cells. Increased protein content of both cells and cell-free extracts could result from conversion of citrate to amino acids. However, the increased number of proteins indicates that this is only part of the answer. When stored in buffer, the loss of soluble protein was greater from cells harvested from citratecontaining media than from control cells. These cells had decreased ability to produce diacetyl and acid compared to control cells (Table 5). Storage for 48 hr in buffer with a pH of 4.5 resulted in a 90% loss in ability of citrate-grown cells to produce diacetyl compared to 54% loss with control cells. Storage at pH 7.0 resulted in 54 and 30% loss of ability of citrate and control cells to produce diacetyl, respectively. Citrategrown cells also produced less acid during incubation after buffer storage (Table 5). Microscopic examination revealed that citrategrown cells had a greater tendency to clump together than did control cells. This was true for both cells stored in buffer and cells in actively growing cultures. DISCUSSION Citrate is a good chelating agent and the presence of this ion adsorbed on the cell surface may enhance flocculation (14). An earlier observation on the effect of citrate on cell growth indicated that this may be the case (2). Although citrate enhances growth during incubation for 24 hr or more, quite early in the growth period it had an inhibitory effect. Magnesium and calcium overcame this early citrate inhibition (3). These metal ions may act by neutralizing the charges on citrate, thus preventing flocculation of the cells. Flocculation would decrease the surface area for entrance of substrates into cells. Results obtained in this study demonstrate that the presence of citrate has profound effects on both the metabolism and physiology of L. casei. It decreases uptake of acetate and reduces the amount of exogenous acetate incorporated into lipids. Cells grown in medium containing citrate have a significantly higher protein content than do control cells. The presence of citrate causes flocculation of cells, and these cells are less stable to cold storage and undergo lysis to a greater extent than do control cells. Citrate itself is accumulated passively by the organism. Small amounts of citrate are utilized in the formation of volatile compounds and lipids. The metabolical changes noted when citrate was present in the medium can be explained in part by its physiological effects on cells. Citrateinduced flocculation which was observed in both growing cultures and isolated, resuspended cells would decrease the surface area available for entry of metabolites. Citrate also appeared to induce lability to sonic treatment and to lysis during buffer storage. This suggests that the presence of the ion in some manner weakens the cell wall, the cell membrane, or both. Increased lysis could also explain the decreased metabolical activity of citrate cells. Decreased utilization of exogenously supplied acetate in the presence of citrate may also be explained by assuming that L. casei catabolizes citrate by cleaving it to oxalacetate plus acetate. The acetate thus produced would dilute the exogenously supplied pool and result in a net decrease of incorporation of radioactivity from acetate-1-14C into diacetyl plus acetoin and cell materials. This explanation is supported by the observation that radioactivity from either citrate-1,5-14C or acetate-1-14C was incorporated into diacetyl and lipids. It thus appears that L. casei formed acetyl CoA from the radioactive acetate which was produced by cleavage of citrate or added to the medium and then incorporated the acetyl CoA into cellular lipids and diacetyl plus acetoin. Based on DNA content, growth in the presence of citrate increased the quantity of protein per cell. This can be explained by assuming that citrate was converted to amino acids, thus allowing for more protein synthesis. Most of the increased protein content was accounted for in the soluble fraction after sonic treatment of cells, again suggesting that citrate increases release of bound protein. More protein bands were observed on electrophoresis of soluble fractions from citrate cells, and the gel filtration profiles of these soluble fractions were altered compared to those from control cells. Although proof of this will require characterization of the additional proteins observed in soluble fractions, it appears that citrate weakens the cell wall complex, thus permitting the release of the additional proteins from this complex. ACKNOWLEDGMENTS This investigation was supported by a grant from the Nutrition Foundation, Inc., and by a National Science Foundation Fellowship to the senior author. This paper is published as Purdue University Agriculture Experiment Station Journal Paper no. 4253.

v3-fos

2018-04-03T03:29:23.372Z

1971-11-01T00:00:00.000Z

5572115

s2

Fate of carbon passing through the glucose pool of rumen digesta. The metabolism of the free glucose pool in rumen digesta from sheep fed roughage rations was studied by adding an insignificant quantity of glucose as uniformly labeled (14)C-glucose of high specific activity to in vitro incubation systems. In all experiments wherein only trace quantities of glucose were added to digesta, most of the (14)C-glucose entered acetate. This was true whether label was presented either as a single dose or by continuous addition over a period of 2 hr. Digesta collected at all times after feeding either once daily or at hourly intervals gave similar glucose dissimilation patterns. If, however, a relatively large quantity of carrier glucose was added together with the tracer, the (14)C-acetate: (14)C-propionate ratio was reduced by a factor of about 10. Physical removal of most of the protozoa from digesta generally had little effect on the dissimilation of (14)C-glucose added in tracer amounts, but in one experiment there was a decreased turnover of the free glucose pool and a marked reduction in (14)C entering butyrate. The paucity of (14)C entering propionate when only trace amounts of glucose were added to digesta suggests that this acid was largely formed from substrates whose carbon did not equilibrate with that in free glucose or with that in intermediates of free glucose metabolism. The metabolism of the free glucose pool in rumen digesta from sheep fed roughage rations was studied by adding an insignificant quantity of glucose as uniformly labeled '4C-glucose of high specific activity to in vitro incubation systems. In all experiments wherein only trace quantities of glucose were added to digesta, most of the '4C-glucose entered acetate. This was true whether label was presented either as a single dose or by continuous addition over a period of 2 hr. Digesta collected at all times after feeding either once daily or at hourly intervals gave similar glucose dissimilation patterns. If, however, a relatively large quantity of carrier glucose was added together with the tracer, the '4C-acetate: 4C-propionate ratio was reduced by a factor of about 10. Physical removal of most of the protozoa from digesta generally had little effect on the dissimilation of '4C-glucose added in tracer amounts, but in one experiment there was a decreased turnover of the free glucose pool and a marked reduction in 14C entering butyrate. The paucity of 14C entering propionate when only trace amounts of glucose were added to digesta suggests that this acid was largely formed from substrates whose carbon did not equilibrate with that in free glucose or with that in intermediates of free glucose metabolism. A number of studies have been published concerning the utilization of soluble carbohydrates by the mixed populations of organisms from the rumen (1,2,5,(11)(12)(13)(14)(15)(16)20). Without exception, however, massive amounts of carbohydrate were added to the system in comparison to the size of the carbohydrate pool which normally occurs within the rumen. Since, under these conditions, the fermentations become abnormal in that, for instance, lactic acid accumulation frequently occurs, it seems that the results of previous work may not reflect the fate of carbon arising from the degradation of complex plant polysaccharides and passing through the free carbohydrate pools within the rumen of normally fed animals. This report concerns the utilization of carbon passing through the free glucose pool in rumen contents from hay-fed sheep. MATERIALS AND METHODS Bacteriological. Incubations were done in vitro with samples of rumen digesta obtained from a mature sheep fed a daily ration of 750 g of wheaten hay chaff plus 250 g of lucerne hay chaff given as a single feed. In one experiment, digesta samples were obtained from a sheep fed the same ration, but provided to the animal in equal portions hourly throughout the day. Water was freely available at all times. Digesta sampling was as described by Walker and Forrest (18), and about 10 times the amount required for in vitro incubation was withdrawn from the rumen. This was thoroughly mixed and subsampled, and the excess was returned to the rumen. To 15 g of digesta was added 1 ,uCi of glucose-U-14C (300,MCi/fAmole) in 1 ml of solution. The air space above the reaction mixture was briefly flushed out with nitrogen, the vessel was sealed with a rubber stopper, and incubation was carried out at 39 C for 1 hr except where otherwise stated. Solids were removed from the digesta by squeezing through terylene voile, and 5 ml of the liquid was centrifuged at 20,000 X g for 10 min. The clear supernatant fluid was retained for determination of 14C in volatile fatty acids (VFA) and glucose and for determination of total and individual VFA. The packed cells were resuspended in 5 ml of water and 1 ml of 2 N H2SO4 was added. After centrifuging, the cell debris was washed twice with 0.5 N H2SO4 and finally suspended in 5 ml of water for determination of 14C in ceH constituents and of deoxyribonucleic acid (DNA) concentration. Chemical determinations. DNA was determined on duplicate l-ml samples of cell suspension by the method of Burton (3). Concentrations of individual VFA species in rumen fluid were determined after separation of the acids on a column of Dowex AG5OW X 12 (200 to 400 mesh, WALKER AND MONK Radiochemical. All radioactivity determinations were done by liquid scintillation counting with a Triton-diphenyl oxazole-p-bis[2-(5-phenyloxazolyl)] benzene phosphor (10). Uniformly labeled 14C-toluene was used as internal standard. Glucose-U-_4C of high specific activity was obtained from the Radiochemical Centre, Amersham, England. Glucose and VFA in 1 ml of rumen fluid were separated on ion-exchange resin as described above, collecting 1.3to 1.4-ml fractions. A 1-ml portion of each fraction was used for determination of radioactivity. '4C in cell debris was determined by mixing 1 ml of the washed debris suspension with 5 ml of Tritonphosphor. Virtually all of the solid material in the suspension was solubilized by the phosphor. 14C in protein and polysaccharide was determined after hydrolysis of cells from 2 ml of debris suspension in 0.4 ml of 30%7a KOH for 3 hr at 100 C. After cooling, 0.4 ml water and 2.4 ml of absolute ethanol were added to precipitate polysaccharide which was removed by centrifuging. The precipitated polysaccharide was washed three times with 70r'7 ethanol, dissolved in hot water, and quantitatively transferred to counting vials for 14C determination. A sample of the supernatant fluid remaining after precipitation of polysaccharide was neutralized with perchloric acid and allowed to stand for several days at 4 C before before removing the precipitated KC104 by centrifuging. By using the cysteine-sulfuric acid method of Dische (4), it was found that the amino acid-containing supernatant fluid contained carbohydrate. Therefore, the neutral supernatant fluid was placed on a column of Amberlite CG 120 exchange resin (Na+ form, 350 to 400 mesh) measuring 10 cm long by 0.5 cm diameter and equilibrated with 0.2 N sodium citrate buffer (pH 2.0). The columns were then washed with three bed volumes of citrate buffer (pH 2.0) and the washings were dried, redissolved in 1 ml of water, and counted. Qualitative tests with ninhydrin showed a virtual absence of amino acids in this fraction and the radioactivity present was included with that for the precipitated polysaccharide. The amino acids were eluted from the ion-exchange resin with three bed volumes of 2 N NH40H and the eluates were dried, redissolved in 1 ml of water, neutralized with 0.4 N H2SO4, and counted. Radioactivity in this fraction was assumed to have arisen from cell protein. RESULTS Effect of glucose concentration on distribution of glucose carbon in VFA. Since preliminary results with 14C-glucose added in amounts such that the glucose added did not materially affect the size of the glucose pool had shown that most of the label recovered in VFA was present in acetate, whereas previous reports (2,11,20) indicated substantial conversion to propionate, the effect of normal and high glucose concentration was studied. Digesta (15 g) were incubated for 1 hr with I uCi (0.6 ,ug) of glucose-U-14C and with In the presence of carrier glucose, the recovered '4C in acetate and propionate was 60.2 and 24.9% of label in the VFA, whereas in the absence of carrier the corresponding values were 94.6 and 3.9%,,ve,. Diurnal variation in distribution of glucose carbon in VFA and cell material. Walker and Nader (19) have shown considerable variation in intraruminal metabolism of energy-yielding substrates during the day in sheep fed once daily. Such variations might be expected to be mirrored in the metabolism of carbon passing through the glucose pool. Accordingly, digesta samples were removed at intervals of 2 hr from a sheep fed once daily and allowed 4 hr to consume its ration. In all incubations, glucose-U-1IC was added without carrier. Figure 1 shows the distribution of recovered label between the VFA, C02(+ methane), and cell material. C02(+ methane) was calculated as being half of the total label entering acetic and butyric acids since both must arise by decarboxylation of pyruvate formed from the uniformly labeled glucose pool. Mean recovery of label was 87.5 + 6.4%, which was considered adequate since no attempt was made to assess radioactivity associated with the organisms left with the solids in the digesta after removal of the fluid. The proportion of "'C appearing in propionate rose, and that in acetate fell during feeding but readjusted rapidly after cessation of feeding. At all times, by far the largest portion of the 14C derived from the glucose pool appeared in acetate. The proportion of label appearing in butyrate did not alter appreciably. The molar proportions of VFA in the rumen contents used were 68:19:13 and 69:20:11 for acetate, propionate, butyrate at 2 and 20 hr after feeding. The labeling of cell material rose proportionately during the feeding period, and Fig. 2 most of the increase was due to the fo carbohydrate materials rather than prc radioactivity per unit of DNA present higher in carbohydrate than in protein. Effect of frequent feeding. Samples were removed just prior to delivery of a portion of feed to an animal fed hourly by an automatic dispensing device. Three such samples were taken followed by one 15 min after the consumption of a 1/24th portion of the daily feed, and all were incubated with glucose-U-'4C without added __4--* carrier. Table 2 shows that a fairly constant proportion of the label appeared in the VFA and that, compared to the sheep fed once daily, rather less appeared in acetate and more in the cell material. Again, of the radioactivity incorporated into cell material, most was in the carbohydrate 0 :' fraction (mean of 7.7%7 of recovered activity), a lesser amount being in protein (mean of 3.2% of recovered activity). Effect of removal of protozoa. The conversion of the bulk of the glucose carbon in acetate was recognized as corresponding to the reported activities of the rumen protozoa metabolizing soluble 16 20 sugars (1,8). Consequently, experiments were done to assess the effect of removal of protozoa from from 14C-rumen digesta. Protozoa were removed by squeez-?ep fed onice ing digesta collected 20 hr after feeding through dose withlout terylene voile and centrifuging the fluid at about r 1 hr. 500 X g for a few seconds. The solids were gently washed twice with clarified rumen fluid from the same animal, and the washings were discarded. The supernatant fluid from the centrifuged fluid was then added back to the solids, and the reconstituted digesta were thoroughly mixed and divided into two equal portions. Half of the protozoa from the centrifuged fluid were then added to one portion and an equal volume of clarified rumen fluid was added to the other. This procedure has been shown to remove about 90% of the protozoa and less than 10%/ of the bacteria in rumen fluid (unpublished data). Three such treatments were done on separate occasions. Incubations were as described above except that a shorter incubation period was used in two of the experiments. Results are given in Table 3. In the first experiment, glucose pool * . turnover was unusually slow when protozoa had 2 been removed and was accompanied by a de- 16 20 crease in 'IC appearing in butyrate. However, the second and third experiments showed no sigeim and cell nificant effect of protozoal removal on distribu-" 14C-glucose tion of 14C. A parallel determination of rate of once daily. total VFA production, done in conjunction with experiment 3, disclosed an 8%o fall due to removal of protozoa. shows that Two control experiments were done to compare ,rmation of untreated rumen contents with contents fully )tein, since reconstituted after undergoing all steps of the was much protozoa removal treatment. There were no differences in distribution of 14C between the of digesta VFA species, or in rate of total VFA production, indicating that the treatment used to remove protozoa had no deleterious effect on any of the organisms present. Continuous infusion of glucose-U-'4C in vitro. Since one particular organism or group of organisms may have contributed substantially to the metabolism of the glucose pool labeled by a single dose of 1C and since turnover of the pool appeared to be relatively rapid, there existed the possibility that the added glucose did not equilibrate with all of the free intracellular pools available. Therefore, an experiment was done with 200 g of digesta (collected 20 hr postfeeding) to which a priming dose of 2 ,Ci of carrier-free glucose-U-14C was added. Further 'C was infused at a rate of 12 ACi (in 12 ml) per hr while stirring the reaction mixture fairly rapidly. To assist in keeping pH relatively constant during incubation, sodium bicarbonate at a concentration of 0.2 mmoles/ml was included in the glucose solution infused. Samples (5 ml) of rumen fluid were withdrawn at 30-min intervals for 2 hr and promptly acidified with 1 ml of 1 N perchloric acid. After 2 hr, a 20-ml sample of fluid was obtained; 10 ml was treated with 2 ml of 1 N HC104 and 10 ml was placed in an ice-cold tube and centrifuged at 0 C. This last sample was used to attempt the estimation of specific activities of the total free glucose pool and the extracellular pool. Rate of total VFA production was linear over the 2-hr period, being 7.5 ,umoles per hr per ml of rumen fluid. Radioactivity in the total glucose pool rose over the first 60 min and remained constant thereafter. Accumulation of radioactivity in individual VFA species was linear (Table 4), and all of these factors show that steady-state conditions were established. Distribution of 14C in the individual VFA species again showed that most of the glucose carbon was converted to acetate carbon. In addition, there was five times as much activity in butyrate as there was in propionate (Table 4). Specific activities of glucose in the total and extracellular pools appeared to be the same, being 2,370 and 2,450 dpm/,g, respectively. However, no significant difference in total and extracellular pool sizes was observed, so the question of equilibration of all available pools with the added glucose remains unresolved. A repeat of the continuous infusion experiment gave substantially the same results (Table 4). Since attempts to demonstrate equilibration between intraand extracellular glucose pools directly were not successful, it was decided to try to assess the rate at which glucose entered the microbial cells and was converted to products. Rumen fluid and "4C-glucose without added carrier were pumped into a mixing cell and magnetically stirred together, and the effluent was led through a length of plastic capillary tubing marked at points corresponding to known times after mixing. Effluent was collected first from the point most distant from the mixing chamber into dilute H2SO4 and, thereafter, the capillary was cut at points progressively closer in time to the time of mixing (P. R. Monk, unpublished method). By this means, it was established that glucose was metabolized very rapidly. In one experiment, the label in acetate was 30% of that in glucose after 6.2 sec, rising to 106% in 123 sec; the disappearance of 14C glucose was first order and yielded a rate constant for glucose pool turnover of 0.32 per min. A second experiment showed label in acetate after 1.2 sec to the extent of 8 % of that remaining in glucose. In both experiments, label appeared in a compound, tentatively identified as lactate from the position of its elution from the ion-exchange resin column, in greater amount than in acetate at times close to mixing. At times further removed from the point of mixing, radioactivity in lactate decreased as that in acetate increased. DISCUSSION Previous investigators of the metabolism of carbohydrate by the mixed microbial population of the rumen have used very large quantities of carbohydrate substrate in comparison to the sizes of the free pools normally existing in rumen digesta (1, 2, 5, 11-14, 17, 19). Even when isotopically labeled substrates have been used, the concentrations of substrate were quite high. (15). The results reported in this paper clearly demonstrate a very large effect of substrate concentration on the distribution of glucose carbon among the VFA end products of fermentation. When only a trace amount of glucose was added to rumen contents (about 1/100th the pool size), glucose carbon appeared in acetate and propionate in the ratio 24:1. On the other hand, when glucose was added in sufficient quantity to increase the glucose pool size by about 300-fold, glucose carbon appeared in acetate and propionate in the ratio 2.4:1. No doubt, a high concentration of glucose leads to the rapid generation of large amounts of reducing power, the disposal of which is reflected in greater propionate production. In addition, relatively large concentrations of lactate transitorily accumulate when high concentrations of glucose are used and this substrate may preferentially give rise to propionate (17,20). The value of 2.4:1 for the ratio of radioactivity in acetate and propionate in the presence of excess glucose is close to other values obtained by using excess glucose-U-'4C and organisms from roughage-fed cattle of 3.1:1(2) and 5.2:1 (20). In pure culture, Selenomonas rwninantiwn has been shown to yield differing ratios of fermentation end products over a range of glucose concentrations between 50 and 5,000 ,ug per ml (7). However, in this case, decreasing glucose concentration resulted in an increase in the proportion of propionate relative to acetate, and lactate was always found in large quantity. The fate of carbon derived from the free glucose pool does not alter greatly during the day whether the animal is fed only once or 24 times daily. During feeding in the animal fed once daily, the proportion of glucose carbon reaching acetate falls by some 10%, whereas that reaching propionate rises slightly. Presumably, the presence of soluble carbohydrates in the feed raised the concentration in the free sugar pools in the rumen (15), and this is the nearest approach under natural conditions to the concentrations used in previous investigations on carbohydrate metabolism. It is apparent, however, that the small change in distribution of glucose carbon to individual VFA species occurring during feeding is quickly reversed upon cessation of feed intake. A rather more marked change observed during feeding once daily is the greatly increased incorporation of glucose carbon into reserve and possibly structural carbohydrate within the microbial cells. This phenomenon has previously been studied in this laboratory (17,19) by using chemical determinations and is confirmed by using isotopes. Carbon derived from the glucose pool appearing in microbial protein accounted for about 3% of the glucose metabolized when feed was offered hourly. Accepting Hungate's (8) estimate of a cell yield of 10%0 of the substrate fermented, 65% of this being protein, it would seem that, of the microbial growth associated with glucose fermentation, about half of the carbon entering cell proteins is derived from glucose itself. In one experiment where physical removal of protozoa showed a distinct effect, a relatively large amount of 14C was left in free glucose after 1 hr. In addition there was a lowered incorporation of 14C into butyrate when protozoa were removed, an observation in keeping with studies showing acetate and butyrate as the chief products of protozoal carbohydrate metabolism (1,8) and consistent with the finding of lowered butyrate concentrations in the VFA species in the defaunated sheep rumen (9). Since, however, two other experiments demonstrated no difference in 1'4C distribution due to removal of protozoa, it is not yet resolved whether the protozoa play a major role in the metabolism of free glucose. Intracellular glucose pool size appears to be too small to be measurable by taking the difference between extracellular and total pool sizes, so it has not been possible to determine whether equilibration of the added '4C-glucose with all available glucose pools was achieved. Nonetheless, the distribution of 14C among the VFA end products was the same whether label was added as a single dose or by continuous infusion over a 2-hr period, indicating that all pools available for equilibration with added glucose were being reached. Appearance of 1C in acetate was very rapid, again indicating fast transport of extracellular glucose into the cells and prompt metabolism. Rapid appearance of label in a compound which is probably lactate as well as in acetate suggests that intracellular glucose concentration would be very low under the normal rumen conditions of low extracellular glucose pool size. Overall, it seems reasonable to suppose that the metabolism of the added '4C-glucose was similar to that of glucose arising from feed component breakdown. The mean results of 15 experiments on distribution of 14C from the labeled glucose pool indicate the following equation for ruminal glucose utilization in roughagefed animals: 1.0 glucose --1.76 acetate + 0.04 propionate + 0.10 butyrate + 1.95 CO2 + 3.48 [2H]. The major feature of this equation is the relative paucity of propionate compared to that proportion normally present in the rumen. This suggests that most of the propionate formed arises from sources whose carbon does not equilibrate with that in the free glucose pool or with that of intermediates of free glucose metabolism. A further apparent anomaly is the excess of reducing power available. However, in terms of the total rumen fermentation, the excess of reducing power would be quite small since unpublished preliminary estimates based upon pool size and turnover rates suggest that less than 10% of the VFA produced, in experiments so far completed, arises from the free glucose pool. Further studies are in progress to evaluate the contribution of free glucose to the VFA produced at various times after feeding.

v3-fos

2020-12-10T09:04:11.090Z

1971-02-01T00:00:00.000Z

237233322

s2

Pathological Response of the Chicken Embryo to an Agent Which Causes Acute Leukosis (Marek's Disease) A laboratory test system specific for Marek's disease was developed by using the pathological response of the chicken embryo. Chicken epidermal scales (dander) and feather calami from infected chickens contain an agent(s) which after a 3- to 4-day incubation period caused gross or microscopic pathological changes (or both) in the embryo. A cell-free inoculum was obtained from infectious dander by 5-min sonic treatment, differential centrifugation, and membrane filtering (0.45 μm). Evidence for the cell-free existence of this agent(s) was obtained when membrane filtrates of dander preparations were shown to cause Marek's disease in 10-day-old chickens and in chickens inoculated at 1 day of age. Transmission of acute leukosis (Marek's disease, MD) was first achieved experimentally by inoculation of suspensions of infective nerves into susceptible chickens (12,14). Although clear differentiation between MD and the myeloid, erythroid, and lymphoid forms of leukosis was not made, transmission of these diseases was not consistently achieved when infective blood was passed through Seitz and Berkefeld N filters and injected into the yolk sac of 1-day-old chicks. Subsequently, blood plasma, tumor suspensions, and peripheral nerves (4) have all been successfully used in the experimental transmission of this disease. Confficting results concerning the filterability of the MD agent have been reported (3,13,16,18). Cell-free tumor extracts, media from infected tissue cultures, and infective plasma were used in these studies. Transmission of MD by cell-free inoculum was first achieved on a repeatable basis when infected chicken epidermal cells (dander) were subjected to 30-and 45-sec sonic treatment and injected into day-old chicks (2). Subsequently, virus antigens in the epithelium of feather follicles of MD-infected chickens have been localized by fluorescent-antibody technique (6). In other work (9), tissue culture filtrates [filtered through 0.45-and 0.25-,um membrane I Published with the approval of the director of filters (Millipore Corp., Bedford, Mass.)] induced a disease similar to MD when inoculated into chicks. This agent did not, however, resemble the virus previously described in the etiology of MD. Chick embryo inoculations of suspensions of infected embryo tissue, filtrates of embryo tissue, and serum of leukotic chicks gave evidence of producing leukosis in the embryo (11). Transmission of MD has been achieved by inoculation of embryonated eggs with citrated blood, spleen, thymus, or tumor cells from chicks infected with MD (19). In other work (5), infective whole blood inoculated intravenously in 10-or 17-day embryos produced MD in the hatched chicks after an incubation period of the same order as that for chicks treated at 1 day of age. Allantoic fluid collected from embryos which had been inoculated with heparinized whole blood from birds with MD was responsible for production of MD when injected intracerebrally into 4to 10-day-old chicks (18). Lyophilization of this infective allantoic fluid destroyed its infectivity. In the present study, attempts were made to show that the chicken embryo can be used as a laboratory test system for detection of MD, that the agent which produces the pathological effects in the embryo is filterable, and finally that this agent is present in the epidermal tissues of chickens which have MD. MATERIALS AND METHODS Source of infectious inoculum. Infective material consisted of epidermal cells (dander) and feather calami obtained from paralyzed birds with MD. 321 Infective whole blood was obtained from paralyzed birds by cardiac puncture. Control inocula consisted of the suspension components, without infectious dander or calami, and noninfected whole blood. Donors were of the Athens-Canadian strain of chickens which had been inoculated intra-abdominally with infective blood at 1 day of age. Birds were kept in Horsfall-Bauer isolators throughout the experimental period. Preparation of the inoculum. A flow chart of the procedure is presented in Fig. 1 5 min (intensity setting of 8) in a Sonifier (Branson Instruments, Inc.). The suspensions were then filtered through filter paper (Whatman no. 1) to remove all large debris, and the filtrates were centrifuged for 15 min at 7,000 X g (4 C). The supernatant from the first centrifugation cycle was equally divided; one-half was filtered through a 0.45-,um membrane filter (Millipore Corp.), and the remaining half was unfiltered. The filtrate supernatants were then centrifuged for 60 min at 200,000 X g (4 C). The pellets were then suspended in 10 cc of HBSS for injection into embryonated eggs. Inoculation of 10-day embryonated eggs. Three strains of white leghorn 10-day embryonated eggs were used in the experiments. They were inoculated via the allantoic cavity with 0.2 cc of the resuspended pellets. In the blood transmission experiments, the same inoculation procedure was followed. All inoculated eggs were checked daily for mortality and all dead embryos were examined for lesions. Preparation of embryo tissue. White leghorn chickens (Athens Canadian strain) were inoculated intraperitoneally at 1 day of age with infected embryo tissue preparations. Samples of embryo tissue which were injected into day-old chickens were tested for bacterial contamination. Brain Heart Infusion slants were inoculated with 0.2 cc of homogenized liver and spleen or chorioallantoic membranes from both infected and control embryos. No bacterial growth resulted from this inoculation of the slants with any of the control or infected tissue. Chickens were then put in Horsfall-Bauer type isolators. The isolators were not opened from the time of injection until the termination of the experiment (6 weeks). Tissue preparation for microscopic examination. All tissue prepared for microscopic examination was fixed in 10% buffered Formalin, embedded in paraffin, processed overnight, sectioned at 6 ,um, and stained with hematoxylin eosin. RESULTS Embryo response. Chicken epidermal scales (dander) subjected to sonic oscillation for 5 min followed by subsequent differential centrifugation (7,000 and 200,000 X g) resulted in the preparation of an infectious MD agent as observed by intra-allantoic inoculation into 10-day embryonated eggs (Table 1). Additional purification of this material by filtration [0.45-,um membrane filter (Millipore Corp.)] resulted in a slight but insignificant loss in infectivity as assayed by the embryo response (52 to 55% lesions, compared to 42% for the membrane-filtered inoculum). When infective whole blood from paralyzed chickens was injected into embryos and when infective embryo tissue was serially passed in the embryo (Table 2), a similar pathological response was observed in the embryo. One group of embryos inoculated with infective blood was allowed to hatch in Horsfall-Bauer type isolators with resultant development of MD (appearance of gross tumors) after approximately 6 weeks. Gross lesions. The gross pathological responses of the chicken embryo to the MD agent were typically characterized by the appearance of small foci to gradually converging perilobular lesions with hepatomegaly (Fig. 2), small granular appearing white foci on the surface of the spleen with splenomegaly and liver discolorations. Occurring less frequently were diffuse, white, granular regions on the surface of the kidney. Other typical lesions were thickenings of the chorioallantoic membrane with the appearance of small, white, granular clumps on the membrane surface. Microscopic appearance. Areas of focal necrosis occurred in the livers of many infected embryos. Although no inflammatory reactions were associated with these areas, in some instances proliferation of lymphocytes and granulocytes occurred in the periportal areas. Splenic enlargement due to reticuloendothelial cell hyperplasia was observed. Chorioallantoic membranes were edematous and thickened and contained areas of focal hematopoesis (Fig. 3). These foci were composed of lymphocytes, granulocytes, and red blood cell precursors. Many cells were in the process of mitosis. Occasional proliferation of membrane epithelium was also noted. Size differential of infected and control embryos. Many infected embryos were considerably smaller in size than the controls (Fig. 4). This size differential was manifested by a significant decrease (P = 0.001) in head-to-tail length of infected embryos as compared to controls. Transmission to 1-day-old chicks. Preparation of tissue from the embryos which were inoculated with the MD agent (in dander) were infective for 1-day-old chickens (Table 3) ment I, only birds which had been injected with embryo tissue from the 7,000 X g pelleted dander had gross lesions. In experiment II, birds which were injected with embryo tissue and membranes from the twice-centrifuged, membrane-filtered groups developed MD. DISCUSSION Useful laboratory diagnostic techniques for the detection of MD have not been adequately developed. Measurements of antibody activity against various MD antigens have provided inconsistent evaluations of the disease state. Indirect hemagglutination of antigen prepared from duck embryo fibroblast tissue cultures with tanned horse erythrocytes (10) has suggested a relationship between antibody titer and recovery of birds infected with MD. Others (7) have indicated that chickens infected with MD have antibodies with specific precipitins to tissue culture-produced antigens. According to one study, the presence of maternal antibodies as assayed by reaction with tissue culture antigens was associated with decreased morbidity and mortality (8). Passive transfer of immune serum resulted in only a small decrease in mortality. The specificity of these tests for the agent causing MD has not been demonstrated. At present, injection of day-old chickens with infectious material and subsequent postmortem examination for gross tumors after a 4to 6-week incubation period provide the most accurate means of detection of the agent. Previous studies (5,11,18) have shown that infective whole blood and embryo tissue could be used to infect embryos with MD. A yolk sac test (19) was developed in which 4-day embryonated eggs were inoculated with infectious material via the yolk sac, and pock-like foci were observed from the 11th day after inoculation. Attempts to transmit the infection to 1-day-old chicks from the infected embryos were successful. The reactions in the yolk sac test were dependent on the source of the embryonated eggs and the age of the embryos inoculated. In the present study, the pathological response of the embryo to the infectious agent occurred after an incubation period of 3 to 4 days postinoculation. This response was independent of breed (no graft versus host reaction); because the results of infectivity could be scored after a short latent period, the requirement of allowing the infected embryo to hatch and subsequently develop MD after a 6-week incubation period was abolished. The chicken embryo has been more routinely used as a growth medium for many viruses (1,15,17) than as a test system for oncogenic viruses. The changes, however, elicited by this MD agent provided several criteria by which to judge the reliability of this technique. Macroscopically, the combination of reduced embryo size, thickenings of the chorioallantoic membrane, and liver and spleen lesions along with the microscopic appearance of these tissues provided a repeatable means of MD diagnosis. Demonstration that these infected embryo tissues contained the MD agent was provided by passage of this tissue to day-old chickens which subsequently developed MD. Certain physical characteristics of the MD agent were determined in the process of preparing various infectious inocula. The agent demonstrated high resistance to extended periods of sonic oscillation. Five-minute sonic treatment released a significantly greater quantity of infectious material from dander, as assayed by the embryo response, than did 45-sec sonic treatment. Evidence for the cell-free existence of this agent was obtained when membrane filtrates of dander preparations were shown to cause MD in embryos and in 6-week-old chickens. The pathological responses of the chicken embryo to other agents such as Newcastle disease virus and encephalomyelitis virus (18), reticuloendotheliosis virus (T-virus) (16), mycoplasma (PPLO), and various bacteria have demonstrated only a small degree of specificity, none of which could be confused with those lesions produced by MD. The ribonucleic acid-containing leukosis viruses are normally found in the embryo (maternal transfer; reference 16) but cause no gross or microscopic lesions when experimentally passed in the embryo. Compared with these agents, the response of the embryo to MD is specific when response parameters are evaluated in terms of both gross and microscopic lesions. Also, the embryos could be examined as soon as 3 to 4 days postinoculation for these changes. Thus, utilizing the pathological response of the chicken embryo as a model for determination of the presence of the MD agent, it has been shown that chicken epidermal scales (dander) contain a agent which cause MD. These scales are constantly being shed by the chicken and represent a potentially important means of natural transmission of the disease. It has been shown previously that sonically treated infectious dander produces MD when injected into day-old chicks (2) and that virusrelated antigens are present in feather follicles of infected chickens (6).

v3-fos

2018-04-03T01:46:04.745Z

1971-12-01T00:00:00.000Z

22677967

s2

Lysine decarboxylase activity in broth and agar media. Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. TIhe nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. Amino acid decarboxylase activities are useful tests in determinative microbiology. Since Moeller (7) in 1955 described practical tests for determining decarboxylases among the Enterobacteriaceae, microbiologist have used Moeller's methods, or modifications, with increasing frequency. Today, these are standard tests in clinical and public health microbiology laboratories. Unfortunately, some of the modifications that were aimed at making the test easier and more convenient are not as accurate as Moeller's methods. This is true whether one is attempting to demonstrate lysine, ornithine, or arginine decarboxylase. The modified Moeller medium as described by Difco was simplified by having one indicator, and the technique required no extraction or addition of reagents, in contrast to some other methods. Johnson's (6) semisolid medium (Moeller base) was modified by the addition of 0.3% agar, which avoids the necessity of the oil overlay. Bachrach (1) incorporated lactose into the Moeller base medium to permit differentiation of the Salmonella-Arizona strains from Escherichia, Klebsiella, and certain Enterobacter strains, which also decarboxylate lysine. The lysine-lactose-agar medium was a modification of the medium of Edwards and Fife (3) because of the addition of lactose and the omission of iron salts. It was useful in differentiating most Salmonella, nonlactosefermenting Arizona, Edwardsiella, Serratia, and certain Enterobacter from Shigella, Escherichia, Klebsiella, Enterobacter aerogenes, and Citrobacter. In the course of identifying a number of microorganisms isolated from biological specimens in the clinical laboratory, procedures for determining lysine decarboxylase activity were evaluated. The problem of interpreting results developed when inconsistent readings from lysine decarboxylase broth medium were obtained. Sometimes these results were positive, and at other times they were negative. At times, no change in indicator (purple color) can be seen. This can be confused with a positive result (purple color), when a change of indicator from yellow to purple is not noticed. This is partially due to the rapid pH shift resulting from the breakdown of glucose substrate and low hydrogen ion concentration. Also, a change ofindicator from purple to yellow in the fermentation of glucose may be missed during the overnight incubation period. The biggest problem seems to involve nonlactose-fermenting gram-negative bacilli that fail to ferment glucose. Although published reports indicate the advantage of using a lysine-agar medium (3,6), decarboxylase broth is more commonly used. It therefore seemed worth while to examine four lysine decarboxylase methods in the hope of arriving at some conclusion regarding a method of choice. MATERIALS AND METHODS The bacterial strains included recent isolates from 149 urines, 104 sputa, 25 wounds, 6 body fluids, 4 ear swabs, and 2 fecal specimens as well as strains taken from our culture library. Original isolations from biological specimens were made on human blood-agar and MacConkey agar. An individual isolate was planted on Kligler iron agar (KIA). When a question of purity arose, one half of the colony was transferred to MacConkey agar for purification. Tests for hydrogen sulfide; urease; phenylalanine deaminase production; indole formation; citrate utilization; lysine decarboxylase activity; glucose, dulcitol, and lactose fermentation; gelatinase; and motility were made from KIA as described by Edwards and Ewing (2). With nonfermenters, the carbohydrate oxidative-fermentative (0-F) test medium of Hugh and Leifson (5), the nitrate test, Kovacs oxidase test (4), and the gluconate test as described by Haynes (4) were added. These biochemical tests were used for identification of the wild strains. Occasionally, a more elaborate series of tests was necessaryto identify wild strains. Identified cultures were inoculated into four lysine decarboxylase test media. The four test media employed here were as follows. Lysine decarboxylase medium (Moeller base). This medium contained decarboxylase basal medium (Difco) with 1% L-lysine and bromocresol purple indicator. An inoculated control made up of the above ingredients minus L-lysine was included with each strain tested. The results were read after incubation for 24 hr and for as long as 4 days. Johnson's seniisolid medium. This medium consisted of decarboxylase basal medium (Difco) with 1% Llysine. Agar (0.3%) was added as indicated by Johnson (6) in the ornithine decarboxylase semisolid medium. The indicator was bromocresol purple. The semisolid medium was inoculated by a single stab with a straight wire to the bottom of the tube. The inoculated control was made up of decarboxylase basal medium in 0.3% agar without L-lysine and was included with each strain tested. Results were read after 24 hr of incubation. Bachrach's lysine-lactose broth. This medium was that described by Bachrach (1) with L-lysine made up to 1%. The indicator used was bromothymol blue. The inoculated control consisted of decarboxylase basal medium and lactose without L-lysine and was included with each strain tested. Results were read after 16 to 24 hr of incubation. Lysine-lactose-agar slants. The test medium contained lysine decarboxylase basal medium (Difco) with 1 % L-lysine, 1 c lactose, 1.5% agar, and bromocresol purple as the indicator. An inoculated control made up of the above ingredients minus L-lysine was included with each strain tested. Results were read at 24 and 48 hr. These media were dispensed in 5-ml samples in sterile screw cap tubes (125 by 16 mm) and were sterilized in the autoclave for 15 min at 15 lb of pressure (121 C). RESULTS Initial studies were done with a series of 305 Enterobacteriaceae and 42 nonfermenters. The latter group included 40 Pseudomonas aeruginosa organisms and 2 Herellea strains ( Table 1). The two Alkalescens-Dispar strains were positive in Bachrach's broth after the prescribed 24-hr incubation period. Whether this may represent a very weak decarboxylase manifesting itself late is open to question. In Table 3, these results were recorded as negative. These two strains were positive with all of the other methods except Johnson's semisolid medium. Repeated tests produced the same results. One of the atypical Escherichia strains was negative in Johnson's semisolid agar but positive in lysine decarboxylase broth. Of the seven atypical Escherichia strains, six were positive by the lysine decarboxylase broth and Johnson's semisolid method, although all were negative by the lysine-lactose media. As expected, Bachrach's broth with lactose and the lysine-lactose-agar slants gave negative reactions with most of the Escherichia and Klebsiella strains. Poor results were obtained with Enterobacter aerogenes with the nonlactose medium of Johnson. Of 14 E. haJniae strains which were positive in lysine decarboxylase broth, 7 were positive in Bachrach's broth, 8 in Johnson's semisolid medium, and five in the lysine-lactose-agar slants. When the results of lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, 8 of the 16 (50%0) strains tested in the latter medium were in agreement with the same strains tested in the former medium. When the results of Bachrach's broth and lysine agar slants with lactose were compared, 5 of the 16 (31 %) strains tested in the latter medium were in agreement with the same strains tested in the former medium. The results of the four lysine decarboxylase methods with the Proteus-Providence group were good except for two P. rettgeri strains which gave falsepositives in the lysine-lactose-agar slants. The Salmonella test results with the four methods correlated very well. Two of the Citrobacter strains negative in lysine decarboxylase broth were not tested with the other test media. With the 20 remaining Citrobacter strains, six discrepancies were found between the results obtained in lysine decarboxylase broth and the test medium of Bachrach and Johnson, whereas five were found LYSINE DECARBOXYLASE ACTIVITY between lysine decarboxylase broth and the lysineagar slants. These discrepancies represent six of the nine Citrobacter strains positive in lysine decarboxylase broth. One of the six strains was positive in lysine decarboxylase broth in 24 hr, five were positive only after 48 to 72 hr of incubation, whereas all six were negative in the media of Bachrach and Johnson in the prescribed incubation time of 24 hr. One of the latter strains produced a weak reaction in a lysine-lactose-agar slant in 24 hr. Of 40 Pseudomonas aeruginosa strains tested, 7 failed to grow in the lysine media, whereas 31 of 33 strains produced no change in the indicator by all methods used. One of the two remaining strains changed the indicator to yellow (acid) only in Bachrach's broth, whereas the other strain produced an acid reaction in both lysine decarboxylase and Bachrach's broth media. In these two instances, it is probable that the rate of metabolism was increased to such a point that the acid formed changed the color of the media to yellow. Both of these strains produced acid in conventional glucose fermentation tubes. Both of the Herellea strains utilized glucose on 0-F medium and in conventional broth but failed to use it in any of the lysine decarboxylase media. DISCUSSION All four methods were reasonably reliable when dealing with nonlactose fermenters, whereas the lysine decarboxylase broth method and the semisolid method of Johnson were better for all of the types of bacteria studied. A major explanation for the great difference in the test results with lactose fermenters such as Escherichia and Klebsiella has been the incorporation of lactose into the medium (Bachrach's broth and lysine-lactose-agar). The high acidity produced from lactic acid usually cannot be overcome by lysine decarboxylase activity in this type of medium. Lysine-agar slants as originally designed by Edwards and Fife (3) are used mainly to separate Salmonella and Arizona strains from the Citrobacter group. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend further the range of usefulness of this medium to other members of the enteric family. For example, this would in- crease the positive results with lactose-positive Arizona strains. When the results of lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, the latter gave 6% fewer positives ( Table 2). The poor results that were obtained with E. aerogenes strains in Johnson's semisolid medium could not be explained. The results with lysine decarboxylase broth and Johnson's semisolid medium were identical with Klebsiella, except for one strain which produced a positive reaction in lysine decarboxylase broth. False-positives produced by Voges-Proskauerpositive Klebsiella did not occur with our strains after 24 hr of incubation. The addition of agar to Moeller's basal medium did not seem to affect our results, even though the addition was suggested by Johnson et al. (6) to decrease the chances for false-positives amont these strains. The difference in results between lysine decarboxylase broth and Johnson's medium with Citrobacter strains might be accounted for by the longer incubation period afforded the decarboxylase broth method. An advantage to Johnson's semisolid medium is that it does not require a paraffin oil seal added to the medium in the tube; however, a tightly secured screw cap tube works equally well. A 1% difference existed between the results obtained with Bachrach's broth and the lysine-agar slant with lactose (Table 3). Most lactose fermenters will give a negative reaction in lysine decarboxylase medium containing lactose even in the presence of enzyme activity. The physical state of the media (broth versus agar) seems to have no effect on decarboxylase production. In the lysine decarboxylase media with bromocresol purple indicator, the color range extends from a deep purple to yellow to purple. At times, reading was found to be difficult with intermediate degrees of color change, espe- cially with the Proteus group; similar colorchanging problems were also encountered to some extent with our controls. Nonetheless, the controls were generally useful in reading and interpreting results. Occasicnally, the change from purple (alkaline) to yellow (acid) was not discernible. This is mainly due to the fact that Enterobacteriaceae fermented glucose during the overnight growth period, since these bacteria usually utilize glucose in the first 6 to 8 hr of incubation. On occasion, we have experienced negative reactions involving glucose in which the yellow (acid) color was absent or faint and the medium had a colorless appearance (reduction of the indicator). Sometimes this happened in the bottom two-thirds of the tube. In some cases, the decoloration changed very little from 6 to 24 hr. This occurred mostly in Johnson's semisolid medium. An inexperienced individual could fail to recognize the first color change brought about by the utilization of glucose, expecially when weak reactions occur. Determining the pH of the colorless medium with pHydrion (Micro Essential Laboratory, Brooklyn, N.Y.) paper usually revealed a change to acid. One of the discrepancies of Bachrach's broth with bromothymol indicator was that quite often the lysine decarboxylase-positive bacteria changed the color of the indicator from yellow to green rather than blue. Another problem has been the shift in color from pale yellow to purple in the lysine-agar slants from 6 to 48 hr of incubation which may result in erroneous positives. Sometimes this was evident in the lysine-containing medium but not in the controls. When tests depend on pH changes as a result VOL. 22,1971 LYSINE DECARBO) of the activity of bacterial enzymes on a substrate, a high percentage of discrepancies occur. With any of the methods, a positive reaction usually is reliable although false-negatives can occur. A pH change was not seen in the lysine decarboxylase media with the two Herellea strains. The pH of the broth media did not fall appreciably over a 10-hr period. Repeated tests showed the concentration (milligrams/100 ml) of glucose in lysine decarboxylase broth before inoculation and during and after a 24-hr incubation period to be about the same. Several P. aeruginosa strains were checked in the same manner and gave similar results. The inability of P. aeruginosa or Herellea to utilize glucose under the anaerobic conditions of the medium is a feature which makes the lysine decarboxylase test an undesirable procedure for these organisms. In conclusion, of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color. A (YLASE ACTIVITY 1095 test method the results of which can be interpreted in 24 hr would be a more suitable one for diagnostic work. LITERATURE CID

v3-fos

2020-12-10T09:04:10.763Z

1971-05-01T00:00:00.000Z

237235292

s2

Microbiological Spoilage of Mayonnaise and Salad Dressings Saccharomyces bailii was isolated from two-thirds of the spoiled mayonnaise and salad dressing samples examined. Most of the rest were spoiled by Lactobacillus fructivorans. However, one sample contained large numbers of both S. bailii and L. plantarum. Two of the spoiled samples also contained small numbers of bacilli. Bacillus subtilis, B. pumilis, B. polymyxa, B. megaterium, and B. licheniformis were found in one sample and B. subtilis and B. pumilis in another. Small numbers of B. subtilis and B. licheniformis were also present in one unspoiled sample. Several media were evaluated for the isolation of L. fructivorans. S. bailii and L. fructivorans vigorously fermented glucose. The concentration of glucose in the spoiled samples ranged from 0 to 38.5 g/kg and from 1.3 to 17.8 g/kg for the unspoiled samples. Spoilage in mayonnaise and salad dressings results from a variety of causes including separation of the emulsion, oxidation and hydrolysis of the oils by chemical or biological action, and growth of microorganisms that produce gas or off-flavors (9). Microbiological spoilage of these products is generally caused by yeasts and bacteria. Williams and Mrak (30) reported gassy spoilage of a starch-based salad dressing to be caused by a yeast similar to Zygosaccharomyces globiformis. Fabian and Wethington (7) found samples of salad dressing and French dressing to be spoiled by an unidentified species ofZygosaccharomyces. Similarly, Appleman et al. (3) observed large numbers of an unidentified species of Saccharomyces in spoiled mayonnaise but also found Bacillus subtilis to be abundant. Pederson (19) reported B. vulgatus to be responsible for spoilage in a Thousand Island dressing. The work of Charlton et al. (5) appears to be the first report of salad dressing spoiled by lactobacilli. The species involved was considered new and described as Lactobacillus fructivorans. The question of survival of pathogenic bacteria in mayonnaise and salad dressings has previously been investigated, and the studies indicate that the products themselves generally represent no health hazard because of survival or multiplication of pathogenic bacteria (4,8,16,24,27). Our work was undertaken because the micro-I To be presented in part at the 71st Annual Meeting of the American Society for Microbiology, Minneapolis, Minn., 2-7 May, 1971. biological spoilage of mayonnaise and salad dressing still occurs frequently and the microorganisms responsible for spoilage have generally not been well studied. Considerations are also given to methods of detecting the spoilage microorganisms and reasons for their growth in these products. MATERIALS AND METHODS Source of samples. Samples of spoiled mayonnaise and salad dressings were received from the Mayonnaise and Salad Dressings Institute and represented various brands manufactured throughout the United States. Unspoiled samples were obtained from local markets and also represented various brands. Plating procedures. An 11-g sample was placed in 99 ml of 0.1% sterile peptone water and mixed in a Waring Blendor for 10 min at low speed. Subsequent higher dilutions with peptone water were made from this mixture. Preliminary experiments with diluents showed that 0.1% peptone water, as recommended by Straka and Stokes (22) for other food products, gave two to five times more colonies than distilled water and was comparable to solutions of glucose (15 and 30%), glycerol (5, 10, and 25%), and sodium chloride (5 and 10%). Plate count agar (PCA; Difco) with 100 ,ug of cycloheximide per ml for inhibiting fungi was used to determine the number of aerobic bacteria. Anaerobic and microaerophilic bacteria were detected by placing a loopful of undiluted sample into a tube of thioglycollatebroth (BBL). After growth was observed, the broth was streaked onto plates of anaerobic agar (BBL) which then were incubated in a nitrogen atmosphere. The cultures from these plates were maintained on APT agar (BBL). Later, samples were plated with Lactobacillus Selective (LBS) agar (BBL) instead of inoculating into thioglycollate broth. The samples were examined for yeasts and molds by plating on malt-glucose-agar (7) and on YXT agar (0.4% yeast extract, 0.4% glucose, 1.0% malt extract, and 1.5% agar, in distilled water). Tetracycline HCI (30 pg/ml) was added to both media to inhibit bacteria. The number of yeasts recovered on YXT agar and malt-glucose-agar were comparable, and only counts from the YXT agar are reported. All plates were incubated at 28 C. Yeast counts were made at 5 days, and bacteria on PCA plates were counted at 3 days, but those on LBS agar were counted at 10-14 days. Identification of microorganisms. The yeast isolates were identified by using the culture techniques of Wickerham (29) and the classification system proposed by van der Walt (26). The lactobacilli were identified by using criteria given by Bergey's Manual, 7th ed., Rogosa and Sharpe (20), Charlton et al. (5), and Vaughn et al. (23). The medium used for carbon assimilation studies of the lactobacilli was that given for gas detection by Gibson and Abd-el-Malek (10). Growth in the presence of bile salts was determined by the method of Wheater (28), and the Voges-Proskauer tests were made with paper test-strips (PathoTec, General Diagnostics Division, Warner-Chilcott Laboratories, Morris Plains, N.J.). Bacilli were identified on the basis of the scheme in Bergey's Manual, 7th ed. Determination of glucose. Glucose was extracted from the mayonnaise and salad dressings by the following procedure. A 5-g sample was mixed with S ml of distilled water and added to 30 ml of cold 95% ethanol. The mixture was centrifuged at 10,000 X g for 20 min at 5 C. The clear supernatant was reduced to 5 to 8 ml in vacuo at 55 C, the pH was set at 7.0 with 1 N NaOH, andthevolumewasbrought up to 10 ml with distilled water. Recovery of glucose by this method was estimated to be 75 i 5% based on addition of known amounts of glucose to selected samples. Glucose was detected chromatographically by using descending paper chromatography (Whatman no. 1 paper) with a butanol-pyridine-water (6:4:3, v/v) solvent system. The spots were located by first spraying the chromatograms with AgNOs in acetone and then spraying with NaOH in ethanol. Quantitative determinations of glucose were made as outlined in the glucose oxidase-peroxidase method of Hill and Kessler (12), but the buffer was changed to 0.8 M RESULTS Microbiological analyses of 17 spoiled samples and 10 unspoiled samples are given in Table 1. Eleven of the spoiled samples contained yeasts, four had bacteria, but two had both bacteria and yeasts. Only one of the unspoiled samples contained microorganisms, a small number of bacilli. Molds were not detected in any of the samples. Thioglycollate broth was used to detect lactobacilli throughout most of the study, but it was then found that LBS agar allowed good growth and a plate count could also be made. Although a variety of samples from different parts of the country were spoiled by yeasts, Saccharomyces bailii Lindner was the only species isolated. All of the isolates formed abundant ascospores on malt extract and yeast-malt-agars (29), and there were usually four spores in each conjugated ascus (Fig. 1). These isolates of S. bailii gave a vigorous gaseous fermentation of D-glucose but no fermentation of D-galactose, maltose, lactose, or raffinose. Sucrose was fermented latently by isolates from nine of the 13 samples containing yeasts. The gaseous fermentation of sucrose began 12 to 56 days after inoculation of the fermentation medium and was quite vigorous. All isolates assimilated D-glucose, D-galactose, ethanol, glycerol, ribitol, D-mannitol, D-glucitol, and acetate (weak), and some isolates also assirnilated L-sorbose, trehalose, and calcium 2-keto-D-gluconate (weak); the other carbon sources used in standard assimilation tests (29) were not utilized. There was no liquefaction of gelatin. Two of the spoiled samples contained small numbers of bacilli in addition to the other micro- Apparently lactobacilli were responsible for spoilage in the rest of the samples. L. plantarum was isolated (on PCA) in high numbers from spoiled sample 1, but S. baiii was also abundant. This was the only sample with high numbers of both bacteria and yeasts. L. fructivorans was isolated from spoiled samples 2, 15, 16, and 17. The number per gram in sample 2 was not determined since isolation was in the thioglycollate broth. The other three samples were plated with LBS agar, and the numbers ranged from 8,100 to 12,600,000 per g. The amount of filament production differed considerably among the isolates, but all had the curious property of producing curves or coils in the filaments of cells (Fig. 2-4). These isolates of L. fructivorans and the type culture NRRL B-1841 produced gas from Dglucose, D-fructose, sucrose (weak), maltose (weak), and malate (weak) but did not utilize D-galactose, L-arabinose, D-xylose, cellobiose, melezitose, acetate, or soluble starch as judged by the failure to produce gas or change the pH of the culture medium. The isolates gave a negative Voges-Proskauer reaction and did not grow in the presence of bile salts. Sucrose is commonly used as a sweetener in mayonnaise and salad dressings (31), but the presence of other fermentablecarbohydrates was suggested since L. fructivorans ferments sucrose only weakly and not all isolates of S. bailii are able to ferment this sugar. The use of corn syrup, invert sugar, and other such sugar sources is permitted in food dressings (6), and they are frequently less costly and more convenient to handle than sucrose. Paper chromatography showed that all samples, except spoiled sample 2, contained glucose. Most of the samples also contained other reducing sugars with concentrations about as great as that of glucose. The concentration of glucose in the spoiled samples, excluding sample^2, ranged from 3.0 to 38.5 g/kg and from 1.3 to 17.8 g/kg for the unspoiled samples. Representative strains of L. fructivorans, NRRL B-3796 through B-4003, and of S. bailii, NRRL Y-7253 through Y-7262, have been retained in the ARS Culture Collection. DISCUSSION PCA has been recommended for the detection of bacteria causing spoilage in mayonnaise and salad dressings (2), and, although the bacilli and L. plantarum were detected on this medium, L. fructivorans was not. Thus, it becomes apparent that LBS agar or some similar medium must also be used if lactobacilli, such as L. fructivorans, are to be detected in these products. L. fructivorans was first isolated from spoiled salad dressing by Charlton et al. (5) and described as a new species. Later, Vaughn et al. (23) studied this species and regarded it as valid, but more recently it has been considered a synonym of L. brevis (Index Bergeyana, 1966). Data presented here indicate that it may indeed be a distinct species. Bergey's Manual, 7th ed. lists L. brevis as forming acid from D-galactose, Dxylose, and L-arabinose. Neither the strain originally described as L. fructivorans nor the present strains form acid from these three sugars. In addition, L. fructivorans was described as forming curved filaments, a characteristic shared by our strains but not one typical of L. brevis. The role of L. plantarum in the spoilage of the blue cheese dressing is difficult to assess since a large number of yeasts also were present. Similarly, the occurrence of small numbers of bacilli in two of the spoiled samples and one unspoiled sample may not be significant. However, Iszard (13) reported B. petasites to cause gaseous fermentation in a salad dressing; Appleman et al. (3) reported large numbers of B. subtilis and an unidentified yeast to be present in spoiled mayonnaise, and Pederson (19) attributed spoilage of a Thousand Island dressing to B. vulgatus. The sources of the spoilage microorganisms have been generally attributed to contaminated ingredients (1, 11,17,32) and to unsanitary manufacturing equipment and surroundings (3,7,30). Zuccaro et al. (33) studied the ability of salad dressing spoilage microorganisms to survive in oil-water mixtures at different pasteurization temperatures. Iszard (14,15) suggested controlling spoilage through addition of lactic acid to the dressings; Shapiro and Holden (21) proposed various antibiotic and chemical dips to decrease the microflora of a packaged salad mix. One facet brought out in our study, and apparently not previously considered as a factor influencing spoilage, has been the sugar concentration in the products. Traditionally, sucrose has been used as a sweetener and, although it is only slowly fermented by L. fructivorans, many isolates of S. baiiii produce a latent but vigorous fermentation of this sugar. Because of this delay, the products may not show gaseous fermentation until quite sometime after they have left the manufacturing plant. Delayed fermentation of sucrose also has been reported for many other species of Saccharomyces (18). KURTZMAN, ROGERS, AND HESSELTINE The practice of substituting syrups and other glucose-containing sweeteners for sucrose seems to further complicate the problem of spoilage since both L. fructivorans and S. bailii readily ferment glucose. One would also expect other spoilage microorganisms to make good growth and perhaps form gas in the presence of glucose where they might not if only sucrose were present. Control of spoilage in mayonnaise and salad dressing thus becomes a problem of manipulating ingredients. The concentration of acetic acidin the products is usually relied upon to prevent the growth of spoilage microorganisms (25), but there is little difference in pH between spoiled and unspoiled samples ( Table 1). The judicious use of sweeteners might also help decrease the amount of spoilage that occurs. ACKNOWLEDGMENTS We thank Helen Gasdorf for the quantitative determination of glucose in the mayonnaise and salad dressing samples. We also thank the Mayonnaise and Salad Dressings Institute, Chicago, Ill., for supplying samples of spoiled mayonnaise and salad dressings.

v3-fos

2022-02-23T16:12:05.263Z

1971-04-01T00:00:00.000Z

247039678

s2

A study on the occurrence of drought at Hebbal Rainfall data ohevents -one yean ofAgricultural Research Sta tion, Heb bal , Be ngalore have been made UICl to atud, the ralnfalldiltribution and ccourrence oCdrought. Out oC severa l ell\llll ifioa.tionll and groupings of rainf&1l. "decile' grouping found to be tbe bolt for any analys is. The pcreent ege of rai ny daya. number of dry IpellA during tbe cropping &eaSOD and coefficient of va.riability in t he amoun t of ra inCall were found to be the importan t f6Cton atrfICt ing the aeverity of drought. V. ing t hese factora along with crop-soll-weter relatiOnships a tenta tive dflf1nlt ion of drought for south eeete m parh of Myaofe State has been drawn. to t ime awl space , Glover and Robi nson (1953i opined tha t the rainfall ,lnta could he used without tr ansformation for most of the studies. ) [anning (1950) on tho other hand has suggested tho transformation of the raw data. The rainfa ll distribut ion has been studied by several workers with more than one objective, Predicting the occurrence of drought based on the ana lysis of rainfall is one of the major objectives in these studiee, r. Introduction In order to study tho frequency of occurrence The quantity and distribution of rainfall of any of drought and its severity, the rainfall data of the place depends upon its geogmphical position. It Agricultural Research Station, Hebbal WllS used. has been pointed out by Hiatt and Schlomer It was also intended to specify the factors that (1955) tha t places near sea shore, usually receive contr ibute to the severity of drought and to define good rainfall associated with good distribution. a drought year based on the observations made. Tannehill (1955) reviewing the work on the cyclic 2. Material and metbod. nature of rainfall, classified the studies into two groups. First group was to identify a cycle based Agricultural Research Station, Hebbal, Bangaon the datil for a very long number of years . He lore is situated at Lat. 13°7' N, Long. 74°37'E and pointed out that in similar studies Edward Brukner at an elevation of 899 m above mean sea level. of Vienna identified a thirtyfivc yea", cycle in the The mean temperature during thc year varies rainfall distribution of Euro pe, based on the severifrom 19·0°C during winter season to 27' O°C during ty of winters. The second group of studies confined hot weather season. to a few years data and in these, thc larger changes The annual rainfall (mean annual 828 mm) is wcre explained by relating them to some physical received in two distinct monsoons, that is, southfactors. Of the several factors responsible for the west monsoon (J une to September) and northeast ohanges in precipita t ion, variation in solar radiamonsoon (October and November). Daily rainfall t ion is of more importance as it is supposed to datil of the station from 1897 have been utilized. follow a definite eycle. In order to interpret tho rainfall data, Cocheme In order to classify the years into bad and good and Frequin (1967) grouped the years into quintiyears in terms of total rainfall and distribut ion les; and fitted a theoretical frequency distribution. three systems were followed. In the first, the dat~ Gibbs and Mahar (1967)have used " Decile" groupwere classified into three groups using mean and ings for rainfall studies and have drawn the maps standard deviation. In doing 50 It WllS observed of Australia, depietmg the zones of drought . that there were ouly 8 years of below one sta ndard deviation. From practical experience, it was obeerStudies on the rainfall pat tern posos a special ved that there were lIlany more number of drought problem as, it is non-continuous both with respect years. Therefore, in tho second, the data was • A88tt. Meteorologist , A88tt. Agronomist. Al8t t. BiomctrioiaA end AgronomiBtrespectively 213 to t ime awl space , Glover and Robi nson (1953i opined tha t t he rainfall ,lnta could he used without tr ansformation for most of the st udies. ) [anning (1950) on th o other hand has suggested tho transformation of the raw data. The rainfa ll distribut ion has been studied by several workers with more than one objective, Predi cting the occurrence of drought based on t he ana lysis of rainfall is one of t he major objectives in th ese studiee, r. Introduction In order to study th o frequency of occurrence The quantity and distribution of rainfall of any of drought and its severity, the rainfall data of th e place depends upon its geogmphical position. It Agricultural Research Station, Hebbal WllS used. has been pointed out by Hiatt and Schlomer It was also intended to specify the factors that (1955) tha t places near sea shore, usually receive contr ibute to th e severity of drought and to define good rainfall associated with good distribution. a drought year based on the observa tions made. Tann ehill (1955) reviewing the work on the cyclic 2. Mat erial and metbod. nature of rainfall, classified the studi es into two groups. First group was to identify a cycle based Agricultural Research Station, Hebbal, Bangaon the datil for a very long number of years . He lore is situa ted at Lat. 13°7' N, Long. 74°37'E and pointed out that in similar studies Edward Brukner at an elevation of 899 m above mean sea level. of Vienna identified a thirtyfivc yea", cycle in the The mean temperature duri ng th c year varies rainfall distribution of Euro pe, based on the severi-from 19·0°C duri ng winter sea son to 27' O°C during t y of winters. The second group of studies confined hot weather season. to a few years data and in th ese, thc larger changes The annual rainfall (mean annual 828 mm) is wcre explained by relating them to some physical received in t wo distinct monsoons, that is, southfactors. Of the several factors responsible for the west monsoon (J une to September) and northeast ohanges in precipita t ion, variation in solar radia-monsoon (October and November). Daily rainfall t ion is of more importance as it is supposed to datil of the station from 1897 have been utilized. follow a definite eycle. In order to interpret th o rainfall data, Cocheme In order to classify the years into bad and good and Frequin (1967) grouped the years into quinti-years in terms of total rainfall and distribut ion les; and fitted a t heoretical frequency distribu tion. t hree systems were followed. In the first, t he datG ibbs and Mahar (1967)have used " Decile" group-were classified into thr ee groups using mean and ings for rainfall studi es and have drawn the maps sta ndard deviation. In doing 50 It WllS observed of Australia, depiet mg the zones of drought . that t here were ouly 8 years of below one sta ndard deviation. From practi cal experience, it was obeer-Studies on the rainfall pat tern posos a special ved that there were lIlany more number of drought problem as, it is non-continuous both with respect years. Therefore, in tho second, the data wa s

v3-fos

2018-04-03T02:11:30.344Z

1971-08-01T00:00:00.000Z

32599471

s2

Effect of Soy Proteins on the Growth of Clostridium perfringens' Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridiwn perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an Eh of below - 300 mv. Viable populations were enumerated by agar plate techniques on Tryp-ticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N2-10% CO2) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an Eh of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N2-10% CO2) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease. Clostridium perfringens foodborne disease continues to be a major concern to the food industry (2), especially that portion associated with cooked meat products which may be handled improperly and will support growth of C. perfringens. Increases in commercial production of synthetic meats and increases in protein supplementation of natural meat systems directed our interest into this area. There is little current literature to determine what significance this addition of proteins has on the potential growth of C. perfringens. Our objective has been to determine whether protein fractions from sources such as soy protein enhance, retard, or do not affect the growth of C. perfringens. Protein fractions that are used to fabricate imitation foods such as synthetic meats and to supplement standard meat formulations were evaluated. A preliminary report of these findings has been presented (F. F. Busta, D. J. Schroder, and M. W. Ewers, Bacteriol. Proc., p. 2,1970). MATERIALS AND METHODS C. perfringens strain S40 was obtained from H. E. Hall (National Center for Urban and Industrial Health, Cincinnati, Ohio). Stock cultures were maintained in Cooked Meat Medium (BBL) at room temperature. Inoculation cultures were grown at 45 C for 18 hr in Thioglycollate Medium without added dextrose (BBL). The culture inoculum was centrifuged at 4,080 X g for 10 min and decanted. The pellet was dispersed in 200 ml of sterile 6.25 X 1(4 M phosphate buffer (pH 7.0) and centrifuged. The pellet was dispersed in buffer, and the procedure was repeated twice. The final pellet was resuspended in 20 ml of phosphate buffer and serially diluted to obtain the proper inoculum. Microscopic examination indicated negligible clumping. All procedures were carried out under aseptic conditions. The generation time was calculated by the formula The stirring assembly and the inoculation needle were autoclaved in the medium at 121 C for 15 min. The platinum and glass electrodes were sterilized by immersion in a 0.05% sodium hypochlorite solution for 20 min followed by rinsing in sterile distilled water before inserting in the growth medium. Studies showing the effect of added protein to meat loaf were carried out by the methods previously outlined (D. J. Schroder and F. F. Busta, J. Milk Food Technol., in press). Growth media. Various test proteins were substituted for Trypticase (BBL) in the thioglycolate medium without added dextrose (Table 1) and autoclaved at 121 C for 15 min. The soy protein and sodium caseinate protein concentrations were adjusted to contain the same protein concentration (w/w) as the Trypticase control. Manufacturers' specifications were used for protein concentrations. This medium was selected as the growth medium because no sugar was present and the sole protein or peptide source was Trypticase. The meat loaf medium has been previously described (Schroder and Busta, J. Milk Food Technol., in press). One part protein additive was added to five parts meat unless stated otherwise. The growth media were steamed before inoculation to ensure a low oxidation-reduction potential. The pH of each medium was adjusted to 7.0 + 0.1. The growth vessels were placed in a constant-temperature water bath maintained at 45 C. The Eh and pH were monitored throughout the incubation. Samples were taken at appropriate times to determine the extent of the lag, the log, and final stationary growth phase. These were serially diluted in 0.1% peptone dilution blanks, and the viable cell populations were estimated by plate count in triplicate with freshly prepared Trypticase-sulfite-yeast-citrate-agar. This medium contained Trypticase (BBL), 15.0 g; yeast extract (BBL), 10.0 g; sodium sulfite (Allied Chemical). 0.5 g; iron citrate (Mallinckrodt), 0.5 g; agar (Difco), 13.9 g; water (deionized), 1,000.0 ml; final pH 7.0 + 0.1. This is similar to SPS agar (1) with the selective agents deleted. The agar plates were overlaid and incubated anaerobically at 35 C for 18 hr in an atmosphere of 90% N2 and 10% CO2. The t test was employed to test the significance of differences between growth in the presence of the soybean proteins, sodium caseinate, beef, turkey, or combinations and growth with the standard Trypticase or meat control (4). RESULTS The reproducibility in population, E1,, and pH in three trials with C. perfringens S40 in thioglycolate medium is demonstrated in Fig. 1. Initiating growth at several levels had little effect on the apparent growth rate, maximum population, or generation time. The relative pH and Eh values for each of the three trials are represented in the lower portion of the figure. The pH remained between 7.5 and 7.0, and the Eh generally registered below -300 mv down to -500 mv. The influence of eight soy protein supplements and one sodium caseinate protein supplement on the growth of C. perfringens was tested. Generation times of two trials are presented in Table 2. The two right columns of the table relate the ratio of generation time in the protein under test to that observed in Trypticase during the same trial. Isolated soy protein batch 1, whipping protein, and soy glycinin resulted in significantly shorter generation times in comparison to Trypticase (t test, 5% level). The soy flour brand B, soy protein concentrate, textured soy flour, and isolated soy protein batch 2 sources resulted in generation times similar to Trypticase. Sodium caseinate and soy flour brand D were significantly inhibitory to the growth of C. perfringens (5% level). Data presented in Fig. 2 show that, when isolated soy protein batch I was substituted for Trypticase, the generation time of C. perfringens S40 was reduced by more than one-third and the maximum extent of growth was increased 10-fold. thioglycolate medium. In the case of batch 1, maximum populations of approximately 108 were obtained in each of the four trials. In batch 1, all four trials of the isolated soy protein resulted in a higher population. The ratios of generation times in isolated soy protein to Trypticase ranged from 0.61 to 0.85 in the four trials. With batch 2 of the isolated soy protein, the growth was essentially the same in either medium. Data in Table 4 show the influence of substitut-ing beef, soy beef, or combinations of beef plus soy beef or beef plus textured soy flour for the Trypticase protein source in liquid media. Data on growth in turkey, soy chicken, or combinations of turkey plus soy chicken or turkey plus textured soy flour in liquid media are also shown in Table 4. With Trypticase as the control, the use of beef or beef plus soy beef, soy beef alone, or beef plus textured soy flour as the sole source of protein produced significantly shorter generation times. The use of turkey, soy chicken, and combinations of turkey and soy chicken or turkey plus textured soy flour produced generation times that were not significantly different from those observed with Trypticase. Data used in Table 4 were reevaluated as shown in Table 5. These data show that soy beef or textured soy flour plus beef did not significantly influence growth of C. perfringens when compared to beef alone as the protein source. Generation times observed with soy chicken or textured soy flour plus turkey were not significantly different from those observed in turkey alone (Table 5) when these were used as sole protein sources in liquid media. Note that growth rates in beef or soy beef were more rapid than in turkey or soy chicken. Table 6 summarizes data on growth of C. perfringens under actual meat loaf conditions. The effects of the addition of soy beef, soy protein, and sodium caseinate to beef are shown as they relate to the beef control. Soy beef was used alone as a meat loaf but did not support growth to any extent. These data show no significant effect on growth by the additives to beef. The data also show that there was a significant difference between beef and turkey and between beef and turkey plus Trypticase, i.e., growth in turkey and turkey plus Trypticase was slower than growth in the beef control. The addition of sodium caseinate and soy glycinin to turkey increased the growth rate of C. perfringens to the level observed in beef. Under meat loaf conditions with turkey as the control, the addition of one part soy chicken to five parts turkey had no significant effect on the growth of C. perfringens (Table 6). The addition of a 1 :1 mixture of soy chicken and 1: 5 additions of isolated soy protein, sodium caseinate, Trypticase, or soy glycinin to turkey all significantly increased the growth rate in the turkey meat loaves. Soy chicken alone supported little or no growth. The Eh remained at +100 to +200 mv, and there was slow death or no growth until about the 6th hr of incubation. Some growth did take place when the Eh was reduced to approximately -30 to -50 mv. This is in contrast to the meat systems which quickly dropped the Eh to -300 mv and reached -500 mv after 7 hr of incubation. DISCUSSION Stimulation of the growth of C. perfringens was observed with some soybean protein products when compared to Trypticase. These include an isolated soy protein. A basic difference between the products that cause stimulation and those that do not is the amount of refinement by the removal of a major portion of the nonprotein components. Manufacturers' literature states that isolated soy proteins are over 92% protein and the whipping protein and soy glycinin are over 80% protein. The soy products that did not appear to stimulate growth ranged from 50 to 55% protein, with the exception of the soy protein concentrate which is approximately 70% protein. The processes involved in the concentration or isolation of the soy protein apparently improve it as a growth medium for C. perfringens. This could be due to the removal of some growth inhibitors or to some protein modification which could make the protein more available to the organism or to some change in availability of certain amino acids. The whipping protein has undergone some enzymatic modification which may effect some breakdown of the protein and facilitate easier uptake by the organism. Another factor may be the further concentration and isolation of the soy protein which in turn may affect the solubility of the soy protein and thus its availability to the organism. This study has shown that, in the case of the isolated soy protein, some batches may have stimulative activity whereas others may not. This suggests that inadvertent changes in the process may modify the end product and cause a change in solubility or in some modification of the soy protein which may affect the growth of C. perfringens. Another possibility is that some factor may be added to the product to increase its functional properties or to prevent some undesired reactions. Similarly, the converse may be applied to the soy flour brand D which caused some inhibition Generation time for beef appears in parentheses. b Generation time for turkey appears in parentheses. of the growth of C. perfringens. Sodium caseinate showed a major inhibition on the growth of the organism. Again protein availability and the presence of some inhibitory factors may play a part in its effects on growth. The rate of growth of C. perjringens in liquid medium in which beef or soy beef was the sole protein source was more rapid than in Trypticase. This may be due to readily available growth factors in beef. Under meat loaf conditions, the rate of growth was rapid in beef and the additions of various amounts of soy beef, soy glycinin, isolated soy protein, and sodium caseinate had little effect on the growth rate. This indicates that beef is an excellent medium for the growth of C. perfringens and added protein sources have little effect. Soy beef alone as a meat loaf supported little or no growth. Under meat loaf conditions, as in the liquid medium, the growth of C. perfringens was slower in turkey than it was in beef. The additions of soy chicken, isolated soy protein, sodium caseinate, Trypticase, and soy glycinin significantly stimulated the rate of growth of this organism in turkey meat loaves. The additions of sodium caseinate or soy glycinin to turkey brought the rate of growth to a level equivalent to that of beef. As with meat loaves containing soy chicken alone, soy beef alone did not support any substantial growth of C. perfringens. Eh values in these meat loaves remained high throughout the 6-hr incubation period. These observations with soy meat loaves may be due to the physical nature of the meat loaf (i.e., somewhat more porous than regular meat loaves) or due to some other inadequacy of the soy meats. In summary, these results show that certain soybean proteins were stimulative or inhibitory to growth of C. perfringens. Natural meat systems are highly stimulatory. Under actual meat loaf conditions, beef appears to be an excellent growth medium and the addition of soy meat, soybean, or other protein additives does not appear to affect its potential for fast growth of this organism. These observations are consistent with earlier findings (Schroder and Busta, J. Milk Food Technol., in press). This is not the case for turkey, in which protein additives did significantly enhance the growth of this organism. The stimulation may be due to the addition of certain factors which enhance the growth of C. perfringens, i.e., factors which are present in beef or soy protein and not in turkey. The presence of stimulative or inhibitory factors in some soybean products or other proteins could be of public health concern and warrant their identification. Food processors fabricating synthetic meats or supplementing natural meats with protein preparations should be aware of their potential influence on growth of C. perfringens.

v3-fos

2020-12-10T09:04:11.145Z

1971-05-01T00:00:00.000Z

237234201

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Effect of Sodium Chloride and pH on Enterotoxin C Production Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 108 cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 108 cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 108 cells per ml and 6.38 to 7.30 for 3.6 × 106 cells per ml. In repeated experiments in which the inoculum contained 108 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts. The application of knowledge gained from studies on the environmental factors that influence the production of staphylococcal enterotoxins is important in the prevention of staphylococcal food poisoning. The effect of two components of the food microenvironment, pH and NaCl, on the production of the enterotoxins has not been studied extensively. A previous communication (6) reported the effect on the production of enterotoxin B in Brain Heart Infusion broth and reviewed the limited literature on the subject. Since then more information has become available, mainly concerning the production of enterotoxins A and B (5,8,11,12,14,15). There is only one report providing data on the effects of pH, medium, and incubation time on the production of enterotoxin C (14). The present study extends our knowledge of the combined effects of NaCl and pH on the growth of staphylococci and on the subsequent production of enterotoxin C. I Present address: School of Veterinary Medicine, Aristotelian University, Thessaloniki, Greece. MATERIALS AND METHODS Enterotoxin C production and purification. Enterotoxin C was produced by growing Staphylococcus aureus strain 137 (ATCC 19095) in a broth medium containing 3% + 3% protein hydrolysate powder (PHP) and N-Z Amine NAK (1). The enterotoxin C was later purified by the method of Borja and Bergdoll (2). A 32-mg amount of highly purified enterotoxin C was obtained from 10 liters of broth. The specific enterotoxin C antiserum to strain 137 and the crude enterotoxin C used during the purification steps were supplied by M. S. Bergdoll of the Food Research Institute of the University of Wisconsin. Production of antisera. Two young New Zealand white rabbits (4 lb) were inoculated with the purified enterotoxin C. At weekly intervals each rabbit received in order, 11, 23, 112, and 575 ,ug of enterotoxin C dissolved in 1 ml of saline and later mixed, with a Vortex mixer, with 1 ml of complete Freund's adjuvant. The antigenic preparation was injected intramuscularly and subcutaneously. Two months after the last injection, the immunity of the rabbits was challenged with 2.3 mg of enterotoxin C in saline injected intramuscularly. Beginning 1 week after the last injection, three 50-ml samples of blood were drawn from the heart over a 3-week period. Additional challenges with 1.15 mg of enterotoxin C followed at 3-month intervals. The antiserum used in the present study came from the first blood sample and had a microslide titer (3) of 1:200 against 10 ,Ag of enterotoxin C per ml. A single line of precipitation was obtained with as much as 2,500 jAg of enterotoxin C per ml, treated by the microslide procedure, even against undiluted antiserum. These results indicated the high purity of the enterotoxin C preparation. Growth media. The 3% + 3% PHP-NAK medium (1) was used throughout this study as the basic medium to which different amounts of NaCl were added and in which the pH was adjusted to various values. Thirty grams of N-Z Amine NAK (Sheffield Chemical, Norwich, N.Y.) and 30 g of protein hydrolysate powder (Mead Johnson and Co., Evansville, Ind.) were mixed with distilled water to make 600 ml. The powders were dissolved with mild heating. The medium was cooled and then distributed, in volumes of 30 ml, to 20 beakers. Sodium chloride in amounts of 0, 2, 4, 5, or 6 g was dissolved in each beaker, and the pH of each broth was adjusted to the desired value with 1 N NaOH or 1 N HCI. Next, each broth was transferred to a 50-ml volumetric flask, autoclaved for 15 min at 121 C, and cooled to room temperature. Then 0.5 ml of a solution containing 500 ,Ag of both thiamine and niacin, and sterilized by filtration, was added. The volume of the broth in each flask was brought to 50 ml by adding sterile distilled water. A 9-ml sample of each broth was placed individually in a 25-ml Erlenmeyer flask (micro-Fernbach style). The flask was then covered with a glass cap, and later the broth was inoculated with the desired number of staphylococcal cells. Inoculation and incubation. Applying the antiserumagar plate technique of Sugiyama et al. (16), colonies of strain 137 producing large amounts of enterotoxin C were selected on PHP-NAK agar (pH 6.2) containing 1:40 enterotoxin C antiserum and lyophilized on porcelain beads (9). One bead was added to a test tube containing PHP-NAK broth (pH 6.2, 0% salt), and the broth was incubated for 24 hr. A flask containing 9 ml of the same medium was later inoculated with freshly grown cells and incubated overnight. The next morning the broth was centrifuged, and the supernatant fluid was kept for enterotoxin analysis. The cells were washed twice in saline and then resuspended in saline to a desired optical density (OD) at 650 nm by using a Spectronic-20 colorimeter (Bausch & Lomb). Each experimental flask was inoculated with 0.1 ml of the cell suspension, placed in a waterbath shaker (Precision Scientific Co., Chicago, Ill., model 66900) at 37 C, and incubated for as long as 8 days at a speed of 160 rev/min. Control flasks were inoculated with 0.1 ml of distilled water. The number of cells in each flask was estimated by plating in duplicate on blood-agar. The initial OD and pH of the broths were determined and recorded at zero time. The pH was measured with a Beckman Expandomatic pH meter, by using a Q15 Thomas combination electrode. Analysis of broths. Samples of broth, usually 2.5 ml, were taken at 2, 6, and 8 days of incubation. Of the 2.5 ml, 0.5 ml was used for determination of OD after dilution with similar broth. After its pH was determined, the remainder of the sample was placed in a cellophane tube and dialyzed at 2 to 4 C against 20 volumes of distilled water. The water was changed once. After dialysis, the samples were lyophilized in a VirTis lyophilizer (model 10-145-MRBA). To test for the presence and amount of enterotoxin C, the lyophilized samples were first rehydrated with 0.6 ml of phosphate-buffered saline (pH 7.2, 0.02 M) plus 2% NaCl. Next the amount of enterotoxin C was measured by the single-gel, diffusion-tube test (6,7), with an antiserum diluted 1:60 with gel. The gel diffusion tubes were incubated for 24 hr at 30 C, and diffusion band measurements were evaluated for the amount of enterotoxin C by using a previously constructed, standard enterotoxin C curve. The curve was prepared from data obtained by using various amounts of pure enterotoxin C dissolved in the phosphate-buffered saline. The specificity of the diffusion bands in the gel diffusion tubes as due to enterotoxin-antienterotoxin C precipitation was tested by the microslide, double-gel diffusion test (3). Leftover sample material was heated at 100 C for 15 min and then tested for the presence of heatresistant nuclease (Lachica and Genigeorgis, unpublished data). RESULTS Both NaCl and pH affected growth of staphylococci and production of enterotoxin C. The rate of growth of strain 137 decreased as the NaCl concentration was increased gradually to 12% (Fig. 1B). Optimum pH for growth of this strain appeared to be between 5.00 and 6.50. In the presence of 0, 4, 8, and 12% NaCl, the respective pH limits for initiation of growth were 4.00 and 9.83, 4.20 and 9.43, 4.40 and 8.55 (Table 1). When broths with 10% NaCl were inoculated with 3.5 X 106 cells per ml, growth was permitted, as this was demonstrated by plating when the pH range was 4.50 to 8.50. The effects of NaCl and pH on the production of enterotoxin C by strain 137 are indicated in Fig. 1A. Enterotoxin C was produced in broths inoculated with more than 108 cells per ml and having an initial pH range of 4.00 to 9.83, 4.20 to 9.43, 4.50 to 8.55 and NaCl concentrations of 0, 4, and 8 %, respectively (Fig. 2). In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for the inoculum level of 108 cells per ml and 6.38 to 7.30 for the level of 3.5 X 10 cells per ml. In repeated experiments in which the inoculum contained 108 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The optimum pH for the production of enterotoxin C appeared to be between 5.50 and 6.5 (Fig. IA). When the OD of the broth and the yield of enterotoxin C after 48 hr of incubation were plotted as ordinates on semilog paper and the NaCi concentration was plotted as the abscissa, an essentially linear relationship was evident between concentration of NaCl in the broth and both OD and yield of enterotoxin C. Both OD and yields decreased with increases in the NaCl concentration. This relationship was true at least for pH 5.50, 6.50, and 7.50 and 0 to 8% NaCl. Data for higher NaCi concentrations and pH values outside the range of 5.50 to 7.50 were not sufficient to permit evaluation. DISCUSSION At first glance the present findings indicate that production of enterotoxin C in foods heavily contaminated with staphylococci cannot be prevented by the proper manipulation of pH, NaCl concentration, or combinations of the two within levels acceptable by the consumer. Yet in view of the ideal laboratory conditions used, it is difficult to extrapolate the findings in broths to food products or environmental situations that might exist in food processing plants. The medium used for these experiments was favorable for the growth of staphylococci and free from natural inhibitors and competing microorganisms that may be present in food. The broths were heavily inoculated with staphylococci and incubated at or near optimal temperature with heavy aeration. Consequently the limiting pH values and NaCl concentrations represent the minima and maxima below or above which there is no enterotoxin C production. Therefore, a processor could feel safe in the knowledge that no enterotoxin is produced in a given product if it is more acid, alkaline, or salty than the limiting pH and NaCl concentration values presented above. Initiation of staphylococcal growth at a pH as low as 4 has been observed for the first time. This is not surprising in view of the heavy inoculum used and the recent reports on the probability of initiating staphylococcal growth in various environments (4; Genigeorgis, Abstr. 131, 3rd Int. Congr. Food Sci. Technol.). These studies indicated that, for each combination of pH and NaCl, there is a minimum number of cells required for growth in a new population. The inocula with greater numbers of cells thus have a higher probability of initiating growth. Whether the growth at such a low pH was due to inadvertent selection of a few acid-tolerant cells, or to the shifting of the pH to higher values by metabolic products of nonreproductive living cells or decomposition products of dead cells, has not been determined. Shifting of the pH to higher values will eventually initiate growth and production of enterotoxin. Markus and Silverman (13) have demonstrated release of about 600 ,ug of protein per ml, including enterotoxin B, by 3 X 10's nonreplicating staphylococcal cells per ml in a nitrogen-free medium incubated for 10 hr at 37 C. In the present study, there was an inverse linear relationship between growth of staphylococci and concentration of NaCl at pH levels of 5.50, 6.50, and 7.50. In other studies, statistical analysis of the data indicated a direct linear relationship between concentration of NaCl and the probability of initiating growth by five staphylococcal strains in broths with different pH. When statistically untested data for each strain were plotted, the relationship varied from linear to sigmoid, depending on the pH of the broth (4; Genigeorgis, et al., unpublished data). The inhibitory effect of NaCl on the total viable population, regardless of the pH and temperature of incubation of the medium, has been reported previously (10). Production of any of the enterotoxins has not been reported before in media, including foods, with pH values as low as 4 or as high as 9.83. Tatini et al. (Bacteriol. Proc., p. 10,1969) reported production of enterotoxin A in sterile, reconstituted, nonfat milk solids with an initial pH of 4.50. Detectable amounts of enterotoxin A were associated with 3 to 5 million cells per ml regardless of the initial inoculum (10U to 105 cells per ml). We have demonstrated enterotoxin B production in Brain Heart Infusions at pH 5.05 (6). Reiser and Weiss (14) recently reported on the production of enterotoxins A, B, and C in four media at three pH levels (5.30, 6.00, and 6.80). With strains 137 and 483, they obtained maximum yields of enterotoxin C at pH 6.00 and 6.80, respectively, in the medium we used in this study. They found that neither the medium nor the pH in the range used materially affected production of enterotoxin A by strains 100 and 196 but that there was considerable effect of pH and medium on production of enterotoxins B and C. Kato et al. (11) studied the effects of pH, composition of broth (various protein hydrolysates), and aeration on production of enterotoxin A. The pH of their broths ranged from 5.0 to 8.0 and the toxin yields ranged from 2.9 to 4.2 ,ug/ml. In contrast to its effect on production of enterotoxins B and C, pH did not appreciably affect production of enterotoxin A. The effect of NaCl on production of enterotoxin C aerobically appears to follow the same pattern as with enterotoxin B (6,8,12,15). As the concentration of the salt increases from 0 to 10%, the yields of enterotoxins B and C decrease VOL. 21, 1971 to undetectable amounts. Enterotoxin C was also produced aerobically in cured meats having up to 10% NaCI in the brine (Genigeorgis et al., unpublished data). We have obtained similar results with enterotoxin B (5). The minimum final OD of a culture positive for enterotoxin C was 1.35. Although high yields of toxin are always accompanied by high OD values, there were broths containing 10 and 12% NaCl with OD values as high as 8.7 but no enterotoxin. Such results indicate that the mechanism of enterotoxin production is more sensitive to osmotic changes than the mechanism controlling cell multiplication. Similar findings have been reported for enterotoxin B (5,6,8,12). Adjustment of pH and addition of NaCl after sterilization of the basic broth did not significantly affect the growth of staphylococci and the production of enterotoxin C. Therefore, for practical reasons this adjustment was done before sterilization.

v3-fos

2020-12-10T09:06:57.598Z

1971-05-01T00:00:00.000Z

237230611

s2

Initiation of Staphylococcal Growth in Laboratory Media The effects of pH and NaCl concentration on the probability of aerobic growth initiation in Brain Heart Infusion broth at 30 C by five staphylococcal strains producing enterotoxins A, B, C, and D were studied in a factorial design experiment. Statistical analysis of the data indicated: (i) significant effects of pH, NaCl, and strain on the probability of growth; (ii) diverse effects of NaCl with various pH levels and strains; (iii) essentially a linear relationship between NaCl concentration and probability of growth initiation when data for all strains were pooled; (iv) the relationship between NaCl concentration and probability of growth initiation varies from linear to sigmoid, depending on the pH of the broth, when the statistically untreated (raw) data are plotted for each strain. Equations were derived which relate the decimal reductions of the number of cells of a staphylococcal population to the concentration of NaCl and pH of broth to which the population was exposed. From the equations, the probability that one cell is capable of initiating growth can be calculated. The impact of these types of studies on the development of realistic staphylococcal standards in foods is discussed. The effects of pH and NaCI concentration on the probability of aerobic growth initiation in Brain Heart Infusion broth at 30 C by five staphylococcal strains producing enterotoxins A, B, C, and D were studied in a factorial design experiment. Statistical analysis of the data indicated: (i) significant effects of pH, NaCl, and strain on the probability of growth; (ii) diverse effects of NaCl with various pH levels and strains; (iii) essentially a linear relationship between NaCl concentration and probability of growth initiation when data for all strains were pooled; (iv) the relationship between NaCl concentration and probability of growth initiation varies from linear to sigmoid, depending on the pH of the broth, when the statistically untreated (raw) data are plotted for each strain. Equations were derived which relate the decimal reductions of the number of cells of a staphylococcal population to the concentration of NaCl and pH of broth to which the population was exposed. From the equations, the probability that one cell is capable of initiating growth can be calculated. The impact of these types of studies on the development of realistic staphylococcal standards in foods is discussed. The effects of heat and radiation on bacterial destruction have been studied extensively, and probabilities for survival and growth, especially of Clostridium botulinwr spores, have been estimated. As a result of such studies, a standard heat treatment for low-acid canned foods has been established (18) which will reduce C. botulinum spores by a factor of 1012 (12 decimal reductions or 12D values). This 1:1012 probability of C. botulinwn spore survival is considered a minimum safety requirement. The effect of other preservation methods, such as acidification, drying, curing, and smoking, have not been expressed in terms of decimal reduction or inactivation values. If such values were known, it would be possible to estimate minimum standards for cured and smoked vacuum-packed foods that would achieve a safety level comparable to that of canned foods. Several researchers (2, 6, 10-16) have described the effect of NaCI and pH on Staphylococcus aureus; but quantitative data are still lacking. Some preliminary work which included C. botulinum has been published (6,17). The present study is concerned with estimating the probability of initiation of aerobic staphylococcal growth in broths. Similar studies of growth and enterotoxigenesis in various meats are published separately in the following note (8). MATERIALS AND METHODS Preparation of experimental broths. To prepare the broths, 37 g of dehydrated Brain Heart Infusion (BHI) broth (Difco) was dissolved in 700 ml of boiling distilled water. After the broth was cooled to room temperature, an additional 100 ml of water was added to make the basal medium. To provide the broths with various concentrations of salt, 80-ml portions were placed in beakers, and NaCl was then added in amounts of 0, 4, 8, 12, and 16 g per beaker. After the NaCl was dissolved, the pH was adjusted to the desired values by adding 0.5 N NaOH or 0.5 N HCI. The values were checked with a Beckman Expandomatic pH meter equipped with a Beckman H2 glass electrode. The broths were autoclaved in 100-ml volumetric flasks (15 min at 121 C under 15 psi of pressure) and then cooled. The volume was brought to 100 ml with sterile distilled water. Overnight BHI broth cultures of these strains were inoculated into 25 ml of BHI broth containing 0.25% Tween 80. The fresh cultures were incubated at 37 C on a reciprocal shaker for 4 hr. The cultures were then centrifuged, the cells were washed once with saline, and concentration of cells was adjusted to an optical 934 density (OD) of 0.3 to 1.0 at 660 nm, by using a Spectronic-20 colorimeter (Bausch & Lomb). Nine tubes each containing 9 ml of broth were prepared from each type of experimental broth. A 1-ml amount of the cell suspension was added in the first tube, and 10-fold serial dilutions of the suspension were added to the other tubes. Three portions of 2 ml each were transferred with a sterile syringe from each of the nine tubes to 2-ml screw-cap vials. The caps were put on loosely and the vials were placed in 2-lb coffee cans. In each can, there was a vessel containing a brine of the same NaCl concentration as the broth. The cans were closed with plastic lids and placed in the aerobic incubator at 30 C where they were left for 20 days. Every other day, vials with growth (turbidity) were removed and recorded. From the presence or absence of growth in the 27 vials prepared for each salt-pH combination, the most-probable number of cells which had initiated growth was calculated by the technique of Fisher and Yates (5). The number of staphylococcal cells present in the cell suspension used as inoculum was determined by plating on cow blood-agar (BHI base) in duplicate. This number was always between 107 and 9 X 107. Statistical methods. The experiments, arranged in a factorial design (19), involved five staphylococcal strains, five NaCl concentrations (1,4,8,12, and 16%), and six pH values (4.7, 5.1, 6.1, 7.0, 7.8, and 8.9). For the statistical evaluation of the effects of strain, pH, NaCl, and their interaction upon staphylococci, the logarithm (log) of the ratio RI/RG was used for each broth and strain condition, where RI is the number of cells in the inoculum and RG is the number initiating growth. This log represents the number of decimal reductions of a staphylococcal population resulting from its exposure to a particular broth environment. The probability that one cell will initiate growth in such an environment can be calculated from the formula P = 1/antilog [log (RI/RG)] = RESULTS Statistical analysis of the data indicated the following effects and interactions. (i) Strain, pH, and concentration of NaCl all significantly affected the size of the log decrease of the staphylococcal population. (ii) The effect of NaCl varied with pH levels and by strains. (iii) When data for all strains were pooled, NaCl concentration and size of log decrease were related linearly. (iv) When the raw data for each strain were plotted, the relationship between NaCI concentration and log decrease of the population varied from linear to sigmoid, depending on the pH of the broth (Fig. 1). The above four observed effects and interactions apply also to the probability of growth. From the factorial and multiple regression analysis, the following five equations were derived for the five staphylococcal strains used, .47, respectively. Approximate 95% confidence contours for a specified log reduction can be obtained by using Ye i 2 SE. Using the equations, response curves have been constructed for each strain relating pH, NaCl, and log reductions. In Fig. 2 the curves indicate the combinations of pH and NaCl which will reduce populations of strains S-6, 137, 243, and 472 by 1, 2, 3, 4, 5, and 6 logs. The range of NaCl and pH combinations which will decrease populations of all five strains by 3, 4, 5, and 6 logs is indicated in Fig. 3. It is obvious from the figure that the response of the various strains to NaCl concentrations is becoming more uniform (narrow) as the pH approaches values between 7 and 8. This more uniform response of strains exposed to optimum pH is also apparent in Fig. 1. Response curves and the approximate 95% confidence limits for 6-and 3-log reductions of strain 243 are presented in Fig. 4 for comparison. As indicated in this figure, there is a zone of pH and NaCl combinations which will result in 3to 6-log reductions, whereas the other two zones combinations are likely to result only in large (6-log) and in small (3-log) reductions of populations of strain 243. DISCUSSION The effect of various environmental conditions on staphylococcal growth in culture media and in foods has been studied extensively (2,7,(9)(10)(11)(13)(14)(15), and certain limited standards for staphylococci in foods have been established (1,4). The present study is one of a series aiming toward the accumulation of data which eventually will help in establishing realistic staphylococcal standards. To obtain basic information, we have studied the effects of two important factors, NaCl and pH, on the log reduction of staphylococcal populations inoculated in Brain Heart Infusion broth. The probability that one cell can initiate growth can be calculated from the derived equations which relate NaCl concentration and pH of the medium to log reductions of a staphyllococcal population exposed to this environment. Thus, for a broth at pH 6.0 with 5% NaCl, the log decrease for the strain S-6 incubated aerobically is 1.69, the antilog is 49, and the probability of initiating growth is Y9 or 2.04%. On the basis of data reported previously (7), the same strain incubated anaerobically in the same broth will have a log decrease of 2.92, and the probability of initiating growth is only 0.12%. The effects of aerobiosis (present findings) versus anaerobiosis (7) on the combinations of pH and salt which will decrease populations of strains 137, 243, S-6, and 472 by 3 and 6 logs are further compared in Fig. 5. In general, there is close agreement between the present findings and the data published previously on the effects of NaCl and pH on staphylococcal growth, indicating that the experimental techniques are comparable. The findings of the present and earlier studies are summarized as follows. (i) There is a decreased rate of growth of food-poisoning staphylococci when exposed to media with concentrations of NaCl increased from 0 to 20% (7,10,13,14). (ii) Increasing the NaCl concentration of a broth requires more concentrated inocula for initiation of growth (14). (iii) Higher concentrations of NaCl and extreme pH values prevent growth, or delay it, or diminish the total amount, depending on concentration of inoculum and time of incubation (6). (iv) Smaller concentrations of NaCl are required to inhibit initiation of staphylococcal growth at pH values remote from optimum (12). (v) There is better staphylococcal growth aerobically than anaerobically. The magnitude of the effects of the two conditions varies with strain and, at some limited pH values, appears to be reversed. (vi) The effects of pH and NaCl on staphylococcal growth vary with the strain and the type of medium used (2,(14)(15)(16). (vii) Different investigators have used different staphylococcal strains, inocula, and media (2,7,10,11,(13)(14)(15)(16). Consequently, minor disagreements in present and past findings in the reported values of upper NaCl concentrations and of pH limits permitting staphylococcal growth are understandable. Certain limited standards for staphylococci in foods have been established (1,4). These standards tolerate 0 to 1,000 staphylococcal cells per g of food. However, they vary with agency and with state and country and are based on research data not analyzed statistically. A suggested tolerance of less than 1,000 cells/g probably represents a contamination level that can easily be met in most commercial food preparations. Any NaCl-pH combination (level) that will reduce the probability of growth initiation by a factor of 106, therefore, seems reasonable. The problem then is to find what combinations of NaCl and pH will have such an effect with a probability of safety of over 95%. Some results concerning only strain 243 are presented in Fig. 4. In this figure, any combination of NaCl and pH outside the outer curve has a 95 % probability of giving at least a 6-log decrease in the number of staphylococcal cells. Such calculations may be biased, of course, because we have extrapolated results from our data to an area where we do not have experimental data. In the experiments, our most concentrated inoculum was 9 X 107 cells/ml, and here we are talking about log decreases in the range of 6 + 2 SE (7.83 to 8.78 logs) for all five strains used. This was the reason that decimal reductions due to the effect of 16% NaCl were not considered in the derivation of the equations. The maximum inoculum used was not enough to measure the full effect of 16% NaCl concentration on the initiation of staphylococcal growth in broths. The present data can only indicate, with 95% confidence limits, the combinations of NaCl and pH which will decrease populations of all five strains by 6 logs, less 2 SE (6 logs -2 SE = 3.22 to 4.17, depending upon strain). To obtain more realistic results, the present studies should be supplemented not only with other studies in which concentrated inocula have been used but also with studies in which appropriate food items serve as culture media. The use of such media will eventually allow the development of equations based on quantitative characteristics of foods and permit accurate predictions of the probability of initiating staphylococcal growth. The results of the first series of these types of experiments have been reported (8). Studies on a third food factor, nitrite, have been interrupted until further data are accumulated on the reactions and stability of nitrite in laboratory media and foods. ACKNOWLEDGMENTS This investigation was sponsored by the Food Protection and Toxicology Cetnter of the University of California and was supported by Public Health Service grant FD 00103 from the Food and Drug Administration.

v3-fos

2020-12-10T09:04:11.834Z

1971-03-01T00:00:00.000Z

237232111

s2

Isolation and Identification of Psychrophilic Species of Bacillus from Milk Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures. Antarctic soil. The first report of psychrophilic sporeforming bacteria in food was that of Grosskopf and Harper (Abstr., J. Dairy Sci. 52:897, 1969), who reported isolating some from milk. We have found species of Bacillus capable of growing at 7 C or less to be present in 25 to 35% of raw milk samples. This paper reports their isolation and identification. MATERUILS AND METHODS Samples. Ninety-seven raw milk samples were collected from several areas of California. The samples were refrigerated during transit to the laboratory and tested immediately. The samples (200 ml of each) were held at 80 C for 10 min in a water bath, cooled, stored at 4 to 7 C, and examined for microbiological spoilage after 3 to 4 weeks. Media. The following media (from BBL) were used: Trypticase soy agar (TSA), Trypticase soy broth (TSB), nutrient broth, nutrient agar, and AK sporulation medium. Sporulation was determined with each of the above media, with nutrient agar plus 0.05% MgSO4 and 0.05% MnSO4 and with soybean agar (17). Microscopic examination. Cell morphology, cell arrangement, cell size, motility, presence of spores, and presence of cell granules were determined by phase microscopy. Stained mounts were used for determining the Gram reaction and spore formation (18). Identification tests. Isolates were identified accord-ing to Bergey's Manual. Analyses for biochemical and microbiological characteristics were done by the procedures of Smith et al. (17). Measurement of growth. For determining ability to grow at different temperatures, the isolates were streaked on plates of TSA, and the plates were incubated at predetermined temperatures from 4 to 50 C. Before the streaking, the plates were preheated or precooled to the incubation temperature. Plates were discarded if colonies did not develop in 1 week. Isolates that grew at 4 C were tested for growth within 2 weeks at 0 C in TSB in an antifreeze-water bath thermostatically controlled at 0 i: 1 C. Experiments for determining specific growth rates were conducted in a G76 gyratory water-bath shaker (New Brunswick Scientific Co., New Brunswick, N.J.) adjusted to predetermined temperatures (5 to 35 C) and an agitation rate of 120 rev/min. A 250-ml Erlenmeyer flask containing 50 ml of TSB was inoculated with 0.2 ml of culture (grown at 10 C for 2 to 3 days in TSB), and the culture was transferred periodically (inoculum, 0.1 ml) until it reached the steadystate of growth (13,14). Growth was determined by periodically measuring optical density at 600 nm with a Beckman spectrophotometer (model DB) with 5-cm cuvettes. RESULTS About 25 % of 50 milk samples collected in the summer spoiled in 3 to 4 weeks in the refrigerator (4 to 7 C). This percentage was about 35% for 47 samples collected in the winter. Portions of the spoiled samples were plated on TSA, and the plates were incubated at 21 C for 2 to 3 days. Colonies were transferred to TSA slants, incubated at 21 C, and stored in a refrigerator. Forty cultures isolated from morphologically different colonies by the above procedure were found to be sporeforming, aerobic, gram-positive Minimal (except 0 C) and maximal growth temperatures were determined on Trypticase soy agar. Tests at 0 C were in Trypticase soy broth in an antifreeze-water bath at 0 1 C. bSpore formation was tested in a variety of media. c Lowest temperature tested. or gram-variable, rod-shaped bacteria. They grew in refrigerated milk and produced off flavors, including rancid, fruity, and sour. Fifteen isolates that were morphologically different and indicated by preliminary experiments to have low minimal growth temperatures were selected for further study and identification. These isolates could be grouped into two different groups on the basis of minimal growth temperatures (Table 1). Those in group A were able to grow at temperatures as low as 0 C, whereas those in group B had minimal growth temperatures of 5 to 7 C. More recent experiments in broth have indicated a doubling time of about 30 hr at 0 C for the isolates of group A. Although the isolates of group A grew at 0 C within 2 weeks, they did not sporulate well at any temperature tested (3 to 25 C). In contrast, the isolates of group B, which had higher minimal growth temperatures, sporulated well at each temperature tested. The growth kinetics of one isolate (RH3) of group A are shown in Fig. 1. Growth was slow at 5 C, in comparison to higher temperatures, and growth did not occur at 35 C. Specific growth rates and doubling times at different temperatures for this isolate are given in Table 2. The doubling times at 5 C for the other three isolates of group A (TS3, TS4, and RH22) were 6.75, 6.75, and 9.8 hr, respectively. The biochemical and microbiological characteristics of those isolates of group A did not match completely any of the species of Bacillus described in Bergey's Manual. All four of these isolates were gram-variable and had growth temperature ranges considerably below those specified for species of Bacillus. One isolate, RH22, was considered to be a variant of B. subtilis since the few sporangia observed were not definitely swollen, the protoplasm of young cells grown on glucose agar were not vacuolated, growth on glucose or soybean-agar was good, and the organism hydrolyzed starch, produced nitrites from nitrates, liquefied gelatin rapidly, and grew in broth containing 4% NaCl. However, with ammonium salts as the source of nitrogen, this organism failed to ferment manitol, sucrose, lactose, or glycerol, and it did not grow in broth containing 7% NaCl. The characteristics of another isolate, RH3, indicated that it was a variant strain of B. circulans, though there was 21,1971 good growth on soybean-agar and acid was produced from each of the above four carbon sources or arabinose or xylose. Two isolates, TS3 and TS4, resembled B. coagulans in biological and microbiological tests. They produced acid from glucose and from each of the above six carbon sources. DISCUSSION Psychrophilic bacteria have been defined by Ingraham and Stokes (10) as bacteria that grow well at 0 C within 2 weeks and by Witter (22), based partly on a standard method for determining psychrophilic bacteria (1), as bacteria that grow at a relatively rapid rate at 7.2 C, i.e., that lorm visible colonies on plates at this temperature in 10 days. Foster et al. (6) defines them as bacteria that grow relatively rapidly at 1.7 to 10 C, the temperature normally used in commercial holding and distribution channels. Witter (22) suggested that a generation time of about 15 hr or less at 7.2 C would be a reasonable requirement for organisms that are to be called psychrophiles, and Ingraham and Stokes (10) suggested a generation time of 48 hr or less at 0 C. Two of the psychrophilic sporeformers isolated from mud and studied by Larkin and Stokes (11) had generation times of 8.5 and 11.5 hr at 5 C, and Ingraham (9) reported generation times of 7 to 10 hr at 4 C for psychrophilic pseudomonads. Some investigators prefer to describe such bacteria as psychrotrophic rather than psychrophilic (12,21), and there seems to be merit in using some term that indicates that they merely are able to grow at low temperatures rather than that they are cold-loving. In any event, all of the isolates we studied grew under refrigeration at 7 C or less, but they grew faster at higher temperatures (25 to 35 C). Those in group A (Table 1) were able to grow within 2 weeks at 0 C and had generation times of about 30 hr at 0 C and 6.5 to 9.8 hr at 5 C. Of the isolates we studied, at least these four fit each of the above definitions proposed for psychrophilic bacteria. The important difference between the bacteria we isolated and known characteristics of mesophilic bacilli is that those we isolated were able to grow at lower temperatures. Mesophilic bacilli normally have not been found to grow below about 8 C, and 10 C often has been found to be their minimal growth temperature (5,10,19,22). We suspect that the organisms we isolated are variants of mesophilic bacilli adapted to lower growth temperature ranges (2,7,16). Simultaneously with this adaptation, there apparently developed a tendency toward poor sporulation that was particularly evident in the isolates of group A. We did not attempt to reverse this adaptation, but Grosskopf and Harper (Abstr., J. Dairy Sci 52:897, 1969) reported that the psychrophilic sporeforming bacteria they isolated could be adapted to higher growth temperature ranges and that they then were unable to grow at low temperatures. Spoilage of pasteurized milk and milk products often results from the growth of heat-sensitive, gram-negative, nonsporeforming bacteria that enter products after pasteurization. In regard to this, failure of the isolates of group A to form spores readily on laboratory media and the fact that they were gram-variable are important. Possibly some instances have occurred in which gram-variable bacteria such as those we isolated have been mistaken for heat-sensitive species, e.g., Pseudomonas, Alcaligenes, or Aerobacter. The doubling times of the four isolates of group A at low temperatures are not greatly different from those of some species ofPseudomonas known to be important in the spoilage of foods (4,8,9,20,22), but the isolates of group B grew more slowly. In regard to this, it is important that the organisms of group A were found in only four of 97 samples of raw milk. Obviously, it would be a mistake to relax efforts to prevent nonsporeforming psychrophilic bacteria from getting into milk and dairy products after pasteurization. However, as attempts are made to extend the shelf-life of fluid dairy products, psychrophilic sporeforming bacilli will become a greater potential problem. This is also important in regard to the use of dairy products in other foods, the development of aseptic filling, and the probability of a shift toward "sterilization" of milk and fluid milk products.

v3-fos

2014-10-01T00:00:00.000Z

1971-01-15T00:00:00.000Z

9978567

s2

Cytogenetic considerations in animal breeding Some fundamental aspects of cytogenetics, a brief review of work done with farm animals, and an assessment of the known effects of chromosomal aberrations on animal population are summarized in this paper. The basic chromosome number and other detailed features of the karyotype have been reliably established for all the farm animals within the past 10 years. Intra-species variants, i.e. chromosomal polymorphisms have been found in cattle, pigs, and goats. Two primary types of aberrations are recognized. The first, an alteration of numbers of whole chromosomes is known as heteroploidy. The second type, rearrangement of chromosomal segments, results following physical breaks of chromosomes. Factors affecting the frequency with which aberrations occur include both genetic and non-genetic elements. Among the most important in the latter category are certain virus groups, some drugs and other chemical compounds, ionizing radiations, age of gametes, particularly ova, and in some animals, maternal age. Presence of chromosomal aberrations are apparently responsible for a large proportion of the embryonic deaths in mammals and birds. In man, perhaps as many as one-third of spontaneously expelled abortuses are afflicted. Early indications from work with pigs indicates that the situation may be similar. Very little cytogenetic work has been done with congenitally malformed livestock, although a number of aberrations have been reported. In man, somewhat less than 1 p. 100 of new born babies carry chromosomal abnormalities. Some aberrations, although they cause only slight if any somatic alteration, can be the source of reduced fertility or sterility in their bearers. Isolated cases of this phenomenon have been reported but a quantitative assessment of its importance has not yet been made. The presence of centric fusions as chromosomal polymorphisms in cattle, pigs, and goats has been reported, and work is being conducted to ascertain how they effect fitness of the populations in which they occur. It is concluded that the occurrence of chromosomal aberrations causes a serious reduction of potential productivity. The extent of this loss should be accurately determined and means found to reduce it. Two primary types of aberrations are recognized. The first, an alteration of numbers of whole chromosomes is known as heteroploidy. The second type, rearrangement of chromosomal segments, results following physical breaks of chromosomes. Factors affecting the frequency with which aberrations occur include both genetic and non-genetic elements. Among the most important in the latter category are certain virus groups, some drugs and other chemical compounds, ionizing radiations, age of gametes, particularly ova, and in some animals, maternal age. Presence of chromosomal aberrations are apparently responsible for a large proportion of the embryonic deaths in mammals and birds. In man, perhaps as many as one-third of spontaneously expelled abortuses are afflicted. Early indications from work with pigs indicates that the situation may be similar. Very little cytogenetic work has been done with congenitally malformed livestock, although a number of aberrations have been reported. In man, somewhat less than 1 p. 100 of new born babies carry chromosomal abnormalities. Some aberrations, although they cause only slight if any somatic alteration, can be the source of reduced fertility or sterility in their bearers. Isolated cases of this phenomenon have been reported but a quantitative assessment of its importance has not yet been made. The presence of centric fusions as chromosomal polymorphisms in cattle, pigs, and goats has been reported, and work is being conducted to ascertain how they effect fitness of the populations in which they occur. It is concluded that the occurrence of chromosomal aberrations causes a serious reduction of potential productivity. The extent of this loss should be accurately determined and means found to reduce it. -INTRODUCTION Interest in the chromosomal complements of farm animals has been high since early in the zoth century when it was first established that the chromosomes were the vehicles on which the genes were carried. Particularly European and Japanese workers pursued the question of the normal chromosome numbers of the domesticated species. It is a tribute to the astuteness and perseverance of the pioneers that although techniques were laborious and inadequate, they managed, sometimes with uncanny accuracy to describe the karyological make-up of a number of vertebrates. (For a historical review of ruminants see H ULOT and I, AUVERGN!, 1967.) Three technical developments of the 195 o's enabled cytological studies of mammals to be made much more readily, accurately, and on larger samples of specimens. These were ( I ) Hsu and P OMERAT ' S ( 1953 ) discovery of the salutary effect of the treatment of cells in hypotonic solution prior to fixation; ( 2 ) the use of mitosis arresting agents, most notably colchicine, to arrest cell division at a stage most suitable for observation of chromosomes; ( 3 ) the development of simplified techniques for cell and tissue culture, particularly the startling finding of M OORHEAD et al. ( 19 6 0 ) that certain substances would induce mitosis of lymphocytes. Although various modifications of these new found methods have been widely used to study variations in the chromosomal complement of man, they have only quite recently been used by workers interested in increasing productivity of farm animals. II. -CHROMOSOMES OF LIVESTOCK The normal karyotype of all domesticated mammals and most of the birds has, by now been well established (Hsu and BE NIRS C HK E, 1970 ). In Table i this information is summarized for the more common ungulates. There are a number of interesting features characterized by the data on this table. First note the extreme evolutionary stability in the number of chromosomal arms in a wide spectrum of species of Bovidae. This point has been well made by the studies of a number of taxa by Wuxs'r!R and B ENIRSCHKE (ig68). In contrast is the wide variability exhibited by karyotypes of the various equine species. This fact would be even more notable if I had listed data for some of the zebras (see Hsu and BE NIRS C HK E, ig 7 o). Secondly, it is interesting to note that although relatively few animals of any of these species have been examined, already chromosomal polymorphisms have been found. The centric fusion in cattle is in reference to the exhaustive study made by G USTAVSSON ( 19 6 9 ) of Swedish Red and White Cattle. A similar type of polymorphism is being studied by M C F EE and others ( 19 66) in a group of wild pigs descended from an importation to the U.S. from Germany. It appears that Zebu cattle possess an acrocentric Y chromosome while the Y of European cattle is submetacentric (K IEFFER and C ARTWRIGHT , 19 68). Undoubtedly as more animals are looked at from a wider geographical spectrum more variants will be found. They are interesting in their own right and because they shed some additional light on the evolutionary history of our livestock. III. -TYPES OF ABERRATIONS Before proceeding with a discussion of the causes and effects of aberrations of chromosomes, it might be well briefly to review the types of things that can happen to chromosomes and to establish some terminology. A more complete discussion was previously published (FE CHH E IM E R , 19 68). In general there are two types of alterations that can occur. The first is an alteration in the number of whole chromosomes present in a cell and the second is a structural rearrangement of chromosomal segments following the occurrence of physical breaks. Alteration of the normal 2 N somatic number by whole multiples of a gametic set is known as euploidy. The two primary categories are haploidy and polyploidy, of which triploidy ( 3 N) and tetraploidy ( 4 N) are the most frequently seen types. Aneuploidy refers to a change in the genome of one or a few chromosomes. The most frequent types of hypodiploidy and hyperdiploidy are monosomy and trisomy respectively. These terms refer to karyotypes with one missing chromosome and one additional chromosome. In mammals all heteroploid conditions with the exception of some trisomics and monosomic X are lethal and result in the premature death of the animal, usually prior to birth. Structural aberrations may be classified upon the number of breaks that necessarily preceeded the rearrangement of material. Thus one break results ultimately in the loss of a terminal segment. If the break is through the centromere, an isochromosome, i.e. one with two identical arms, may arise. Two breaks may occur in the same chromosome. This can lead to an interstitial deletion, an inversion, or formation of a ring. Should one break occur in each of two chromosomes, the most likely recovery products would be translocation chromosomes each of which possessed a centromere. Products resulting from other types of refusions of broken ends are mostly unstable. Quite obviously it is possible to have multiple aberrations, either in the same cell line or in separate cell lines of the same organism. It may seen unlikely, given the low frequency of occurrence of most aberrations, that one would find multiple aberrations, but such is not the case. The physical event giving rise to one error in many instances produces two new aberrant cells at the same time. An example of this would be nondisjunction of a chromosome at an early cleavage division. In this case two new lines, one monsomic and one trisomic for the chromosome in question would be generated simultaneously by the single event. Finally, in discussing organisms which possess more than one cell line, it is useful to distinguish between mosaics and chimerae. The former is the of two or more cell lines derived from the same zygote. Chimerism is the presence presence of an additional cell line in an organism, derived from another zygotic product. Freemartins are therefore chimerae. IV. -VARIABLE EFFECT OF ABERRATIONS The effect that any of these chromosomal aberrations will exert on an organism or on a population depend upon a number of interrelated factors, some of which are briefly discussed. a) Quite obviously if a large chromosomal segment were lost (or perhaps duplicated) the expected effect would be greater than if a small segment were involved. This would be particularly so if the lost segment were composed primarily of heterochromatin and thus contained little genetic activity. b) Some aberrations, e.g. inversion or translocation, when carried as heterozygotes, will exhibit no somatic manifestation on their bearers. However, at meiosis complications do arise and aberrant gametes will be produced. Secondary aberrations are, as a consequence to be expected in some of the progeny of parents carrying a structural aberration. c) The later in embryogenesis that an aberration occurs, the more attenuated will be its influence on the organisms. If, for instance it occurs at the first cleavage division then all (or at least half) of the zygotes cells will eventually contain an aberrant genome. On the other hand if it first occurs in one cell in an already differentiated tissue, only a small proportion of the organisms cells will contain an altered genome and no noticable alteration of phenotype need be produced. ' d) Some aberrations are self limiting in that they produce such profound effects that they severely alter the mitotic activity of the cell containing them. In this case the aberrant cell line will remain small and may not manifest itself at all. Very little is in fact known about competition and selection among genetically different cell lines. e) Other phenomena of embryogenesis can serve to attenuate the phenotypic expression of an aberration. ( I ) It was pointed out by BE AT T Y ( 1957 ) that if an event occurs during the first few cleavage divisions, the aberrant cell line might be entirely included in the extra-embryonic tissues, because only a few cells in the inner cell mass are destined to contribute to the embryo itself. ( 2 ) The presence of dosage compensation mechanisms is another factor that alters the effect of an aberration. Inactivation of one X chromosome in each cell of developing mammals is such a mechanism that was first postulated by I, YON ( 19 6 2 ). When an aberrant X is present, it has been shown that it is preferentially inactivated so that most cell lines are left with the normal X as the fully functional one. f) Just as with gene mutations, the same event even in otherwise genetically identical populations will produce variable effects depending upon the environment in which the population is found. The fact has been elegantly demonstrated by D OBZHANSKY and his group. They have observed that the frequency with which particular inversions are observed in populations of Drosophila is influenced by a number of environmental factors. Some inversions, detrimental to the population in one ecological niche, enhance its fitness in others. V. -CAUSATIVE FACTORS OF ABERRATIONS Explicit in the preceding discussion is the concept that mitosis and meiosis are not invariably regular processes that always give rise to the expected end products. Both are subject to a variety of irregularities, each of which has its own expected array of aberrant end products. In Table 2 are listed a number of agents that have been implicated as etiological factors of chromosomal aberrations. It will be seen that, like any other characteristic of living organisms both genetic and non-genetic factors are involved. Let me very briefly relate the nature of some of the evidence implicating the factors listed. In regard to genetic control, the evidence is quite clear that incidence of heteroploidy differs in genetically distinct stocks of mice. Furthermore, selection for increased incidence yielded a positive response (see B EATTY , 1957 for review). In cattle inbred lines derived from a common source have been shown to differ in the frequency of cells with hypodiploid or hyperdiploid chromosome complements (Z AR T MAN and F!CHH!IM!R, ig6 7 ). At least one gene is known in man that produces chromosome breaks and also brings about a relatively high rate of somatic pairing (GERMAN and A R C HIBALD , 19 65). That meiosis is primarily a genetically controlled process is clear from studies of actions of many mutant genes in bringing about variations that lead to production of aberrant gametes. Most of such work has so far been done with plants and lower animals (S ANDLER et al., 19 68). Primary aberrations, both numerical and structural, cause complications at meiosis that result in new secondary aberrations in the gametic genomes. Frequently however the relationship cannot be discovered because the primary aberration will have been cryptic. An example of this would be paracentric inversions which can be discovered only by linkage studies or by viewing germ line cells at fist meiotic prophase. They cannot be seen in somatic cell preparations. The relationship between autoimmune disease and aberration is not yet clear. It has been noted that parents of babies with monosomic or trisomic conditions have a higher incidence of autoimmune disease than paired controls (BuRCH et al., 19 66; Vnr,!,oTOrr and FORBES, 19 67). A multitude of specific external agents appropriately applied to cells will bring about aberrations. The entire spectrum of ionizing radiations, many chemical compounds (Fvnrrs, 1970 ), certain virus groups (NrcHOr,s, 19 66) and heat shock (BE AT T Y , 1957 ) produce the indicated effects. The action of some of these agents is fairly well understood while for others nothing at all is known except that results are repeatable. The maternal age effect manifests itself in man where there is a precipitously increased frequency of trisomic children born to mothers over 40 years of age (P!ENROS!, 19 66). While the same phenomenon was not observed in mice (G OODLIN , 19 65), it may be that an unfortunate choice of lines was used for the study. E DWARDS ( 197 o) has shown that chiasma frequency is lower in oocytes of older females, both human and mice. He argues that this fact could be a causative agent for increased trisomy. When mating is delayed much beyond the time of ovulation, mammalian ova either lose their mechanism of defense against penetration by more than one sperm or do not undergo a second meiotic division. In both instances the zygote contains more than two haploid sets of chromosomes, i.e. is polyploid. Polyploidy resulting from delayed mating has been demonstrated in mice (V I C K E R S, 1 9 6 9 ), rats (BUTCHER and FuGO, 19 6 7 ) rabbits (SHAVER and C ARR , 19 6 7 ) and pigs (HArrcocx, zg5g; HuNT!R, ig6!). It has not yet been demonstrated in a monotocous species but the relatively high occurrence of triploidy in man, where there is no estrus exhibited, might be considered indirect evidence. S A I, ISBURY ( 19 68) has repeatedly observed a correlation between time of storage of sperm prior to use in A.I. and estimated death rate of the embryos resulting from its use. Such observations are certainly suggestive of the increased presence of aberrations in stored gametes. Finally, it can be shown that aberrant cells accumulate in certain mammalian tissues at specific times in the life of the individual (S WARTZ , 195 6) or can be induced to accumulate (or not to) by appropriate hormone treatment. The physiological function of polyploid cells in liver, pancreas and other tissues is not known. Their appearance can be hastened by sex hormone treatment and can be repressed by castration or thyroidectomy (S WARTZ et al., ig6o; S WAR T Z and FORD, ig6o). ' It is clear from the foregoing that enough is now known about the etiology of various types of aberrations that a researcher can produce, almost at will, any type of aberration that he wishes to study. Not nearly enough is known however to be able to discern how the spontaneous incidence of chromosomal aberrations might be reduced by the animal breeder or clinical practitioner for economic or humanitarian purposes. VI. -EFFECTS OF CHROMOSOME ABNORMALITIES Just as with gene mutations, the effect of chromosome aberrations is harmful in the overwhelming majority of instances in that they reduce the fitness of their bearer in one or more of a variety of ways. Embryo and fetal deaths, congenital malformation, intersexual phenotype, other forms sterility or reduced fertility are the costs of chromosome aberrations. Following is a brief review what is known regarding the incidence of chromosomal abnormalities and their effects in animal populations. A. -Chromosome aberrations and embvyonic death In Table 3 are shown some representative estimates of the frequency of embryos afflicted with one or another type of chromosomal aberration. In man, well over 1 , 000 spontaneous abortions have been examined cytologically with the very surprising finding that about z / 5 , i.e. 20 p. 100 possess an aberration that in all likelihood was the cause of death and abortion (Geneva Conference, ig66). The frequency of embryos with aberrations is higher in younger embryos (C ARR , 1970 ) and it is now thought that among the very early abortions (those occurring in the first month of pregnancy) the incidence may be as high as 30q.o p. 100 . If it is supposed that 20 -25 p. 100 of successfully fertilized human eggs are subsequently aborted, then it can be calculated 4 -5 p. 100 of all zygotes carry a lethal chromosome abnormality. BE A TT Y and F I SC HB E R G (BEATT Y , 1957 for review) looked at more than although the scale of the experiments so far conducted has been small, it appears that about 10 p. 100 of preimplantation embryos are chromosomally defective and destined to die as a result. From preliminary studies of cattle by McFEEi<Y ( 19 68) and HA RP E R ( 1970 ) it appears that the situation is similar. The meaning of all this is that about one third of the prenatal pregnancy wastage of our farm animals is attributable to the presence of chromosome abnormalities. We have found the chicken to be a very useful organism for more detailed studies of this phenomenon. Different strains have different incidences of chromosomal aberrations in very early embryos, one broiler stock having an incidence of z2 p. 100 (MILLER et al., 1970 ). Aberrant embryos in this stock include monosomy, trisomy, ZZW, haploidy, triploidy, tetraploidy and rearrangements. In addition simple and complex mosaics are common occurrences in this stock. In one experiment 15 of 3 q. aberrant embryos all derived from one hen. This observation plus the clear difference in frequency among lines leads one to suppose there is a strong genetic component in the etiology. The distribution of types of abnormalities seen in embryos of diff erent species of animals is shown in Table 4 . It is seen that in aborted fetuses o f man trisomy is the predominant abnormality and monosomy (mostly XO) and po lyploidy are about half as frequent. In the mouse, pig and rabbit polyploidy causes most of the trouble while in chickens, euploid mosaics are by far the most important. Some of these differences reflect the type of material collected and the techniques used but there is no doubt that different mechanisms are involved in causing cytological errors in the different species. Very probably, an important job for those interested in livestock production is learning about the causes of and how to control the induction of spontaneously occurring polyploidy. B. -Chvomosome aberrations in neonates Estimates of the frequency of neonatal animals with chromosome abnormalities are not available for any species other than man. Representative estimates of incidence of newborn babies with aberrations are listed in Table 5 . As a general statement, the overall incidence can be taken as somewhere between o.5 p. 100 and i.o p. 100 . For technical reasons this can be thought of as the minimal frequency. The afflictions are divided rather evenly among three categories: sex chromosome aneuploidy, trisomy and rearrangements. Sex chromosome aneuploidy is higher in males owing to the fact that XXY is more viable and therefore has a greater probability of surviving to birth than does XO. Also XYY is not infrequent in neonatal samples properly ascertained. For the rest, there does not appear to be a difference between the sexes. No similar surveys appear to have been conducted in animals. It is thought that the incidence would be lower because trisomy does not seem to be so frequent in animal populations. From the relatively few laboratories engaged in cytogenetic research with domestic animals, the whole array of expected sex chromosome aberrations are being turned up (see Table 6 for list). XX /XY chimerism has been seen in all the Llngulatas. XXX, XO, XXY and various mosaics containing these cell types have been reported by German, Scottish and New Zealand workers. what is causing them and what are the effects. A comprehensive review of sex chromosome aneuploidy in animals was written by BE A TT Y (i 9 6q). Along this line it should be noted that a number of aberrations can be experimentally produced in mammals now. A number of structural aberrations were found in live born pigs derived from sperm that had been x-irradiated (Z ARTMAN , FEC HH EI M E R and BAKER, r 9 6 9 ). A total of 4 o pigs was born of which 8 contained aberrations including translocations, inversions and deletions. This sort of study is very promising both in terms of the genetic information that can be gained and by being able to produce at will aberrant animals for detailed anatomic and physiological study. C. -Chromosome aberrations and infertility From studies of man it has been learned that numerical aberration of the sex chromosomes are reasonably well tolerated in that they are compatible with survival although, with few exceptions, the bearers are sterile. Those causing complete sterility, e.g. XXY and its variants, are unlikely to be sources of serious loss of fitness in animal populations. However, carriers of structural aberrations are expected to have reduced fertility, so if such mistakes happen at all requently, they can be an important source of loss of reproductive fitness in the population. In mice, semi-sterility of males is a regular concomitant of translocation heterozygosis and most other chromosome anomalies (1 / y ON and ME R E DI T H , 19 66). In man, about 10 per cent of individuals presenting themselves to infertility clinics where karyotyping has been done have been found to carry a chromosome aberration (KJ!ss!,!x, ig6 5 ; Mdi / T R EE et al., zg66). It is not known, of course, what proportion of the total population this represents. In the data presented earlier, it was seen that about 0 . 14 per cent of neonatal babies are carriers of structural aberrations. A large proportion of these will have a reduced fertility or sterility. The picture is even less clear in farm animals. It has been shown by KNUns!rr (1958, ig6ia, z 9 6zb) and other Swedish workers (H!Errxrcsorr and BÄ CKSTR Ö M , 19 6 4 ) that some of the infertility of cattle and pigs is attributable to structural aberrations possessed by otherwise normal animals. Recent observations on infertile rams have disclosed translocations (B RU E RE , 19 6 9 ) as well as the sex chromosome aneuploidy referred to earlier (BRUi;R! et al., 19 6 9 ). It is too early to attempt an assessment of the importance of chromosome aberrations in causing reduced fertility of farm animals. It is clear however that they are a factor to be taken into account. VII. -POLYMORPHISMS The number of chromosome polymorphisms that have been found in farm animals is very surprising in view of the relatively small numbers of animals that have been studied. Centric fusions, i.e. translocation and loss of a centro- ig66). Each of these variants is thought to be present as a stable polymorphism in the population where it was found. It is probable, but not at all certain, that they became established through genetic drift but to be maintained they must not be severely detrimental. In any case, from this plus other evidence, it does appear that the ungulate chromosomes are particularly prone to centric fusion and it is to be anticipated that more will be found. They should be thoroughly studied not only because they are of interest in their own right but because important insights applicable to similar conditions in man can be gained. An important source of duplication in man is unbalanced gametes produced by translocation carriers. Much can be learned in our animals regarding segregation mechanisms in translocation heterozygotes that would be extremely useful to human geneticists. Need f o y mo y e work It is clear from this brief review that cytogenetic research in farm animals has only just begun but promises to contribute answers to some important problems of livestock breeding and genetics. There are a number of things needing to be done immediately. Surveys should be conducted in a number of places to determine the frequencies and types of chromosome aberrations in domestic animals. Attempts should be made to ascertain the expected effects of given aberrations on their carriers and on populations as a whole. The importance of the various possible causes of aberrations should be carefully determined for each of the groups of farm animals. When this is done, it is probable that their frequencies can be materially reduced by adopting appropriate husbandry practices. In addition, when the causes are known, various aberrations can be experimentally produced and their possible beneficial aspects viewed by geneticists. Chromosome abnormalities in domestic animals will be very useful as models of their counterparts in man. The mouse, for technical reasons, has a limited usefulness in this respect. Once this background of information is established, it no doubt will be found that cytogenetic investigations of farm animals have much to contribute to enhancing efliciency of animal production. Chez le Boeuf, le Porc et la Chèvre on a trouvé des fusions centriques et des travaux sont en cours pour préciser leur effet sur les valeurs adaptatives des populations où elles sont apparues.

v3-fos

2018-04-03T05:48:59.614Z

1971-05-01T00:00:00.000Z

44580340

s2

Preliminary Observations on the Pathogenesis of a Virulent Strain of Newcastle Disease Virus in Chickens Development of Newcastle disease, after experimental and natural infection with the virulent strain VLT of Newcastle disease virus, and its growth and distribution in some selected tissues as assayed by the enumeration of plaques are reported. The plaque technique developed by Dulbecco (1) has been extensively used in studies of Newcastle disease virus (NDV; references 2, 6-8). Despite its potential value in yielding precise determination of virus, the technique was little utilized in studies of the pathogenesis of the disease in the domestic fowl or in epizootiological investigations. Earlier workers (3,4,9) have reported successful isolation and enumeration of different strains of NDV in embryonated eggs from various tissues after experimental or natural infection of chickens. In this paper we describe the development of Newcastle disease (ND) in chickens after experimental and natural infection and present data on proliferation of the virus in some selected tissues, as enumerated by the plaque technique. The virulent strain VLT of NDV, isolated during 1968 from an outbreak of highly fatal ND among chickens at Talamara, Lebanon, was plaque purified and used in its third chick embryo passage level for these experiments. Virus stock was prepared by inoculating with approximately 104 plaque-forming units (PFU) in 0.1 ml of seed virus into the allantoic cavity of 9-day-old chick embryos. Infected allantoic fluid was stored in 1-ml amounts at -60 C. Primary chick embryo cell cultures were prepared from minced 9-day-old decapitated chick embryos subjected to repeated trypsinization. The cells were grown in Eagle's minimum essential medium (MEM) containing 5% calf serum and 8% Tryptose phosphate broth. Approximately 5 x 106 cells per 2-oz prescription bottle were seeded, and monolayers were overlaid after infection with 6 ml of overlay medium, which consisted of Hanks balanced salt solution without phenol red, 0.5 j' lactalbumin hydrolysate, 1.0 Noble agar (Difco), 3 %-y, horse serum, 1.5% of 1:1,000 dilution of neutral red, 5% of 4.4%,, sodium bicarbonate solution, 100 units of penicillin and 100 ,ug of streptomycin per ml. Monolayers were used for virus assays 3 days after seeding (10). Seventy White Leghorn six-week-old chicks were divided into seven groups of 10 each. They were caged and placed in a room in which birds were never kept before. To avoid the risk of transmission of infection through feed and water supplies, each group was provided with its own feed and water. One group was maintained as contact controls, and the birds in the remaining groups were inoculated intramuscularly (pectoral muscles) with 0.5 ml of serial 10-fold dilutions of VLT strain of NDV. Chicks were observed twice daily. At postmortem examination, brain, spleen, trachea, and lung tissues were removed aseptically with separate, sterile instruments to avoid cross contamination from dead chickens as well as from sick ones killed by cervical dislocation. A 10%, (w/v) suspension of tissue was made in MEM containing 1,000 units of penicillin and 1,000 ,ug of streptomycin per ml. Tissue suspensions were kept frozen at -60 C until tested. All end points were calculated by the Reed and Muench method (5). The embryo-propagated virus stock had a titer of 109.6 50% chicken lethal doses per 0.5 ml, indicating that the virus strain is highly virulent for susceptible chickens. The incubation period varied from 3 to 4 days, but the majority of birds showed symptoms on the third day. Respiratory symptoms with rales were pronounced in almost all birds. Most curred within 2 to 4 days after the onset of the symptoms. Torticollis and lateral movement of the head were commonly observed. A few birds developed paralysis of both legs. The gross pathological lesions consisted of extensive involvement of the proventricular submucosa and intestinal follicles. Severe hemorrhagic necrotic lesions adjacent to lymphoid plaques were also common. Infection spread easily to the contacts as signs appeared 4.8 days after probable exposure (Table 1). A summary of virus titers in various tissues is presented in Table 2. The VLT strain multiplied extensively in the tested tissue, namely, spleen, trachea, brain, and lungs. Virus titers varied between 10-3 and 108.1 PFU/g of the brain tissue, indicating the relationship between concentration of virus in the brain and occurrence of nervous symptoms. Large amounts of virus were present in the tested tissues even on the day the chickens first showed symptoms, indicating that generalization of NDV in chickens probably occurred before the onset of clinical signs. Virus may therefore be excreted 1 or 2 days preceding the appearance of clinical signs. From the practical standpoint, it seems that probably the trachea is more suitable than other tissues for virus recovery. Bird 1848, which had a moderate quantity of virus in its trachea but not in the spleen, brain, or lung, remained apparently healthy, whereas its cagemates died on the sixth day. In bird 1844, which almost recovered after showing respiratory and nervous signs (paralysis of extremities), a high concentration of virus was found in the trachea and brain when killed on the 10th day. It is possible that such birds can become effective carriers. From our study it can be concluded that the strain VLT is highly pathogenic for 6-week-old chickens. In our experience it spread readily and multiplied extensively in various tissues after experimental and natural infection. Critical organs, damage of which reflects the occurrence of symptoms, were the brain, trachea, and lungs. The virus seemed to generalize before the onset of clinical signs, and we consider that it could disseminate during this period. It has been observed that some birds can recover and may possibly become carriers after infection. The tential value of the plaque technique in primary chick embryo cell culture for the study of pathogenesis of ND has been indicated.

v3-fos

2020-12-10T09:04:17.169Z

1971-09-01T00:00:00.000Z

237231909

s2

Thermal Resistance of Certain Oncogenic Viruses Suspended in Milk and Milk Products Thermal destruction rate curves were determined for adenovirus 12, reovirus 1, and herpes simplex virus in sterile milk, raw milk, raw chocolate milk, and raw ice cream mix. At 40 to 60 C, the curves were asymptotic to the base line. At 65 C, which is near the pasteurization standard, the curves approached a first-order reaction. Thermal resistance studies, by means of in vivo assays, of Moloney and Rauscher leukemia viruses and Moloney and Rous sarcoma viruses indicated that Rous sarcoma was the most resistant. A comparison of the 12D processes of Rous sarcoma virus, reovirus 1, adenovirus 12, and herpes simplex virus in ice cream mix (the most protective of the suspending menstrua studied) with the U.S. Public Health Service pasteurization standard indicated an adequate safety factor in current pasteurization practices. Budding "C"-type viral particles have been observed in short-term phytohemagglutinin-stimulated lymphocytes recovered from the blood of normal and leukemic cattle (13). Similar viruses have been found in lymphocytes in cows' milk (12). Electron microscopic examination of feeder layers inoculated with buffy coats recovered from the blood of leukemic cattle and with long-term bovine lymphocyte suspension cultures revealed the presence of "C"-type budding viruses (4). Viruslike particles have also been found in milk, in biopsies, and in cell cultures derived from leukemic cows (7,8,10,15,20). These reports stimulated interest in viral heat resistance and thus in the adequacy of the pasteurization practices of the dairy industry. Pasteurization times and temperatures recommended by the U.S. Public Health Service have been adopted by the states so that there is uniformity in the required minimal holding times and temperatures (21). The minimal standards are (i) 62.8 C for 30 min or 71.7 C for 15 sec for milk, (ii) 65.6 C for 30 min or 74.4 C for 15 sec for milk products with added milk fat or sweeteners, and (iii) 68.3 C for 30 min or 79.4 C for 25 sec for ice cream mix. Because the viruses demonstrated in milk and the lymphocytes could not be propagated in the laboratory, the assessment of the effectiveness of pasteurization was based on thermal inactivation studies of oncogenic animal viruses that could be assayed either in vivo or in vitro. This report describes the thermal resistance of Rauscher and Moloney murine leukemia viruses, Moloney and Rous sarcoma viruses, adenovirus 12, herpes simplex virus, and reovirus 1 suspended in milk and milk products. Reovirus, although not a known tumor-producing virus, was studied because it has been isolated from human lymphoma (1,17) and from feline leukemia patients (3,16). MATERIALS AND METHODS Virus. Crude mouse spleen extracts of Rauscher leukemia virus, plasma concentrates of Moloney leukemia virus, partially purified extracts of Moloney sarcoma virus, and partially purified extracts of Rous sarcoma virus, Bryan strain, were obtained from various contractors within the Special Virus Cancer Program of the National Cancer Institute. In addition, adenovirus 12 NIAID, herpes simplex virus HF VR 260, and reovirus 1 Lang VR 230 were passaged in Cercopithecus aethiops primary kidney cell cultures. In vitro assay. A modification of the plaque-forming unit assay previously developed was used in these studies (19). Viruses were assayed on primary cell cultures of C. aethiops monkey kidneys under an overlay medium. The medium contained 0.95% clarified lonagar No. 2, Eagle's minimal essential medium with nonessential amino acids in Hanks balanced salt solution without phenol red, 2% fetal bovine serum, 0.19% NaHCOB, 0.0015% neutral red dye, 0.51% MgC12. 6H20, and 1.0% sterile homogenized bovine milk. Bacterial and fungal contamination were controlled by the addition of 1,000 units of penicillin G, 1,000 mg of streptomycin sulfate, 50 ,Ag of tetracycline hydrochloride, and 0.5 ,ug of amphotericin B per ml of the overlay medium. Plaques were counted and marked daily. In vivo assay. The frozen ampoules containing the heat-treated leukemia viruses suspended in milk or ice cream mix were thawed in a water bath at 37 C. The contents of three ampoules were pooled and diluted with an equal volume of phosphate-buffered saline, and 0.1 ml was inoculated intraperitoneally into each of 20 Balb(c/cr) mice. Two additional 10-fold dilutions were made, and 20 mice were inoculated at each dilution. Unheated control samples, with and without virus, plus a standard virus control, were titrated with each sample tested. The test mice were less than 3 days old at the time of inoculation. A toxic effect resulting in death of the mice within 48 hr was encountered when the samples were inoculated into the newborn mice. The minimal dilution that could be tolerated was 10-1 3. Therefore, all murine in vivo results were based on this dilution. The Rous sarcoma virus was diluted to 10-1 for similar reasons. The undiluted inoculum killed the embryos within 24 hr. Small numbers of viruses surviving the heat treatment could have been sufficiently diluted to prevent detection. Mice inoculated with leukemia virus were palpated for splenomegaly or lymphadanopathy at weaning, and then at weekly intervals until they were sacrificed. All surviving animals were killed after 9 months. With preparations containing Moloney sarcoma virus, mice were inoculated (0.1 ml) intramuscularly or subcutaneously in the left hind leg. The mice were examined for tumors at 7 days postinoculation and three times weekly thereafter. Surviving mice were killed after 4 months. Serial dilutions of the Rous sarcoma virus preparations were inoculated onto the chorioallantoic membranes (CAM) of 9-day-old embryonated chicken eggs. After 10 days, the CAM were removed and the pocks were counted. Histopathological examinations were performed on all mice that died or had abnormalities after inoculation with the terminal dilution samples. Thermal inactivation. The virus was suspended in raw milk, sterile milk, raw chocolate milk, or raw ice cream mix, and was processed in a constant-temperature water bath or in a slug-flow heat exchanger (18). Tests at 40 to k0 C were made in the water bath for 5 to 30 min at 5-min intervals. High-temperature tests at 55 to 71.7 C at times ranging from 2 to 30 sec were processed in the heat exchanger. Suspensions of adenovirus 12, herpes simplex virus, and reovirus 1 were diluted to obtain approximately 104 plaque-forming units per ml in the dairy product. The leukemia and sarcoma preparations were diluted 10-2 in the product to be examined. The resulting titers varied from 1023 to 105 98 per ml depending on the lot used in the test. Ten replicates containing 1.2 ml of the suspension were tested at each temperature in the water bath method. Continuous agitation of the sample during the holding time was accomplished by means of a reciprocal shaker built into the bath. At the end of the heating times, the tubes were immediately cooled to 4 C, frozen, and stored at -65 C until tested. All samples for in vivo studies were shipped in dry ice to Microbiological Associates, Inc., Rockville, Md., for assay. Corrections for the heating and residence time distribution effects in the heat exchanger have been previously reported (18) affect the z values. Therefore, the calculations and corrections are not included in this report. RESULTS The destruction rate curves for reovirus 1 and herpes simplex virus at 55 C were asymptotic to the base line ( Fig. 1 and 2). Similar results have been published for erythroblastosis virus, poliovirus 1, and rhinovirus HGP (2,5,9). The "tailing" effect was much less noticeable for herpes simplex virus at 60 C (Fig. 3). The destruction rate of herpes simplex virus appeared to approach a first-order reaction except when the virus was suspended in raw ice cream mix. The asymptotic configuration was quite apparent with reovirus 1 at 60 C (Fig. 4). It was also apparent that the raw ice cream mix was the most protective medium of the dairy products studied. At 65 C, the curves for adenovirus 12 and herpes simplex virus were first-order reactions (Fig. 5), and reovirus 1 appeared to be rapidly approaching this condition. Calculation of the D values, the time required to reduce the viral concentration by 90%, was impossible, and the mechanism of thermal destruction is not known. However, it was feasible to employ the following polynomial mathematical model to evaluate the consistency of plaque formation in repeated runs: where Y equals logio (plaque count/milliliter), So, #3, and 2 are true but unknown regression coefficients, X is time (minutes or seconds), and e is experimental error. This model yielded adequate curve fits (R2 > 0.90) in most experiments. A disadvantage of this model is that the regression coefficients cannot be easily related as a function of temperature. An estimate of the relation between rate of inactivation and temperature was obtained by considering a conservative estimate of D. This D, was based on the use of the inflection point estimate from equation 1. An example of this technique is presented in Fig. 6. The inflection point is XI = -b1/2b2, where b1 and b2 are estimates of ( and (2 in equation 1. Thus, a conservative estimate of D based on the XI above is DI= I 21 (2) Values of z were computed in the usual manner with the use of DI . The DI value was determined for a sufficient number of runs at different temperatures so that z values could be calculated for all of the viruses in the in vitro study. Results of the in vivo study of the resistance of Rauscher and Moloney leukemia viruses suspended in raw ice cream mix (the most protective of the dairy products) indicated that 50 C for 5 100,0OR IJI 10 min or 55 C for 10 sec was sufficient to reduce the viral population so as to prevent leukemia, splenomegaly, or death in the inoculated mice. These results are comparable to data previously reported for Rauscher and Moloney leukemia viruses (14,22). The Moloney sarcoma virus was slightly more resistant than the leukemia viruses. Exposure for 15 min at 50 C or 20 sec at 55 C was sufficient to prevent the development of tumors in the inoculated mice. Rous sarcoma virus appeared to be the most resistant of the viruses studied and survived at 55 C for 20 min and at 65 C for 2 sec. A half-life of 42 sec at 60 C has been previously reported (6). The low initial titers of the viruses tested in vivo and the limitation in the amount of crude virus that could be added to the milk or milk product without adulterating or changing the thermal characteristics of the product presented difficulties when an attempt was made to analyze the results statistically. This problem was compounded by the necessity of diluting the viral inoculum because of adverse reactions in the newborn mouse and the embryonated chicken egg. Sufficient data, however, were available because of the relatively high initial titer of the Rous sarcoma virus preparations to calculate D values at four different temperatures. From these D values, the z value for Rous sarcoma could be plotted. If a 12-log reduction, which is commonly used in industry to determine a food process, is utilized along with the calculated z values for Rous sarcoma virus, adenovirus 12, reovirus 1, and herpes simplex virus, a graphic comparison can be made with the U.S. Public Health Service pasteurization standard for ice cream mix (Fig. 7). It is apparent that the times and temperatures advocated by the U.S. Public Health Service for pasteurization of ice cream mix are sufficient to inactivate any of the oncogenic viruses tested. Theoretically, an ice cream mix suspension containing 12 logs of Rous sarcoma virus would be inactivated in 46 sec at 68.3 C (155 F) and in 1.4 sec at 79.4 C (175 F). A lytic factor or nonspecific inhibitor of Rous sarcoma virus was present in three of the raw milk samples tested. The potency of this factor was such that a raw milk suspension containing 107 pock-forming units/ml was negative when inoculated onto the CAM of embryonated chicken eggs. Similar results were obtained with two other raw milk samples. Interestingly, the raw milk samples used in this study were all recovered from a bulk milk tank of a large milk-processing plant. A similar factor that reduced the titer of murine leukemia viruses has been reported (11). DISCUSSION The asymmetry of viral thermal destruction curves at low temperatures has been reported by other investigators. Whether survival is due to resistant members of the viral population or to the 10 protective effect of the suspending menstruum is debatable. The destruction curves approached a first-order reaction as the temperatures studied neared the pasteurization temperatures. When the z values were calculated for the viruses in the most protective medium, an adequate safety factor was present in the currently recommended pasteurization procedures. The calculated process, incorporating a 12D destruction factor, is greater than would be expected in milk or milk products. Unlike bacteria, viruses do not increase in numbers in milk. Therefore, the possibility of 12-log viral contamination is highly unlikely. Preliminary studies on the viral content of 57 raw milk samples indicated that viruses, if present at all, were present in small numbers. Viruses were found in two of the samples tested. Poliovirus 1 was recovered from one pooled sample and poliovirus 3 was recovered from the other sample (Sullivan et Whether similar results would be obtained with the reported bovine viruses can only be determined with the development of an assay system that will make possible a similar study to deter-mine the thermal inactivation properties of these agents. The presence of an interfering factor for Rous sarcoma virus in three samples of raw milk raises questions as to the possible presence of antibodies to a bovine oncogenic virus or to an avian tumor virus in cows' milk. An attempt should be made to determine a possible relationship of this factor to a subclinical infection of bovine leukemia.

v3-fos

2020-12-10T09:04:16.629Z

1971-06-01T00:00:00.000Z

237229387

s2

Rapid Infrared Determination of the Potency of Chlorinated Bactericides A rapid infrared reflectance method for evaluating the germicidal potency of synthetic materials containing various amounts of two chlorinated bactericides was developed. The dimeric product 2,2′-methylenebis (4,6-dichlorophenol) exhibited a characteristic C=C skeletal inplane stretching infrared absorption band at 1,640 cm−1. The monomeric 2,4-dichlorophenol precursor showed a characteristic absorption band at 1,579 cm−1. These characteristic infrared absorptions may be used for analysis of the potency of the manufactured chlorinated bactericide. For a series of samples known to vary in dimer content, the micrograms per milliliter required for a 100% bacterial kill is first determined by a standard American Petroleum Institute method. Then the area ratio of the infrared absorption bands characteristic of the chlorinated bactericides is measured for each sample and plotted versus the microgram per milliliter required for 100% bacterial kill. The potency of subsequent samples is simply and rapidly determined by measuring this ratio from the infrared absorption curve and calculating micrograms per milliliter required for 100% kill from the calibration curve. Analysis time is approximately 1 hr compared to biocidal tests in current use requiring approximately a 1-month incubation period. A rapid infrared reflectance method for evaluating the germicidal potency of synthetic materials containing various amounts of two chlorinated bactericides was developed. The dimeric product 2,2'-methylenebis (4,6-dichlorophenol) exhibited a characteristic C=C skeletal inplane stretching infrared absorption band at 1,640 cm7l. The monomeric 2,4-dichlorophenol precursor showed a characteristic absorption band at 1,579 cm7l. These characteristic infrared absorptions may be used for analysis of the potency of the manufactured chlorinated bactericide. For a series of samples known to vary in dimer content, the micrograms per milliliter required for a 100% bacterial kill is first determined by a standard American Petroleum Institute method. Then the area ratio of the infrared absorption bands characteristic of the chlorinated bactericides is measured for each sample and plotted versus the microgram per milliliter required for 100% bacterial kill. The potency of subsequent samples is simply and rapidly determined by measuring this ratio from the infrared absorption curve and calculating micrograms per milliliter required for 100% kill from the calibration curve. Analysis time is approximately 1 hr compared to biocidal tests in current use requiring approximately a 1-month incubation period. The Baroid Division of the National Lead Co. has developed a method for the preparation of the bactericide 2,2'-methylenebis (4,6-dichlorophenol) from 2,4-dichlorophenol. Bacterial potency of experimental preparations was measured by a typical test for potency in which the concentration in micrograms per milliliter required for a 100% kill of a standard bacterial culture is used. In the American Petroleum Institute (API) method (1), a sulfate-reducer medium is inoculated with an actively growing culture of a standard sulfate-reducing bacterium, which is added to various concentrations of the chemical to be tested and incubated. Growth is indicated by a blackening of the medium, whereas cultures with no growth remain clear. The API method requires 1 month of incubation, which delays observing the effect of variations in the synthesis conditions on the yield of the reaction. Although pure samples of the dimer were not evaluated, it has been estimated that the dimer is approximately seven times as potent as the monomer precursor. The higher the dimer content, the higher the potency of the product and the lower the amount required for germicidal action. Therefore, the measure of potency is directly related to dimer content or yield of the synthesis reaction. Since the biocidal test is time-consuming, it was highly desirable to develop a rapid method for measuring potency of synthetic bactericide for production control. Before the successful application of the infrared method, various approaches were investigated without success. These included ultraviolet and visible spectrophotometry; gas, thin-layer, and liquid chromatography; and nuclear magnetic resonance spectroscopy. Since the product is not entirely soluble in many transparent solvents used in spectroscopy or chromatography, we chose the multiple internal reflection technique for obtaining infrared spectra. MATERIALS AND METHODS Apparatus. A Perkin-Elmer infrared spectrophotometer (model 621) with a frustrated multiple internal reflectance accessory and a KRS-5 internal reflecting crystal was used. Any infrared spectrophotometer capable of resolving the two peaks of interest (1,640 and 1,579 cm7l) would be satisfactory. An analytical balance for weighing traced peak areas or a planimeter for measuring peak areas was used. Reagents. Isopropanol was used as the reagent. at about 105 C and thoroughly mixed while fluid. The sample is then allowed to cool until it assumes a pasty form and is buttered onto a KRS-5 (thallium bromidethallium iodide salt) internal reflecting crystal. The crystal is placed in the reflectance accessory, and the infrared spectrum is obtained by using a quantitative schedule. Samples are removed from the crystal by washing with isopropanol. The areas under the analytical peaks (1,640 cm-' for the dimer and 1,579 cm-' for the monomer) are measured by tracing and weighing or by planimetry. A single base line is drawn from 1,685 to 1,540 cm-' for both peaks. Since the peaks are not fully resolved, it is necessary to compromise somewhat in defining the specific boundary for each peak. Figure 1 illustrates a typical area determination. The work described in this paper is based on area determination by the cutting and weighing technique. A calibration curve is obtained, by using a series of samples of various bactericidal potencies previously determined by the biocidal test by plotting peak area ratio versus micrograms per milliliter required for 100% kill. Figure 2 shows the calibration curve obtained by the authors. Samples are handled in exactly the same manner as the standards, and the ratios obtained are applied to the previously plotted calibration curve to estimate biocidal potency. RESULTS AND DISCUSSION The multiple reflection infrared method can be used in the rapid estimation of biocidal potency of binary mixtures of the chlorinated phenols. Although the method is not designed to replace entirely the biocidal tests, it affords a rapid means of control for estimation of biocidal potency of products in production. Once a calibration curve has been prepared, with samples whose potency has been determined by biocidal test, an infrared analysis may be completed in approximately 1 hr. The analysis of a group of samples may be completed in a relatively short time by heating in a thermostated oven, mixing to obtain a soft homogeneous melt, and running the infrared spectra over the 1,800 to 1,500 cm-l range in succession. LITERATURE CITED 1. American Petroleum Institute. 1965. Recommended practice for biological analysis of subsurface injection waters. R.P. 38, 2nd ed. American Petroleum Institute, New York.

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2020-12-10T09:04:11.838Z

1971-02-01T00:00:00.000Z

237230424

s2

New Method of Isolating Salmonellae from Milk The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations. The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations. In 1965 and early 1966, an interstate outbreak of gastroenteritis occurred involving 29 laboratory-confirmed cases of Salmonella newbrunswick (2). Epidemiological and laboratory evidence implicated instant nonfat dried milk as the source of infection. S. newbrunswick was isolated from shelf samples of the product and from the milk drying plant in which it was produced. A study of the plant indicated an environmental situation that allowed continual perpetuation of salmonellae within the plant; it was assumed that the initial source of salmonellae was the raw milk supply. As a result of the S. newbrunswick epidemic, a protocol for the examination of milk for salmonellae was recommended by members of a joint committee from the Center for Disease Control, the Food and Drug Administration, and the Division of Environmental Engineering and Food Protection (13). It is a revision of the procedure of North (11). Julseth and Deibel (6) have shown that in the case of nonfat dry milk, nine salnoneilae per liter of reconstituted milk can multiply to a level sufficient to cause disease. Thus it appears that a method of analyzing milk should be sufficiently sensitive to detect fewer than 10 salmonellae per liter of milk. The purpose of this study was to evaluate the recommended method of analyzing raw whole milk for salmonellae and to develop a procedure more applicable to field conditions. MATERIALS AND METHODS Preparation of Salmonella inocula. S. typhimurium isolated in this laboratory from a frozen dessert product was used throughout this study. The inoculum was prepared from a culture grown in Trypticase Soy Tryptose (TST) broth [15 g of Trypticase Soy (BBL) and 13 g of Tryptose Broth (Difco) per liter] for 18 to 24 hr at 37 C. After incubation, 1 ml of the culture was transferred to 8 ml of fresh TST broth, and the inoculated TST was incubated at 37 C for 4 to 6 hr. The growth was quantitated by reading turbidity at 500 nm on a Spectronic-20 colorimeter (Bausch & Lomb). The culture was diluted to a 40% transmission with TST broth, and plate counts on MacConkey agar (Difco) showed this to contain 5 X 108 salmonellae per ml. Final dilutions to obtain the desired inoculum for tests were made in normal saline. Milk. Raw milk used in this study was obtained from a local dairy which produces grade A milk. Total bacterial and coliform counts were determined on the raw milk prior to each experiment by making appropriate dilutions of milk in normal saline, inoculating 0.1 ml of each dilution to duplicate plates, and spreading the inoculum on the plates with a sterile glass rod. Tryptone glucose extract (TGE) agar (Difco) was used to determine total counts. The plates were read after 24 hr of aerobic incubation at 37 C. The coliform count was defined in this study as the number of lactose-fermenting colonies present on MacConkey agar after 24 hr of aerobic incubation at 37 C. Conventional method. The currently accepted method of isolating salmonellae from liquid milk is a modification of the procedure of North (11). The method consisted of adding 20 mg of Brilliant Green per liter of liquid milk. After 24 hr of incubation at 37 C, a loopful of this pre-enrichment was streaked to Brilliant Green agar (Difco) containing 80 mg of sodium sulfadiazine per liter of agar (BGS) and bismuth sulfite agar (BS, Difco). At the time of plating, 10 ml of the milk was subcultured to 100 ml of tetrathionate broth (Difco) containing 10 mg 235 Brilliant Green per liter (TET), and this enrichment was streaked after 24 hr of incubation at 37 C to BGS and BS. Swab culture method. Moore (8) found that suspending a cotton gauze swab in the flowing sewage in a sewer for 1 to 3 days with subsequent culture of the swab was a useful technique for isolating salmonellae. This method was modified in this study for isolating salmonellae from liquid milk. For laboratory evaluation, the swab, which was a piece of cotton gauze 4 ft by 6 inch, was folded (eight times), tied in the middle with a wire, and suspended in a liter of milk. The milk was then stirred on a magnetic stirrer at about 100 rev/min for 10 min. The swab was removed from the milk, placed in 150 ml of TET broth, and after 24 and 48 hr of incubation a loopful was streaked to BGS and BS. Serial transfer experiment. Thirteen beakers, each containing a liter of raw milk, were used. The second beaker was inoculated with the test organism. The gauze swab was suspended in the first beaker (uninoculated), stirred on a magnetic stirrer for 10 min, and then passed sequentially through the remaining 12 beakers in the same manner. This was to simulate a condition in which a swab in a flowing stream of milk would be exposed temporarily to contaminated milk (beaker 2) and washed in noncontaminated milk for a prolonged period (the remaining 11 beakers). After removal from the last beaker, the gauze swab was cultured by the procedure described above. Secondary enrichment. Subcultures, after 1 week of incubation at room temperature, were made in several of the experiments. The initial tetrathionate enrichment broths, after the first culturing, were left at room temperature for 1 week, and then 1 ml was transferred to 9 ml of fresh tetrathionate enrichment broth (secondary enrichment). The latter was incubated for 24 hr, and then a loopful was streaked to BGS and BS. Isolation and identification. The BGS and BS plates were incubated at 37 C: the BGS for 24 hr and the BS for 48 hr. Three salmonella suspect colonies were picked from each positive BGS or BS plate and transferred to triple sugar iron (TSI, Difco) agar slants. All TSI cultures having typical salmonella reactions after 24 hr of incubation were subjected to serological and, when indicated, biochemical tests. Details of the procedures followed the techniques described by Galton, Morris, and Martin (3). Statistical analyses. Statistical analyses were conducted with probabilities based on exact binomial confidence limits. Probabilities of 0.05 or less were considered significant. RESULTS The efficiency of the conventional method for recovering salmonellae from raw liquid whole milk was evaluated by inoculating known numbers of S. typhimurium into the milk, and the results indicated that an inoculum of over 1,000 organisms was required to recover salmonellae by this method. These data indicated that the sensitivity of the conventional method was in- adequate for routine surveillance of milk for salmonella contamination. Subculturing a primary broth to a secondary broth has been shown to be advantageous for isolating salmonellae in a procedure where a lactose broth is subcultured to a selective broth (12) and in a procedure where tetrathionate broth is subcultured to another tetrathionate broth (5; G. K. Morris, J. G. Wells, and C. G. Dunn, unpublished data). Therefore, it was decided to evaluate the usefulness of a secondary tetrathionate enrichment inoculated (1 ml to 9 ml) from the primary tetrathionate broth utilized in the conventional method. Also, since the cotton gauze swab described by Moore (8) for sampling sewer effluent would be very applicable to field conditions for sampling raw milk, it was decided to evaluate in the laboratory the efficiency of this swab for isolating salmonellae from milk as compared with the conventional method. The results of the comparisons indicated that the swab culture technique was approximately equivalent to the conventional method for isolating salmonellae ( Table 1). The secondary tetrathionate enrichment of the conventional method yielded the best results, followed by the primary tetrathionate broth, whereas no isolates were obtained by plating the pre-enrichment. A secondary enrichment also appeared to be advantageous when using the swab culture technique. Total bacterial and coliform counts determined on the raw milk prior to each experiment indicated that the sensitivity of the method was influenced by these counts. The total counts ranged from 9,000 to 172,000 per ml, and the coliform counts ranged from less than 10 to 4,000 per ml. A more sensitive test for Salmonella was observed in the milk with lower bacterial counts. Serial transfer experiments were conducted to determine whether salmonellae were retained by the gauze swab after washing in noncontaminated milk. After 2 hr of serial washing (12 beakers of milk at 10 min each), salmonellae were recovered from the swab even when the swab was inoculated with as few as 100 salmonellae prior to washing. There have been reports of an increased sensitivity in the isolation of salmonellae from various type samples by utilization of the elevated incubation temperature of 43 C (1, 4, 10). All of the milk in studies previously discussed were incubated at 37 C. Incubation temperatures of 37 and 43 C were compared by utilizing the swab culture technique and a secondary enrichment ( Table 2). There were more recoveries made at the 43 C incubation temperature at the inoculum levels of 1,000, 100, and 10 salmonellae per liter. In addition, at 43 C frequent recoveries were made from samples at the level of one salmonella per liter, whereas there were no recoveries made at this level at the 37 C incubation temperature. There was not a statistically significant increase in the recovery rate at either temperature between the primary and secondary enrichment, but recoveries obtained by 43 C incubation were greater than those at 37 C by both primary and secondary enrichment (P < 0.01). Since incubating at 43 C increased the sensitivity of the swab culture technique, an experiment was conducted to determine whether the swab culture technique was more sensitive than the conventional method at this temperature. The results indicated that the swab culture technique was superior (P < 0.001) to the conventional method for isolating salmonellae (Table 3). A comparison was made of the method currently recommended, the conventional method incubated at 37 C, and the method of choice as indicated by these studies, the swab culture technique incubated at 43 C ( Table 4). The swab culture technique incubated at 43 C was superior to the conventional method incubated at 37 C (P < 0.01). The conventional method yielded salmonellae from 10 of 24 samples, whereas the swab culture recovered salmonellae from 18 of 24 samples. In addition, two recoveries by the latter procedure were made at the one salmonella per liter level, whereas no recoveries were made at this level by the conventional method. The BS agar and BGS agar were equally effective for isolating salmonellae in this study. However, recoveries on the two media were not 21,1971 always made from the same sample. Recoveries on the BS agar alone were frequently made from samples with low salmonella inocula. The raw milk analyzed in these experiments appeared to contain natural flora that was highly adapted for fermenting lactose, and with plating medium such as BGS, which contains lactose as a differential agent, the indicator system in the plate was frequently overwhelmed by these lactose fermentors, causing an acid condition over the entire plate. Under these conditions, small numbers of salmonella colonies did not give the typical alkaline reaction. BS agar does not contain lactose as a differential agent; thus small numbers of samonella colonies were not overlooked because of these lactose fermentors. BGS agar, however, was advantageous in that it suppressed these coliforms better than BS agar. DISCUSSION Efficient methods of isolating salmonellae from raw milk are necessary to facilitate surveillance of the milk industry for salmonella and to aid in investigating salmonella outbreaks in which milk products are implicated as the source. Milk products are still a problem as a source of salmonella as indicated by U. S. Department of Agriculture data reported in the Center for Disease Control Salmonella Surveillance Report (14). During 1969, salmonellae were isolated from 44 of 1,697 product samples of dry milk and from 89 of 196 environmental samples. The raw milk from which this milk was manufactured must be considered as a possible source of contamination. Although workers have recovered salmonellae from nonfat dried milk and other milk products with relative frequency, there has been little success in isolating salmonellae from the raw milk when attempts were made to trace the contamination back to its source. The conclusions from these investigations usually are that the problem is one of perpetuation of salmonella in the plants rather than contaminated raw milk supplies. Although perpetuation in the plant may be a major part of the problem, the raw milk cannot be ruled out as a source of contamination without a sufficiently sensitive method of detecting salmonella, especially at very low levels. Contaminated milk from one cow when combined with milk from hundreds of other cows in a bulk tank truck would be diluted to the point that the number of salmonellae present would be extremely low, possibly in the range of 1 to 10 salmonellae per liter. We have shown in this study that the conventional method is inadequate to detect this level of salmonellae. To make a recovery by the conventional method, the presence of 103 salmonellae per liter of raw milk was frequently required. In contrast, the swab culture technique at 43 C recovered salmonellae from liter quantities of raw milk containing 1 to 10 salmonellae. These results indicate that the swab culture technique has an advantage over the present method for isolating S. typhimurium from raw milk. In addition, larger quantities of milk can be examined by the swab technique than by the conventional method. Salmonellae were recovered from the swab inoculated with 100 organisms after 12 successive washings of 10 min each while stirring on a magestic stirrer (2 hr total washing time). This indicates that a swab can be suspended in a flowing stream of milk at a milk drying plant or suspended in milk delivery trucks; thus large quantities of milk can be examined with one swab. In contrast, only 1-liter samples can be conveniently examined by the conventional method. Additionally, many problems are encountered when shipping refrigerated quantities of liquid milk to the laboratory for examination, whereas swabs are easily shipped and require no refrigeration if shipped in tetrathionate broth. The secondary enrichments appeared to yield a better recovery of salmonellae than the initial enrichments with both the conventional method and the swab culture technique. This difference appeared to be greater for the swab culture technique at 37 C than at the 43 C incubation temperature, indicating that the secondary enrichment is of little value when using the swab culture technique at 43 C. The secondary enrichment would appear to be most advantageous when samples are held at room temperature for a period of time before examination (such as one might expect under field conditions). Incubation of the swab culture in tetrathionate at 43 C was found to be superior to incubation at 37 C. Other workers have observed success with the elevated temperature of 43 C for isolating salmonellae from sewage by using selenite broth (4) and feeds and poultry litter by using selenite-Brilliant Green-sulfapyridine broth (1). McCoy (7) found that tetrathionate broth incubated at 43 C was lethal to salmonellae. No evidence of such toxicity was noted in this study nor in other studies in this laboratory involving the isolation of salmonellae from fish meal and pork sausage by using tetrathionate broth (9,10). The use of duplicate plating media was advantageous in this study. Duplicate media, even if they were the same medium, would probably increase the chances of making a recovery, but it appears advantageous to use BS in conjunction with BGS in examining raw milk. Since raw milk contains a large number of lactose fermentors which tend to upset the acid base balance in a medium that contains lactose, a medium that does not contain lactose as a differentiating agent such as BS is advantageous. BGS agar, however, was advantageous in that it suppressed coliforms better than BS agar. The results of this study indicate that: (i) sampling raw milk with a cotton gauze swab and subsequent culture of the swab is a more sensitive method of isolating salmonellae than the conventional method, (ii) salmonellae are retained by the swab even after extensive washing, (iii) an incubation temperature of 43 C is better than 37 C for isolating salmonellae from the swabs, and (iv) both BGS agar and BS agar should be used as plating media. ACKNOWLEDGMENT We thank W. Jere Housworth, Center for Disease Control, for conducting the statistical analyses. ADDENDUM IN PROOF Subsequent to the completion of this study, 45 shipments of raw milk arriving at commercial milk plants were examined by the conventional method incubated at 37 C and the swab culture technique incubated at 43 C. S. typhimurium was recovered from one of these shipments. The isolation was made by the swab culture technique, but not by the conventional method.

v3-fos

2020-12-10T09:04:12.301Z

1972-03-01T00:00:00.000Z

237233096

s2

Semiautomated Method for Microbiological Vitamin Assays A semiautomated method for microbiological vitamin assays is described, which includes separate automated systems for the preparation of the cultures and for the measurement of turbidity. In the dilution and dosage unit based on the continuous-flow principle, vitamin samples were diluted to two different dose levels at a rate of 40 per hr, mixed with the inoculated test broth, and dispensed into culture tubes. After incubation, racks with culture tubes were placed on the sampler of an automatic turbidimeter. This unit, based on the discrete-sample system, measured the turbidity and printed the extinction values at a rate of 300 per hr. Calculations were computerized and the results, including statistical data, are presented in an easily readable form. The automated method is in routine use for the assays of thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, pantothenol, and folic acid. Identical vitamin solutions assayed on different days gave variation coefficients for the various vitamin assays of less than 10%. The first type of system has mainly found application in antibiotic assays (1,5) where short incubation periods are required. In these assays, the production of carbon dioxide (3,10) and bacterial growth (9) serve as parameters. Systems with longer incubation periods present problems in continuous-flow systems, requiring holding coils of considerable length in which the regular air-bubble pattern cannot be maintained because of the high pressure needed. The first example of a discrete-sample system has been described by McMahan (8). In this apparatus, samples in tubes pass a number of stations where the various manipulations are performed mechanically. A discrete-sample system especially suitable for antibiotic assays has been described by Gualandi (4). Recently, Kuzel and Kavanagh (7) described an automated system consisting of a diluter and turbidity reader module. The system which is commercially available can be used for antibiotic and vitamin assays. An extensive survey of the theoretical and practical aspects of the design is given by the same authors (6). In our laboratory, we aimed at the automation of the vitamin assays carried out as a modified two-point parallel line assay in which automation should include most of the laborious manipulations (i.e., the preparation of dilutions and of the test mixture in culture tubes, turbidity reading of the incubated cultures, and the evaluation of assay data). The system should be sufficiently flexible to perform alternately assays of all B vitamins, for example, thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, pantothenol, and folic acid. Finally, the method should be able to cope with preparations of widely varying vitamin content. To meet the latter requirement, automation was introduced from the point where prediluted vitamin samples could be treated identically. It proved to be impractical to include the rather long and variable incubation period in the automated system. Hence a semiautomated method consisting of two independent units was designed. A dilution and dosage unit based on the continuous-flow principle prepares suspensions containing vitamin solution and inoculated test broth in culture tubes ready for incubation. A discrete-sample system performs the turbidity measurement of the cultures after incubation. MATERIALS AND METHODS Growth curves showed that the growth rate of the test organism is related to the concentration of the vitamin within the limits of the test. The growth was measured turbidimetrically after an incubation period of 16 to 18 hr. In the numerous vitamin assays carried out under standardized conditions, it was shown that the log dose response curves for all vitamins were steep and highly consistent between fixed concentration ratios. Hence a vitamin assay was designed based on the principle of the two-point parallel line assay according to Finney (2). In the twopoint parallel line assay, preparations were diluted to two dose levels for which the responses lie on the steep slope of the log dose response curve. In one assay, several preparations with an assumed vitamin content were assayed against the standard. One dilution series of each unknown preparation and 7 to 10 dilution series of the standard were used in one assay to attain maximum efficiency. Each dilution series consisted of two dilutions with the high and two dilutions with the low concentration. A test set-up was chosen for which the dilution series were prepared, and the turbidity of the series was measured in a random order. In agreement with the design of a two-point parallel line assay, a statistical test was carried out to examine whether the regression coefficient of the log dose curve of each individual unknown preparation could be regarded as equal to the regression coefficient of the standard preparation. In case of an erroneous assumption of the potency of the unknown preparation or a dilution error, the test on equality of regression coefficients of unknown and standard (test on parallelity) is significant with a probability larger than 0.05. In the latter case, the unknown should be retested with a new assumption about its potency, if necessary. An indication of the precision of the assay is given by lambda = s/b, where s is the standard deviation of the mean extinction value of the standard, and b is the mean regression coefficient of the standard and of those unknowns that have not been rejected because they are not parallel. The principal data on the assay method of the various vitamins have been compiled in Table 1. For all vitamin assay tests, commercially available media were used. Methods are also available for the preparation of alternative media ( Table 2). The dilution and dosage unit consisted of a main sampler containing the vitamin solutions under test; four auxiliary samplers delivering dilution fluid and test broth (in most cases already inoculated, see Table 1); a proportioning pump with a manifold in which dilution took place and in which two test solutions (one high and one low concentration) from each vitamin solution were prepared; and a combicollector delivering identical amounts of the final mixture to culture tubes. All of these elements were linked to a control unit. The main sampler (MS, Fig. 1) consisted of a tube-rack holder with a carriage carrying the sampling needle and a cup for rinsing fluid. The sampler had a capacity of eight racks each containing 20 tubes with a volume of 3 ml. The carriage automatically moved from one sample tube (and row) to another. With two small handles in the side panel of the carriage, the sampling needle could be moved to any position. Instead of the last rack, a reservoir with rinsing fluid can be placed in the rack holder. The sample tubes in the racks were always arranged in such a way that after the last vitamin sample solution the carriage arrived at the rinsing fluid reservoir. The sampling needle was adjusted to the required height by means of a microswitch. The auxiliary samplers (Fig. 2) supplied either dilution fluid (H1, H2, and H4) or test broth (H3). In contrast to the main sampler in which rinsing fluid was drawn between the samples, the auxiliary samplers drew air when they were not sampling fluid. Hence, for the latter samplers, the cup for rinsing fluid was omitted. The construction of the auxiliary samplers was identical to the sampling device mounted on the main sampler. The manifold (Fig. 3) was built from tygon tubes with diameters of as great as 0.110 inch (approximately 0.28 cm) and glass connections in accordance with common autoanalyzer systems. Determination of the dosages of the final mixtures prepared in the manifold was performed by the combicollector (Fig. 4). The latter consisted of a conveyor belt above which were mounted two solenoid valves (Versa N.V., Apeldoorn, Holland) each provided with a stainless-steel outlet and a bypass. In the "on" position, the valves were open and connected via a bypass to the waste. When the valves were closed, the liquid passed to the outlets. Special racks containing two rows of six culture tubes were carried by the conveyor belt so that two tubes were centered below the two valve outlets. Each time the valves were opened, the next two culture tubes were moved to this position. All of these elements were electrically linked to the control unit. In this unit (Fig. 5), adjustable electromechanical time clocks (Schleicher, W. Berlin, Germany) were grouped together to direct the operation of the various elements. The sampling and rinsing times of the main sampler were controlled by the first pair of time clocks. Each operation of the other individual mechanical parts of the dilution and dosage unit was also controlled by a pair of time clocks. Four pairs of time clocks controlled the three auxiliary samplers (H1, H3, and H4) and the opening of the valves on the combicollector. The remaining two pairs of time clocks regulated the two operations performed by auxiliary sampler H2 (see below). The first time clock in such a pair controlled the lag time and the second controlled the actual operation period. The period between the receipt of the electrical signal and the commencement of the operation is defined as the lag time. Signals derived from the time clocks of the main sampler initiated the clock controlling the lag time for the second operation of auxiliary sampler H2. In this case, the signal was derived from the end of the first operation of the same auxiliary sampler. The starting signal for all time clocks was derived from the start or end of the sampling time of the main sampler. Signal lights on the panel showed which operations were taking place. The time clocks controlling the duration of the actual operations were fixed on constant time intervals (Fig. 5), whereas the lag time for each individual operation was adjustable. In this way, means were provided for synchronization of the various liquid streams in the manifold. In routine use, only minor adjustments of the lag times were necessary to ensure accurate synchronization. The racks with tubes filled with the vitamin cultures on the combicollector were transported in special trays and incubated either in a thermostatically controlled water bath (Marius N.V., Utrecht, Holland) or on a shaker (type SL 69, Marius N.V.) in a thermostatically controlled cabinet. In the latter case, the racks were tilted on the shaker to increase aeration and agitation of the cultures in the tubes. The turbidity measurement unit (Fig. 6) consisted of a modified automatic sample transfer apparatus (AST 100, Vitatron, Dieren, Holland) built over a transport system together with a Unicam SP 600 spectrophotometer (Philips N.V., Eindhoven, Holland) electrically connected via a digilog convertor (DRP 100, Vitatron, Holland) to a printer (Addo-X, Malm6, Sweden). A stirrer (Phillips N.V.) that operated as a sampling needle was mounted on the conveyor belt at a position six tubes before the point at which the sample was taken. The stirrer and the sampling needle were simultaneously introduced into the respective culture tubes. In this way, uniform cell suspensions were obtained before the turbidity measurement. The transport system of this unit was similar to that used in the combicollector. The culture tubes thus remained in the same racks throughout the assay. The sampler was connected to a 1-cm flow cell with a volume of 0.5 ml (type 186, Hellma GmbH, Mullheim/Baden, Germany) mounted in the Unicam SP 600 spectrophotometer. In this system, a wavelength of 1000 nm proved to be optimal for FIG. 2. Auxiliary sampler. turbidity measurements. The spectrophotometer was modified to permit conversion of transmission into extinction values which were subsequently printed out via the digilog convertor on the Addo-X. The vitamin reference standards were weighed and dissolved in the appropriate solvent (Table 1) and diluted to a level of 2,000 times the concentration to be attained in the culture tube (high dose level). Products assayed for vitamin content consisted mainly of coated tablets, aqueous solutions, and freeze-dried preparations. Tablets were homogenized in the solvent by means of an ultrasonic device (S 75 Sonifier, Branson Sonic Power Co., Danbury, Conn). All solutions and freeze-dried preparations were diluted directly to a concentration approximating that of the standards. These prediluted vitamin standard and sample dilutions were prepared the day before performance of the assay and stored in the dark at 4 C overnight. The concentrated solutions of thiamine-HCl, pyridoxine.HCl, calcium pantothenate, nicotinic acid, and pantothenol can also be stored for longer periods without any detectable loss in activity. Before starting, the assay duplicate sample tubes were filled from each of these concentrated solutions together with a number of tubes with distilled water from which the inoculated blanks were derived. Following the statistical design of the assay, these pairs were randomized over the positions in the main sampler. The dilution and dosage unit was a continuousflow system capable of diluting 20 duplicate samples per hr. The main sampler drew sample and rinsing fluid (twice-distilled water containing 0.01% Triton X-405) during 60 and 30 sec, respectively. The auxiliary samplers also sampled liquid for 60 sec, thus providing fluid segments of equal length. By adjustment of the lag times for the individual samplers, the continuous-flow streams could be synchronized so that all liquid parts met precisely at the various intersections in the manifold (Fig. 3). At intersection I and II, the samples were diluted 20-and 50-fold with diluent from auxiliary samplers H1 and H2, respectively. After the second dilution step, the diluted sample was divided into two parts at intersection III in the manifold. One part (4 ml) went to intersection IV and was mixed with an equal volume of test broth from H3 to form the high concentration sample. The second part (2 ml) was mixed at intersection V with 2 ml of diluent (from H4) and with 4 ml of test broth (from H3), thus forming the low concentration sample. Both samples were then dispensed into tubes by the combicollector as follows. The two ends of the tubing circuit were connected with the valves on the combicollector. These valves were controlled in such a way that from each liquid segment of approximately 8 ml the first 3 ml was discarded as waste. The valves were then closed for 30 sec, and approximately 4 ml from the central part of both liquid segments was dispensed into the culture tubes. The remainder of the liquid was led to the waste. Rinsing fluid sampled by the main sampler served to avoid contamination of a sample segment with ternal diameter of tubes. MS = main sampler provided with cup for rinsing fluid; Hi = auxiliary sampler diluent first step; H2 = auxiliary sampler diluent second step; H3 = auxiliary sampler test broth; H4 = auxiliary sampler diluent low potency sample; CC = combicollector. Flow of sample stream is indicated by a thick line. At intersection I, the sample segment was diluted with air-segmented dilution fluid. After resampling at RSi, the second dilution step took place at intersection I. The relatively large volume of dilution liquid was supplied by two tubes with a large diameter. The main part of the resulting diluted sample segment was resampled at RS2. From intersection mU about 4 ml was pumped to IVand 2 ml to V, respectively. At V, the high potency sample was prepared by mixing the diluted vitamin with an equal amount of test broth. At V, 2 ml of the diluted sample was diluted twofold and mixed with test broth, thus forming the low potency vitamin sample. Finally, 4-ml amounts of both final mixtures were dispensed in tubes in racks on the combicollector. the preceding one in the sample line up to intersection II. From that point on, the manifold was rinsed with dilution fluid from auxiliary sampler H2. For this purpose, the latter sampler was programmed to sample dilution liquid during a period of 15 sec between its main sampling periods. To limit carry-over between two consecutive vitamin samples, care was taken to build a circuit with minimal total length. After each assay run, the manifold was rinsed with 5% aqueous formaldehyde solution and subsequently with 0.2% aqueous sodium lauryl sulfate solution. Every fortnight, an additional rinse with 2 N sulfuric acid was carried out. Every two months, the tubes of the manifold were renewed. After each row of culture tubes was filled, the six tubes were covered with one stainless-steel cap. To avoid premature growth in the culture tubes, the racks were placed in cold water (0 C) immediately after filling. The assay procedure continued for the various vitamins as described in Table 1. After an overnight incubation period, growth was terminated by immersion of the racks in cold water (O C). On the turbidity measurement unit, the cultures were mechanically stirred and the turbidity was read at a rate of 300 tubes per hr. On both conveyor belts of the combicollector and the turbidity measurement unit, the tubes were handled in a similar sequence. containing 12 tubes, first the front row of tubes (6) was filled or sampled; then all of the racks were turned, and the other row of tubes was handled. All data required for calculation of the vitamin content of the products were transferred to data sheets. These were transcribed on punch cards which were fed into an IBM 360/50 or a PDP 15 computer, programmed to calculate the vitamin content of the samples expressed in terms of the vitamin reference standard. The computer supplied all input data, statistical data on the assay (Fig. 7a), and the calculated vitamin contents (Fig. 7b, c) of the products together with 95% confidence limits in an easily readable form. As a regular check, the dose response curves for all the vitamins were determined every three months. RESULTS AND DISCUSSION Routine use of this semiautomated method provided reliable results. Table 3 shows the coefficients of variation and the mean values calculated from the vitamin contents of identical vitamin standard solutions assayed on different days. The low coefficients of variation calculated and the mean vitamin contents determined for thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, and pantothenol demonstrated the good reproducibility and high precision of the method. Data for the folic acid assay were not included in Table 3 since only a few assays of this vitamin were performed. Preliminary experience indicated that folic acid could also be assayed automatically if Triton X-405 was omitted from the dilution and rinsing fluids. Riboflavine, cyanocobalamin, calcium pantothenate, and nicotinic acid were assayed without special care to avoid contamination. The test media used for the assay of thiamine and pantothenol were very susceptible to contamination. In these assays, individual inoculation of each tube was necessary after sterilization of the tubes containing the vitamin test medium mixture in an autoclave for 10 min at 110 C. Better assays of pyridoxine were obtained when the tubes were inoculated individually immediately prior to incubation. Flow birefringence of the rod-shaped bacteria caused no problems in the turbidity measurement. In the turbidity measurement unit, one suspension of Lactobacillus leichmannii divided over a great number of tubes was read. The standard deviation in extinction units proved to be so small that this error could be neglected. The system combined the advantages of both continuous-flow and discrete-sample systems. The continuous-flow system with synchronization is eminently suitable for the performance of the large dilution step of all vitamin samples with a high degree of reproducibility. On the other hand, the discrete-sample system is more suitable for the turbidity measurement of large numbers of cultures in a short period (11). With two dilution and dosage units and one turbidity measurement unit, four different vitamin assays could be performed daily. Every assay included 20 to 25 individual unknown vitamin sample solutions yielding a test capacity of 300 to 400 samples per week. Turbidity measurement for four assays took about 4 hr which left the automatic turbidimeter available for other purposes for the remainder of the day. To assay 300 unknown sample solutions per week, four technicians were necessary, three of whom were required for preparation of the prediluted vitamin samples and the test media while one operated the automated equipment. Use of the computer has greatly added to the assay procedure. The only manual transfer of data was in entering the pertinent data of the assay, including the turbidity measurements, onto the computer input data sheets. On a separate sheet (Fig. 7c), the assay results were reported in such a way that the printout can be used as data sheet for the Quality Control Department. Although in our laboratory the above auto-APPL. MICROBIOL. mated system has only been used for vitamin assays, it should also be applicable to automation of turbidimetric antibiotic assays.

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2020-12-10T09:04:17.676Z

1972-03-01T00:00:00.000Z

237231702

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Identification and Aflatoxin Production of Molds Isolated from Country Cured Hams Of 562 molds isolated from country cured hams, 403 isolates were of the genus Penicillium, 121 were Aspergillus, and 36 were Cladosporium, Alternaria, and other genera. Country cured hams (6) are very popular in the southeastern United States. The surface of country cured hams often is covered by the growth of different species of mold, some of which might be considered desirable for improving the flavor; yet undesirable molds could cause spoilage of the product or could be pathogenic or toxigenic for man. Strains of Aspergillus flavus Link ex Fries and A. parasiticus Speare are able to produce aflatoxins (1,14). One aflatoxin-producing strain was recovered from an Italian-type salami (3). Five strains of A. flavus were isolated from a single country cured ham by Strzelecki and coworkers (12). Four of these five strains produced aflatoxin. Experimentally, A. flavus and A. parasiticus were shown to be capable of producing aflatoxin on fresh beef, ham, and bacon (4,5). Since the presence of A. flavus on hams conceivably could constitute a potential health hazard, it was considered of interest to note how often mold isolates capable of producing aflatoxin are found. Samples from country cured hams were taken from different ham processors in the State of Georgia. Four hundred and fifty-five swabs were taken from 356 country cured hams from 11 ham processors. Each swab was inoculated on the following media: Czapek Dox agar, Czapek Dox agar plus 16% NaCl, malt agar, and potato dextrose agar (8). Penicillia were identified to genus only, and all species of aspergilli were identified by the methods outlined by Raper and Fennell (10). Production of aflatoxin by isolated strains of mold was determined by the screening method used by De Vogel et al. (7). Fluorescence under long-wavelength ultraviolet light was checked after 3 and 10 days of incubation at 27 C. Isolates which showed blue or green fluorescence by the screening test were inoculated into 50 ml of YES broth (20% sucrose and 2% yeast extract) and onto sterile moist rice. Inoculated broth and rice were incubated at 27 C for 7 to 12 days. After the mold cultures were extracted twice by shaking for 10 min with 75 ml of chloroform, the chloroform layer was collected with a separatory funnel, filtered, and evaporated to dryness at 40 C with a flash evaporator (12). The residue was dissolved with 5 ml of chloroform, and the mycotoxins were separated by using thin-layer chromatograms (TLC) coated with MN Silica Gel G-HR (Brinkmann Instr., Westburg, N.Y.). Chloroform-acetone (85:15 v/v) was used as the TLC developing solvent for aflatoxin. Production of aflatoxin was determined visually by comparing the sample with aflatoxin standards (Southern Utilization Research and Development Laboratories, New Orleans, La.). From producing stains, the chloroform extract was rechromatographed on a preparative scale, and the suspect spot removed. The aflatoxin was eluted from the silica gel with chloroform and filtered, and the ultraviolet absorption spectra were obtained on a Perkin-Elmer model 202 spectrophotometer. The method of Verrett et al. (13) was used for bioassay. Chloroform extracts of broth were evaporated to dryness and dissolved in 2 ml of sterile propylene glycol. A 0.03-ml amount of this solution was inoculated into the air sac of eggs by sterile syringe. A control was inoculated with the same amount of pure propylene glycol. The development of the embryo was observed after 4, 6, and 8 days. After 8 days, all eggs which failed to develop were discarded. Of the 562 mold isolates taken from 356 country cured hams, 403 Penicillium, 121 Aspergillus, and 36 other mold isolates, mostly members of the genus Cladosporium or Alternaria, were identified ( Table 1). The number of aspergilli depended on the age of the hams and on the amount of moisture in the storage room. All aspergilli were more abundant on the surface of hams aged for 12 months or longer than on 1to 3-month-old hams. Under dry storage conditions, more aspergilli were isolated from 1to 3-month-old hams, whereas under moist conditions these hams yielded more penicillia. The largest number of aspergilli isolated were members of the A. glaucus group ( Table 2). Twenty-one were A. repens, 18 were A. amstelodami, 3 were A. pseudoglaucus, and 2 were A. ruber. The A. versicolor group was the second most common, having 21 of A. versicolor and 13 of A. sydowi. These groups are of fakus. Each of the three possessed distinct morphological and color characteristics. By comparing extracts of these strains with aflatoxin standards on TLC, it was found that only two were able to produce aflatoxin. Only aflatoxin Bl was produced in detectable amounts by either strain. The absorption spectra of the eluted TLC spots for both strains showed the characteristic maxima of aflatoxin B1 at 363 and 265 nm. The embryos of all 48 chick eggs inoculated with extracts of broth cultures of the two aflatoxin-producing strains of A. flavus failed to develop. In the same experiment, 20 of 24 control eggs hatched. These results, together with the screening test, TLC, and ultraviolet spectrophotometry, indicate that two of the three strains of A. flavus isolated from country cured hams produced aflatoxin B. when inoculated on experimental substrates. Of 356 hams examined, only two yielded strains of A. flavus having the ability to produce aflatoxin. Previous work from this laboratory (Strzelecki et al., 1969) has shown that had these two toxigenic strains been provided with appropriate conditions for toxin production, they could have posed a real hazard. There still is no definite evidence to date that such hams contain harmful amounts of aflatoxin.

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2020-12-10T09:04:17.455Z

1972-02-01T00:00:00.000Z

237229993

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Relationship of Pathogenicity to Tobacco Leaves and Toxicity to Chicks of Isolates of Alternaria longipes One hundred thirty-seven single-conidium isolates of Alternaria longipes were tested for pathogenicity to tobacco leaves and for toxicity to 1-day-old chicks. Of 58 isolates pathogenic to tobacco, 43 (74.3%) had a significant effect on test chicks. Of 79 nonpathogenic isolates, 59 (74.7%) were nontoxic, 7 were toxic, and 13 were lethal. A relationship between pathogenicity and toxicity is suggested. Toxicity of species of Alternaria to experimental animals and man has been established. Joffe (5) reported that cultures of A. humicola Oud. and A. alternata (Fries) Keissler (=A. tenuis Auct.) isolated from overwintered small grains that had been the source of several outbreaks of moldy grain toxicosis in man in the USSR during and shortly after World War II were toxic to experimental animals. Forgacs et al. (3) isolated an unidentified species of Alternaria from feed and litter that caused mycotoxicosis in chickens, and Forgacs and Carll (2) found that smoke aerosol generated from hay on which a species of Alternaria was grown caused pulmonary emphysema and other pathological changes in mice. Doupnik and Sobers (1) described an experimentally induced mycotoxicosis in chicks, ducks, and turkeys caused by a metabolite produced by single-conidium cultures of A. longipes (Ell. & Ev.) Mason, the causal organism of brown spot of tobacco (Doupnik and Sobers, Bull. Ga. Acad. Sci. 26: 58, 1968;Phytopathology 58:1048Phytopathology 58: -1049Phytopathology 58: , 1968). Sobers (Phytopathology 58:731, 1968) and Sobers and Doupnik (6) indicated that a relationship apparently exists between conidium morphology and pathogenicity of isolates of A. longipes to tobacco leaves, and they (Sobers andDoupnik, Phytopathology 58:1068, 1968) suggested that pathogenic isolates of A. Iongipes were more likely to be lethal or toxic to chicks than were nonpathogenic isolates. Hamilton et al. (4) showed that 30 (78.9%) of 38 Alternaria isolates from uncured tobacco leaves infected with A. alternata (A. tenuis Nees) and 128 (60.4%) of 212 Alternaria iso-lates from cured tobacco leaves were toxic when cultures of these isolates were homogenized and injected into the peritoneal cavity of mice. The primary purpose of this study was to determine the extent of the relationship of pathogenicity to tobacco leaves and toxicity to chicks of single-conidium isolates of A. Iongipes. Secondary objectives were to substantiate further the relationship between conidium morphology and pathogenicity, and to determine whether there were differences in the effects of the three conidial types of nonpathogenic isolates on chicks. MATERIALS AND METHODS Cultures. The 137 cultures of A. longipes used in pathogenicity and toxicity studies were derived from single-conidium isolations. Each isolation was made at random from a single, typical brown-spot lesion on leaves of one of 137 tobacco plants that included 17 varieties and four breeding lines of flue-cured tobacco, three cigar filler varieties, two cigar wrapper varieties, and one variety of burley tobacco. Cultures of all isolates were maintained at 4 to 8 C on slants of Difco potato-dextrose-agar (PDA), and were transferred at 4-month intervals. Conidium morphology of each isolate was determined after 12 days of growth on V-8 juice-agar (V-8A). All designations of conidium type are based on descriptions presented in a previous study (6). Cultures to determine conidium morphology and for pathogenicity and toxicity tests were started at the same time to minimize variation. Pathogenicity tests. Cultures used in preparing inoculum for pathogenicity tests were grown at 23 to 29 C on V-8A under continuous light provided by two 40-w F40D or F40D/3 Ken-Rad daylight fluores-313 SOBERS AND DOUPNIK cent tubes suspended 30 cm above the surface of the cultures. When the cultures were 7 days old, they were scraped with a square-tipped spatula. Conidia from each plate were suspended in 5-ml portions of distilled water 5 days later. Suspensions containing 0.05 ml of Triton B-1956 (active ingredient, 77% modified phthalic glyceryl alkyd resin) for each 30 ml of inoculum were blended for 30 sec, filtered through a single thickness of cheesecloth, and adjusted to contain a maximum of 30,000 conidia per ml. A solution containing 0.05 ml of Triton B-1956 per 30 ml of distilled water was prepared for spraying on leaves of control plants. Each of the 137 isolates were tested for pathogenicity to ten 3-to 5month-old Coker 187-Hicks tobacco plants. All test and control plants were maintained in a mist chamber for 48 hr after the inoculum was applied to the leaves, and then were placed in a greenhouse where temperatures varied from 21 to 34 C. Toxicity tests. Rations for toxicity tests were prepared as follows. Fernbach flasks containing six parts (500 g) of cracked corn and five parts (417 ml) of distilled water were autoclaved for 1 hr each on consecutive days. Discs (10 mm) of each isolate from 12-day-old cultures grown on V-8A were placed in flasks and incubated at room temperature (23 to 29 C) under continuous fluorescent light for 14 days. The flasks were shaken each day to prevent mycelial matting. At the end of the growth period, the contents of each flask were removed, dried at 50 C for 15 to 18 hr, ground, and mixed with a 36% protein supplement, 6:4 (v/v). Approximately 1,500 g of the corn-fungus mixture was required for ad libitum feeding to groups of ten 1-day-old Babcock B-300 cockerels for 14 days. Control chicks of the same age and number received sterile corn that was similarly treated and mixed. Weights were recorded for each group of chicks at the start of the test and for those that survived the test at the end of 7 and 14 days. All birds were sacrificed, and a gross examination was made at the end of the test period. Those that died during the test were examined as soon after death as possible. Isolates referred to as lethal in this study are those that caused death of 50% or more of the chicks in the group to which they were fed. Toxic isolates are those that caused death of less than 50% of the birds to which they were fed, those that suppressed weight gain by 20% or more of the control birds, or those that had both effects. This level of weight suppression was selected as significant because all but one of the lethal isolates caused weight suppression of 20% or more of the control. RESULTS AND DISCUSSION Pathogenicity tests. Fifty-eight (42.3%) of the 137 single-conidium isolates of A. longipes were pathogenic to leaves of 3to 5-month-old Coker 187-Hicks tobacco plants. Lesions were apparent 2 to 4 days after conidial suspensions were applied to the leaves, and typical brownspot lesions were evident 3 weeks after inoculation. Conidium morphology. All pathogenic iso-lates were distinguished morphologically from nonpathogenic isolates. Conidia produced after 12 days of growth on V-8A exhibited long beaks, whereas beaks of nonpathogenic isolates were 70 to 80% shorter. Conidia of the 79 nonpathogenic isolates were classified as being one of three types based on length, width, and shape. Fifty-five isolates exhibited type I conidia (long, narrow, and cylindric), 16 produced type II conidia (short, narrow, and cylindric), and eight had type III conidia (short, wide, and obclavate). A comparison of the four types of conidia produced by A. longipes is given in Table 1. These results substantiate those of a previous study (6) and indicate a relationship between conidium morphology and pathogenicity. Toxicity tests. A relationship between pathogenicity and toxicity is suggested by the following results. Forty-three (74.2%) of 58 pathogenic isolates had a significant effect on 1-dayold chicks; 34 (58.7%) isolates were lethal, 9 (15.5%) were toxic, and 15 (25.7%) had no effect on test birds. By comparison, only 20 (25.3%) of the 79 nonpathogenic isolates had a significant effect on test chicks. Of the 79 nonpathogenic isolates, 55 were type I; these included 9 lethal isolates, 7 that were toxic, and 39 that had no effect. None of the type II isolates was lethal or toxic. Four of eight type III isolates were lethal, and four had no effect (Table 2). Comparing the average per cent kill for all pathogenic isolates with that of all nonpathogenic isolates shows a 51.2% average kill as compared with an 18.7% kill for nonpathogenic isolates. The average per cent weight of control of surviving birds was -20.5% for pathogenic isolates and -1.1% for nonpathogenic isolates. The effect of nonpathogenic isolates on chicks based on conidium type shows the average kill for type I isolates as 18.6%, 0.6% for type II isolates, and 56.3% for type III. The average effect on per cent weight of control was -1.7% for type I isolates, +1.4% for type II, and -5.2% for type III. These data show that the average kill levels for all pathogenic isolates fall in the lethal and toxic group designations, and that the average nonpathogenic isolate falls into the no-effect group. On an individual basis, all of the nonpathogenic types fall into the no-effect group, with the exception of type III isolates which show a 56.3% kill. The reason for the high average kill in this group is not known, but may reflect the small number of isolates of this type.

v3-fos

2018-04-03T06:14:49.727Z

1972-02-01T00:00:00.000Z

45996220

s2

Heat Resistance of Salmonellae in Concentrated Milk The heat resistance of Escherichia coli, Salmonella typhimurium, and Sal- monella 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 sig- nificant 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 resist- ance of the organism when heated in a fresh 42% solids concentrate. Applica-tion 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. 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 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, DEGA, GOEPFERT, AND AMUNDSON 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 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

2020-12-10T09:04:12.302Z

1972-03-01T00:00:00.000Z

237231674

s2

Enterovirus Concentration on Cellulose Membranes Cellulose nitrate membranes were used as one of the adsorbents in concentrating viruses from water. For adsorption to occur, salts were required. With increase in valency of salt, less salt was necessary for enhanced virus adsorption to membranes. Trivalent salts were more effective because they could be used at only 1% the concentration required for divalent salts. Thus, 0.5 mM AlCl3 was as effective as 50 mM MgCl2. For testing 500 gal of water, only 0.24 kg of AlCl3 was required in contrast to 20 kg of MgCl2. Virus could then be eluted from such membranes, having an area of 486 cm2, with 250 ml of pH 11.5 buffer. Lowering the pH of the eluate and adding AlCl3 permitted the virus to be quickly readsorbed on a smaller cellulose membrane, i.e., 4 cm2. Virus for assay was eluted from the small membrane in 1 ml. This procedure has provided the basis for concentrating minute amounts of virus from large volumes of water. MATERIALS AND METHODS Monkey kidney (MK) cells. Kidneys obtained from immature vervet monkeys were trypsinized and grown as described (4). Virus and virus assays. A plaque-purified line of poliovirus type 1 (Mahoney strain) was used in all experiments, unless otherwise indicated. Other vi-476 ruses used were attenuated poliovirus type 1 (LSc strain), echoviruses type 1 (Farouk) and type 7 (Wallace), and coxsackieviruses A9 (Grigg) and B3 (Nancy). Stock viruses were grown in MK cells using an input of 1 to 10 plaque-forming units (PFU)/cell and stored at -70 C. Virus assays were made by the PFU method as used in this laboratory (4). Virus adsorbents. Cellulose membranes (Millipore Corp.) with a porosity of 0.45 Am were used throughout this study. The diameter of membranes is 25 mm, having an available surface area of 4.0 cm2, unless otherwise indicated. The method used for concentrating viruses on cellulose membranes has been described (10,11). Virus eluents. Proteins, as in serum (10) and beef extract (1), that adsorb to cellulose nitrate membranes, and wetting agents (10,11) exchange for virus and elute it. The membrane-coating components which are used in the eluent are then found in the eluate, where they interfere with reconcentration of virus on smaller surface membranes. Thus, another eluate had to be found. Protein-free salt solutions at pH levels from 2.0 to 8.5 are required for adsorption of viruses to membranes; above pH 8.5 the efficiency of adsorption is decreased. Enteroviruses and myxoviruses can also be eluted with a nonprotein solution at pH 11.5 without detectable loss of infectivity (8,12). The suspension containing eluted virus can then be adjusted to acidic pH levels (4.0 to 4.5), AlCl, can be added (0.0005 M), and the virus can then be readsorbed to cellulose membranes. This cycle can be performed repeatedly with 100% recovery of virus. The recommended eluent is 0.05 M glycine buffer which can be adjusted to pH 11.5 with NaOH or pH 2 with HCl, without inacti-vating enteroviruses in short-term exposures (5 to 10 min). Buffers that have Mg or other cations present which react with NaOH to form a gel should not be used, nor should buffers be used which contain anions which complex with Al ions. Purified water. Water containing no more than 0.01 Mg of dissolved solids per ml and no detectable organics was obtained by passing tap water (see below) through a water purification system (Carborundum Co., Niagara Falls, N.Y.), and was used in place of distilled water. Tap water. Tap water contained 457 ppm dissolved solids and 350 ppm suspended solids. When used, it was dechlorinated with 1-3 ppm sodium thiosulfate. Clarifying filters. To prevent clogging of cellulose membranes, AP20 fiberglass pads (Millipore Corp.) were sometimes used as clarifiers to remove suspended solids and ferric complexes from tap water. To prevent virus adsorption, the pads were first treated with 1% Tween 80 (100 ml for each 100-cm2 surface); residual Tween was removed by thorough washing of the pad. RESULTS Effects of different pH levels on elution of virus from membranes with glycine buffer. Table 1 shows the results of eluting poliovirus (Mahoney) from cellulose membranes at different pH levels. At pH 11 to 12, virtually 100% of the virus was recovered from the membrane. In these tests, 5 ml of glycine-NaOH buffer was used to elute virus from 25mm cellulose membranes; the membrane was then washed with 5 ml of glycine-HCl buffer at pH 2.0 to 4.4, thus yielding a total volume of 10 ml of neutralized eluate. The minimum volume required for elution of virus was found to be 0.5 ml for each 4-cm2 surface. Thus, virus adsorbed to a 25-mm membrane (4 cm2) could be eluted with 0.5 ml of buffer (pH 11.5), immediately followed by 0.5 ml of pH 2 neutralizing wash, to make a final recovery volume of 1.0 ml. Effects of salts on virus adsorption to cellulose membranes. Several monovalent, divalent, and trivalent salts dissolved in purified water were tested to determine the lowest concentration required for virus adsorption. The experimental procedures and results are shown in Table 2. Trivalent aluminum salts proved best, and AlCl3 and Al2(SO4)3 were just as effective at about 1% the concentration of MgCl2. Thus, the amount of salt which would have to be transported to the field is significantly lower for AlCl, than for MgCl2. Only 0.24 kg of aluminum salts would be required for adsorption of poliovirus to membranes possessing 500 gal of water, whereas 20 kg of MgCl2 would be necessary ( Table 3). Effects of MgCl2 and AICI3 on virus suspended in tap water. Since our goal is to concentrate virus in the field from natural water, Houston tap water was used to determine the effects of Mg and Al ions on virus adsorption. Figure 1 shows the experimental procedures and results. Again, AlCl3 was at least 200 times more efficient than MgCl2 in facilitating poliovirus adsorption. Effects of AICIl on large volumes of tap water. The experiments described above required that only 5-ml samples be passed through the cellulose membrane. When large volumes of tap water were treated with AlCl3, the aluminum hydroxide gel which formed clogged the filter. However, enteroviruses can line, and 10-ml samples were filtered through each of six 25-mm cellulose membranes (0.45 Mm pore size). The membrane filtrates were collected, pooled, and assayed (control virus, membrane filtrate). Each membrane was then washed with 10 ml of saline at pH 6.9, and the membrane washes were pooled and assayed. Each membrane was then treated with 5 ml of the eluent at the indicated pH, and the membrane was washed with 5 ml of buffer made acidic with HCl so that the total 10 ml recovered would be neutral. 477 VOL. 23, 1972 be adsorbed to cellulose membranes at low pH levels, and the gels are soluble in acid. Therefore, an experiment was performed to determine flow rates of tap water containing 0.0005 M AlCl3 through membranes at different pH levels. Tap water was filtered through 90-mm membranes (0.45 gm pore size) at constant pressure. Tap water at 5 psi, free from Al ions, required 24.4 min for filtration of 10 liters. Tap water containing 0.0005 M AlCl3 at pH 7 clogged the filter before 1 liter passed the membrane. Similarly, at pH 6 and 5, immediate clogging occurred. However, at pH 4.5, 4.0, 3.5, and 3.0, the flow rate was faster (13.6 min/10 liters) than the control sample free from AlCl3. The increased flow rate is attributable to the fact that the acid dissolved some of the natural gels present in tap water, especially ferric hydroxide. All subsequent experiments were carried out by adjusting virus-tap water mixtures to pH 3.0 with HCl and then adding AlCl3 to give a final concentration of Table 1, except that tap water was used as a virus and salt diluent. Average number of PFU/0.1 ml present in the control was 150 (±15). 0.0005 M AlCl3. The water must be made acid before addition of the Al ions. Effect of AICI3 on adsorption of enteroviruses to cellulose membranes. A number of enteroviruses were studied ( Table 4). All viruses tested (polioviruses, echoviruses, and coxsackieviruses) were adsorbed to the membranes with 0.0005 M AlCl3 and were quantitatively eluted with pH 11.5 buffer. a Viruses were diluted in pH 3.5 tap water containing 0.0005 M AlCl3, and 10-ml samples were filtered through 25-mm cellulose membranes (0.45 tm pore size). The membranes were washed with 10 ml of saline and then treated with 5 ml of pH 11.5 eluent followed by 5 ml of pH 2 neutralizing buffer. Reconcentration of poliovirus adsorbed to cellulose membranes. Virus recovered from membranes as outlined above can be readily readsorbed to new membranes, since the eluate does not contain any membrane-coating components (10). This is done merely by adjusting the pH levels and adding AlCl3. Under conditions which require the processing of large volumes of water at high flow rates, 293mm membranes (0.45 um pore size) require 200 to 300 ml for virus elution. The virus can then be readsorbed on a 25-mm membrane (0.45 gm pore size) at acidic pH levels and eluted from this membrane with as little as 0.5 ml of pH 11.5 buffer. An experiment was conducted to show that by pH control and salt addition poliovirus can be recycled on and off cellulose membranes. A total of 1,300 PFU of poliovirus was adsorbed to a 293-mm membrane in the presence of 0.0005 M AlCl3 at pH 4.0. The membrane was washed with saline to remove residual Al ions to avoid subsequent gel formation with the basic eluent and thus to prevent clogging of membranes. The 293-mm membrane was then treated with 300 ml of pH 11.5 eluent. The eluate was collected in 300 ml of pH 2 buffer to yield 600 ml of virus suspension at pH 6.7. [If the suspensions are not acidic (pH 4.0 to 4.5) at this point, they should be made acidic with HCl before addition of AlCl3.] AlCl3 was added to yield a final concentration of 0.0005 M to enhance virus readsorption to a freshly prepared 25-mm membrane (0.45 lim pore size). Virus was then eluted from the membrane with 0.5 ml of pH 11.5 buffer, and the membrane was washed with 0.5-ml volume of pH 2 buffer. The 1 ml of suspension now con-tained 1,250 PFU of poliovirus, virtually all of the virus initially contained in the 5-gal test volume. This type of concentration and elution of poliovirus was carried out many times with 90 to 100% recovery of the virus in the concentrate. Processing of large volumes of tap water. These new findings were then applied to recovery of virus from large volumes of tap water. Five gallons of 0.05 M AlCl3 in 0.5 M HCl acid was placed in a 5-gal pressure vessel. The salt solution was mixed into running tap water (20 psi) by nitrogen pressure (50 psi) and a metering valve so that the mixture was diluted 1: 100 in the water to give a final concentration of 0.0005 M AlCl3 and a pH of 3.0. A pH meter was used to monitor the effluent, and the metering device was adjusted to produce a flow that gave the desired pH of 3.0. Upstream a second vessel with 5 gal of purified water containing poliovirus, sodium thiosulfate, and 0.04% phenol red was connected to the nitrogen pressure. These fluids were also diluted 100-fold in the running tap water to give a final concentration of virus as indicated in Table 5 (i.e., 0.5 PFU/gal when 250 PFU input was used for 500 gal), 2 Mg of thiosulfate per ml, and a faint pink color. Immediately before the downstream pressure vessel, a sight glass was inserted in the tubing to monitor the color of the fluids, and in this way the quantity of virus and thiosulfate being added was controlled. The results of three consecutive tests in which 500-gal batches of water were processed by adsorption on a 293-mm cellulose membrane (0.45 um pore size) and then reconcentrated on a 25-mm membrane are shown in Table 5. Additional experiments have shown that methyl orange can serve as an indicator to determine pH, since the dye changes from light yellow to bright red at pH 3.0. A solution of 0.5 M HCl, 0.05 M AlCl3, and 0.1% methyl orange was prepared in a 5-gal pressure vessel so that 1:100 dilution would yield a pH of 3.0, The concentrate was forced into running tap water under nitrogen pressure until the color changed from yellow to red. Testing the effluent by a pH meter confirmed that pH 3.0 had been obtained and also assured that the concentrate was being delivered into the running water at a dilution of 1:100. At this dilution, methyl orange was not toxic to the tissue culture cells (thus the effluent containing dye could be assayed without dilution), nor was the dye virucidal to poliovirus. With Houston tap water (pH 8.2), the concentration of HC1 indicated above was required. However, for other areas, the water under test must be titrated to determine the amount of HC1 required to bring the water to pH 3.0. The concentration needed must then be determined so that a 100-fold dilution of the salt-dye-acid stock can be injected into the water to yield the optimal salt-pH concentrations. DISCUSSION The concentration of viruses from large volumes of water in the field is now feasible with the use of aluminum salts for adsorption of viruses to cellulose membranes. Aluminum salts can be used in 1% the concentration of magnesium salts. For virus to adsorb from a 400-gal sample, 45 lb of MgCl2 is needed. On the other hand, with AlCl3, 1,000 gal of water can be processed with only 1 lb of this trivalent salt. In this report, we have also described a method for concentrating enteroviruses by serial adsorption to and elution from cellulose membranes. By manipulating hydrogen ion levels, viruses in 400to 500-gal samples can be adsorbed to 293-mm membrane surfaces at pH 3 to 4 at high flow rates, eluted at pH 11.5 with 250 to 300 ml of protein-free buffer, and readsorbed to a small surface membrane (25 to 47 mm). Viruses can then be removed into a APPL. MICROBIOL. final eluate of 1 to 5 ml, which can be conveniently assayed.

v3-fos

2020-12-10T09:04:12.811Z

1972-02-01T00:00:00.000Z

237229293

s2

Fermentation of Isolated Pectin and Pectin from Intact Forages by Pure Cultures of Rumen Bacteria Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass. Previous studies in this laboratory have investigated the digestion of cellulose and hemicellulose from intact forages by pure cultures of rumen bacteria (9,17). The only other carbohydrate occurring in appreciable quantities in forage is pectin, which can constitute from 10 to 20% of the total carbohydrate complex in grasses and alfalfa, respectively (22,25). Thus, the present study was undertaken to investigate the ability of several species of rumen bacteria to ferment both isolated and naturally occurring pectins. In vivo trials with sheep have indicated that 75 animal (23). The ability of both rumen protozoa and mixed cultures of rumen bacteria to ferment isolated pectin has been reported (1,18,27), and an earlier study from this laboratory revealed that a considerable portion of the pectin present in intact immature alfalfa is digested in vitro by mixed cultures (16). Both the rate and extent of pectin digestion were decreased with advancing maturity of the alfalfa. Dehority (15) used a selective medium, containing purified pectin as the only added carbohydrate source, to isolate bacteria from the rumen contents of a steer fed alfalfa hay. Ten strains were selected for characterization, and subsequently identified as two strains of Lachnospira multiparus, four strains of Butyrivibrio fibrisolvens, three strains of Bacteroides ruminicola, and one strain of Peptostreptococcus 332 sp. Bryant and co-workers have reported that these same species, except Peptostreptococcus, plus Succinivibrio dextrinosolvens and Bacteroides succinogenes, are all capable of fermenting pectin (3,(5)(6)(7). Their cultures were isolated on a nonselective glucose-cellobiose medium and subsequently tested with the pectin medium. The growth response to pectin by eight strains of B. ruminicola, nine strains of B. fibrisolvens, and two strains of L. multipanrs were measured in a medium containing pectin as the only added carbohydrate (15). Based on these results, two strains each of B. ruminicola and B. fibrisolvens, with widely differing growth responses, were chosen for the present study. One strain of L. multiparus and the Peptostreptococcus sp. were also included, thus making a total of six pectin-fermenting strains. Several pure cultures of cellulolytic rumen bacteria have been shown to degrade isolated and intact forage hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources (9,11). This ability appeared to be the result of a nonspecific, constitutive enzyme or enzymes (14), and, on this basis, one strain each of B. succinogenes, Ruminococcus flavefaciens, and R. albus were included in the present study to evaluate a possibly similar action on pectin. MATERIALS AND METHODS Nine pure cultures of rumen bacteria were used in this study. They were strains D31d and 23 of B. ruminicola, strain A3c of B. succinogenes, strain B34b of R. flavefaciens, strains D16f and HiOb of B. Galacturonic acid (Exchange Lemon Products Co.) and purified pectin (Pectin NF, Calbiochem; Pectin NF, Sunkist Growers) were used as substrates. Intact forage substrates consisted of boot and bloom stages of bromegrass (Bromus inermis Lincoln) and prebloom and late bloom stages of alfalfa (Medicago sativia Vernal), which were harvested from pure stands, artificially dried, chopped, ground in a large Wiley mill, and finally ground through a 40-mesh screen of a laboratory Wiley mill. All substrates were added at a level of 0.5%. The anaerobic culture techniques were similar to those described by Hungate (19), except for the mod-ifications described by Dehority (15) in which the medium was autoclaved in individual culture tubes. In general, three different basal media were used. The first was the complete medium of Scott and Dehority (24) plus 0.0002% hemin, with the substrate added prior to sterilization. The second was a 40% clarified rumen fluid (CRF) medium (17), with the substrate added before sterilization. The third media was the same as the first, except that the substrate was sterilized separately in water and then added aseptically to each tube. This latter medium will be designated as "combined" in subsequent sections. Recoveries for galacturonic acid and purified pectin were increased from 66 and 80% to 75 and 92%, respectively, when the substrate was sterilized separately. For those studies with the intact forage substrates, a 40% CRF medium was used (9,17). In this case, the 0.5% forage substrate was added to the medium prior to sterilization. Optical density (OD) measurements were made in culture tubes (16 by 150 mm) at 600 nm with a Bausch & Lomb Spectronic-20 spectophotometer. Growth was estimated turbidimeterically at 0 and 12 hr and every 2 hr until maximum OD was reached. All fermentations were made in duplicate and replicate. Inoculum cultures were grown for approximately 16 hr in an optically clear broth identical to the fermentation media in all constituents except substrate. Strains A3c, B34b, and 7 were grown on cellobiose; strains HlOb and 23 were grown on glucose, and strains D15d, D31d, D16f, and D43e were grown on pectin. These inoculum cultures were diluted with anaerobic dilution solution until an OD of 0.2 was obtained. For all fermentations, 0.2 ml of the 0.2 OD inoculum was added to 5 ml of fermentation media. The fermentations were incubated for 168 hr at 39 C. Inoculum level and fermentation time were chosen to insure maximum rate and completion of the fermentation, as determined by Dehority and Scott (17). Galacturonic acid concentration was determined colorimeterically by the orcinol reaction (2). Readings were taken at 670 nm, which was determined by an absorption curve as the wave length giving maximum absorption of known galacturonic acid. Isolation of pectin from plant materials generally involves extraction and precipitation from an acidified ethanol solution (21,26). Fractionation of fermentation mixtures with 80% acidified ethanol has been used for measuring degradation or solubilization of isolated and intact forage hemicellulose (9,11); however, whether these were optimal conditions for estimating pectin solubilization had to be determined. The heterogeneity of the pectin molecule itself, plus the variation associated with source, make quantitative estimations quite difficult. However, since most pectins contain 75% or more galacturonic acid (21,26), it was believed that colorimetric analysis of this component would provide a reasonable estimate of pectin content. The only other source of uronic acids in the plant is from the hemicelluloses, and their concentration is low enough to be consid-VOL. 23,1972 ered negligible (10% or less of the hemicellulose) (26). Based on preliminary studies, the following procedure was adopted for the analysis of isolated pectin. The entire contents of the fermentation tube (5 ml) were washed into a polypropylene centrifuge tube with 45 ml of a 5% acetic acid-95% ethanol solution. The contents were mixed and allowed to stand at room temperature for 60 min. The mixture was centrifuged for 25 min at 30,000 x g; the supernatant fluid was decanted into a 250-ml volumetric flask and brought to volume with distilled water. The precipitate was hydrolyzed by adding 10 ml of 0.5 N H2SO4 to each tube, covering the tube with a metal cap, and autoclaving for 30 min at 15 psi. After cooling, the hydrolysate was transferred to a 500-ml volumetric flask and brought to volume with distilled water. The supernatant fluid and hydrolysate were analyzed for galacturonic acid as described earlier, and pectin concentration was estimated from standard curves. With a slight modification to remove hexose and pentose interference, the forage fermentations were analyzed by the same procedure. The modification involved treatment of the samples with an anionexchange resin, Amberlite IRA-400 in the acetate form, which removes galacturonic acid (17). Concentration of galacturonic acid was calculated as the difference in absorbance between resin-treated and untreated samples. The overall procedure for forage media analysis included the precipitation with 90% acidified ethanol, centrifugation, and hydrolysis steps, as used with purified pectin, except that the supernatant fluid and hydrolysate were diluted only to 50 ml. Twenty milliliters of the supernatant fluid was placed in a 50-ml volumetric flask and brought to volume with distilled water; a second 20-ml sample was treated with 6 ml of resin. The sample plus resin was allowed to stand at room temperature with occasional swirling for 1 hr, after which the supernatant fluid was transferred to a second tube containing a similar quantity of fresh resin and treated for the same duration of time. The resin was removed by filtration and washed with distilled water, and the filtrate plus washings were diluted to 50 ml. Ten-milliliter samples of the hydrolysate were handled in a similar manner. The resin-treated and untreated samples were then analyzed by the orcinol procedure. In this study, degradation is defined as the solubilization of 90% ethanol-insoluble pectin, whereas utilization is defined as a loss in total galacturonic acid. Degradation and utilization values were based on two replicate fermentations run in duplicate: a total of four tubes. When the coefficient of variation of the four values was larger than 15%, the fermentations were repeated. If the coefficient of variation remained above 15% after repeating the fermentations, a mean digestibility value was calculated from all eight fermentations, duplicates in quadruplicate. The coefficient of variation value of 15% was chosen as the upper limit to coincide with studies reported by Dehority and Scott (17) and Coen and Dehority (9) on the digestibility of forage cellulose and hemicellulose. RESULTS Only three strains of the original nine (B. ruminicola 23 and D31d and B. fibrisolvens D16f) used galacturonic acid as an energy source. Growth response and per cent utilization by these three strains is shown in Table 1. For both strains of B. ruminicola, 23 and D31d, the maximum OD was slightly higher in the combined than in the complete medium; however, the time to reach maximum OD was approximately the same. These results are reflected by the utilization data. Growth and utilization of galacturonic acid were both decreased in the CRF medium as compared to the complete medium. Growth curves revealed a much shorter lag phase with the CRF medium; however, rates of growth appeared to be similar on all three media. These data suggest that the galacturonic acid substrate became partially complexed or unavailable when autoclaved in the medium and that either this effect was more pronounced in the CRF medium or the CRF medium was limiting in a specific nutrient required by these strains. On the other hand, growth of B. fibrisolvens D16f appeared to be much slower in the complete and combined media as compared to the CRF medium ( Table 1). Extent of utilization was quite high in all three media and not closely associated with maximal OD. These data suggested the possibility of a limiting nutrient in the complete and combined media which slowed the rate of growth, and further investigations were made on this point. Suspensions of D16f were grown in CRF and complete media containing either galacturonic acid or cellobiose as the energy source. With the cellobiose substrate (Fig. 1), growing the inoculum in CRF broth reduced the lag phase approximately 12 hr, but the rate of growth did not appear to be influenced. Growth in the CRF medium was of the same magnitude for either inoculum. In the complete cellobiose medium, growth was reduced for the CRF grown inoculum and markedly reduced for the complete inoculum. When galacturonic acid was the substrate (Fig. 2), inoculum from CRF broth again had a shorter lag phase as compared to the complete medium inoculum. However, in this case, lag phase, rate, and maximum growth were all markedly reduced in the complete medium. These data suggest that the complete medium is nutritionally limiting for strain D16f, and that nutrient carry-over in the inoculum could influence the various criteria. The large discrepancy observed between growth and utilization for strain D16f in the complete medium ( growth rate, in which case a higher optical density is not attained because of subsequent cell lysis (4,20). Results on growth response, degradation, and utilization of purified pectin are presented in Tables 2 and 3. No measurable growth was observed with the three cellulolytic strains, B34b, A3c, and 7 or B. fibrisolvens H1Ob, and thus they were omitted from Table 2. In general, the data on the per cent pectin utilization (Table 3) are in agreement with the maximum OD values, the major exceptions being the very low OD observed with strain D43e in the complete medium and the marked utilization obtained with D15d in CRF medium as compared to the complete medium. Strains D16f, D15d, and D43e all exhibited an increased utilization in the CRF medium as compared to the complete medium, whereas 23 and D31d values were decreased. In essence, the pectin growth and utilization results for strains 23, D31d, and D16f are similar to those obtained with galacturonic acid. It appears that most of the differences observed for all strains could be the result of the pectin becoming partially unavailable when autoclaved in the medium, and nutritional limitations in the medium itself, or a combination of these factors. Pectin degradation (solubilization of pectin in 90% acidified ethanol) ranged from 72.5 to 98.5% in all three media for the five pectin uti- lizing strains. Although no measurable growth response in either galacturonic acid or purified pectin medium was indicated for any of the three predominant cellulolytic species found in the rumen, R. flavefaciens B34b consistently degraded about 30% of the purified pectin in all three media and utilized between 4.0 and 19.6%. B. succinogenes A3c was essentially unable to degrade or utilize any pectin, whereas R. albus 7 degraded 44.0 and 28.5% in the CRF and combined media, respectively, and utilized 22.6 and 19.1%. From the results on the degradation and utilization of purified pectin, six cultures were chosen for an investigation of pectin degrada-tion and utilization with intact forages. These were R. flavefaciens B34b, a pectin degrader but limited utilizer; B. ruminicola 23 and D3ld, B. fibrisolvens D16f, L. multiparus D15d, and Peptostreptococcus sp. D43e, all of which could extensively utilize purified pectin as an energy source. Table 4 lists the data on the degradation and utilization of pectin from two maturity stages of intact alfalfa. Degradation and utilization of pectin from alfalfa decreased only slightly with increased plant maturity. Degradation of pectin by R. flavefaciens B34b and B. fibrisolvens D16f was diminished by 10 to 15% as the alfalfa plant matured, whereas de- creases of less than 10% were noted for all other strains. Each strain capable of utilizing purified pectin as a substrate also degraded and utilized intact alfalfa forage pectin. However, in comparison to the other species, both strains of B. ruminicola were extremely limited in their ability to degrade and utilize intact alfalfa pectin. All strains, except R. flavefaciens B34b, were able to utilize almost all of the pectin which they could degrade or solubilize, thus suggesting that degradation is limiting the utilization of pectin from intact alfalfa. It seemed desirable to determine whether those strains which could utilize isolated pectin, but were limited in their ability to degrade and utilize plant pectin, could utilize the material solubilized by the cellulolytic strain B34b. Thus, R. flavefaciens B34b was combined in the same fermentation with either B. ruminicola D31d or B. fibrisolvens D16f. For these studies, 0.2 ml of a 0.2 OD suspension of each organism was used as inoculum. These results are presented in the bottom two lines of Table 4. In both cases, degradation was increased above the extent obtained with either organism alone. The most striking effect, however, was on the utilization of plant pectin by both combinations. Utilization increased from approximately 30 and 25% for either B34b or D31d alone to 82 and 74% for maturity stages I and III, respectively. The combination of B34b and D16f also increased pectin utilization; however, the magnitude of the increase was considerably less. The results from a similar series of forage fermentations using two maturity stages of bromegrass are presented in Table 5. With the possible exception of B. fibrisolvens D16f, both degradation and utilization of pectin from intact bromegrass were found to decrease markedly with increased plant maturity, which is in contrast to the earlier observations with alfalfa. This maturity effect was especially severe with both strains of B. ruminicola. When considering the first maturity stage of bromegrass, pectin degradation and utilization values were similar to those observed with alfalfa I for R. flavefaciens and Peptostreptococcus sp. Values for B. ruminicola were somewhat higher, whereas degradation and utilization values for L. multiparus and B. fibrisolvens were lower than those found with alfalfa. Results of the synergism studies on bromegrass I indicated that degradation was not improved with either combination of organisms above that of R. flavefaciens B34b alone. The B34b and D31d combination resulted in an increased utilization of approximately 30%, whereas only a 5% increase in utilization was observed with the B34b and D16f combination. The combination of B34b and D31d on the bromegrass II substrate resulted in a marked synergistic effect on both degradation and utilization. In contrast, degradation and utilization with the B34b and D16f combination were reduced below that of D16f alone. DISCUSSION The fermentation of galacturonic acid by those strains isolated in a selective pectin medium agreed with the results of Dehority (15), who reported that of the strains used in this study only D16f and D31d were able to grow with galacturonic acid as the sole energy source. These data suggest that different types of pectinolytic enzymes are present in the various species. The possible relationship between types of pectinolytic enzymes and utilization of galacturonic acid has been discussed previously (15). The ability to degrade and utilize the isolated and naturally occurring pectins varied between the pectinolytic strains (D16f, D15d, D31d, and D43e). In only one instance was degradation below 75% on the isolated pectin substrates. However, utilization data varied between 36 and 95% depending on the organism, culture medium, and method of medium preparation. Intact forage pectin was degraded and utilized to some extent by all pectinolytic organisms, with the exception of the two B. ruminicola strains and the mature bromegrass substrate. This ability varied both between organisms and between forages; for example, B. ruminicola D31d degraded and utilized more pectin from bromegrass I than alfalfa I, yet the decreases in pectin degradation and utilization attributed to increased maturity with the alfalfa were minimal, whereas the bromegrass II values dropped to essentially zero. At the same time, Peptostreptococcus sp. D43e degraded considerably more pectin than D31d from both alfalfa I and bromegrass I. The two strains of B. ruminicola, 23 and D31d, were somewhat similar in their ability to degrade and utilize galacturonic acid, iso-lated pectin, and intact forage pectin. Of particular interest was their almost complete inability to degrade and utilize pectin from the mature bromegrass. In contrast, a marked difference was observed between the two strains of B. fibrisolvens. Strain D16f was able to degrade and utilize galacturonic acid, as well as both isolated and naturally occurring pectins, whereas strain HiOb was unable to degrade or utilize any of these substrates. In general, the differences noted between strains and species in the present study tend to parallel results on hemicellulose degradation and utilization (9,11,13). On this basis, it would be desirable to investigate a number of strains of a given species, isolated on various selective and nonselective media, before concluding whether this was a definite species characteristic. Of the three normally predominant cellulolytic species found in the rumen, none showed a growth response in either galacturonic acid or pectin medium. However, R. flavefaciens strain B34b and R. albus strain 7 degraded (solubilized) approximately 30% of the purified pectin and results suggested a slight amount of utilization. B. succinogenes A3c was almost completely inactive in this respect. Although R. flavefaciens B34b was the only cellulolytic species tested on the intact forage substrates, it was capable of degrading as much or more of the pectin from intact forages than the pectinolytic strains. The limited utilization of pectin observed for this strain with the forage substrates might be attributed to a permeability of the cell wall to some of the specific oligouronides produced. It would be of considerable interest to test this strain on isolated alfalfa pectins. The degradation of isolated pectin by R. flavefaciens B34b and R. albus 7 plus the extensive degradation of intact forage pectin by B34b, accompanied by a very limited utilization, might suggest that this ability is similar to the activity previously observed with hemicellulose substrates. Dehority (11) and Coen and Dehority (9) reported that many of the rumen cellulolytic organisms were capable of partially degrading isolated and naturally occurring hemicelluloses from an ethyl alcoholinsoluble form to soluble products. Utilization of the hemicelluloses as an energy source was not associated with degradation, and subsequent studies with resting cell and cell-free systems suggested that the degradation of the hemicellulose occurred as a result of the action of a nonspecific constitutive enzyme (14). Definite synergism was observed by combining a pectin-degrading, but limited utilizing strain (B34b), with the pectinolytic strains D16f and D31d. The most striking synergistic results were observed with the combination of strains B34b and D31d, where utilization of alfalfa I increased from 30% for either organism alone to 82% for the organisms together, and on bromegrass II, where utilization increased from less than 10% for each organism alone to 53% for the combination. Except for the combination of strains B34b and D16f on the bromegrass H substrate, increases in degradation and utilization were observed for each of the other combinations. In general, synergism in the utilization of forage pectin paralleled earlier results on synergism with respect to intact forage hemicellulose utilization (9). The above results strongly suggest that, although the different pectinolytic species vary markedly in the amount of pectin they can degrade or solubilize from a specific intact forage, if the pectin can be freed from the forage either chemically or by another organism, it is then almost completely available for utilization. On the basis of the extensive degradation and marked synergism observed, it appears that the cellulolytic bacteria could contribute to the overall rumen fermentation of forage pectin by supplying the pectin-utilizing bacteria with more soluble substrate than they could degrade alone. No explanation is readily available for the slight decrease in degradation and utilization of bromegrass pectin obtained with the combination of strains B34b and D16f. A somewhat similar pattern was observed in the synergism studies on hemicellulose utilization from alfalfa by using a combination of R. flavefaciens B34b and L. multiparus D15d (9). The degradative end products produced by B34b may differ markedly from those of D16f on this particular forage and, subsequently, cannot be utilized by D16f. Competitive degradation, possibly influenced by rate phenomena, could then result in less available substrate for strain D16f. LITERATURE CITED

v3-fos

2019-03-30T13:12:22.265Z

1972-09-01T00:00:00.000Z

232758477

s2

Occurrence of Aflatoxins and Aflatoxin-Producing Strains of Aspergillus spp. in Soybeans Above average rainfall in Maryland during August, September, and October 1971 resulted in heavy mold growth in soybeans while still in the field. Of 28 samples of soybean seed, aflatoxins were found in 14, 2 of which had been used in poultry feed. Aflatoxins were identified by thin-layer chromatography, spectrophotometry, and chicken embryo bioassay. Aspergillus spp. were isolated from 11 samples, and 5 of these isolates produced aflatoxins when grown in liquid culture. Aflatoxins are not considered a serious problem on soybeans (6). Although aflatoxinproducing strains of Aspergillus flavus will grow on soybean seed in the laboratory, the amount of aflatoxins produced is much lower than on other commodities (5). The occurrence of both A. flavus and aflatoxins in soybeans under field conditions is generally very low. During 1965 and 1966, A. flavus could not be isolated from more than 3,100 samples of soybean seed examined (5), and Shotwell et al. (9) reported that only 2 of 866 soybean samples contained aflatoxins and the samples were in "sample grade." A number of reasons have been proposed for the resistance of soybeans to A. flavus such as unfavorable moisture conditions for the fungus at the time of soybean maturity, development of seeds in a closed pod, or the possibility that soybean seed contains an inhibitor which prevents growth of the fungus (5). An unusually wet growing season in Maryland during 1971 resulted in heavy mold growth in soybeans. Soybean samples were collected and examined to detect either the occurrence of aflatoxins or the presence of aflatoxin-producing strains of Aspergillus spp. MATERIALS AND METHODS Twenty-eight samples of soybean seed were examined; 24 were collected in experimental field plots from different locations in Maryland. Included were the three cultivars "Wayne," "Callard," and "Cutler"; the remainder were experimental lines. The percentage of moldy seed in each sample was determined. Four soybean samples to be used in poultry feed were obtained from the Poultry Department, University of Maryland. Samples were composed of nonground soybeans. To isolate Aspergillus spp., soybeans showing mold damage were surface sterilized with sodium hypochloride, split in half, and placed on Difco potato dextrose agar (PDA) containing 6.0% NaCl and incubated at 30 C. When colonies of Aspergillus spp. developed on the PDA, they were transferred to SMKY liquid medium (3) and the cultures were incubated at 25 C for 10 days prior to extraction of aflatoxin. The Aspergillus spp. were not identified as to species. The extraction and identification of aflatoxins were by the procedures of Pons and Goldblatt (8). Fifty grams of moldy seed, hand-selected from each sample, and the culture filtrate from the Aspergillus spp., isolated growing in liquid medium, were examined. Soybeans were ground in a Waring Blendor, shaken for 30 min with 70% acetone, and filtered to remove solid particles. Pigments were precipitated with lead acetate and centrifuged. The centrifuged filtrate and the liquid culture filtrates were each extracted with chloroform to remove aflatoxins. The chloroform extracts were evaporated under nitrogen and spotted on Adsorbosil I thin-layer chromatography plates along with pure aflatoxin standards obtained from L. A. Goldblatt (U.S. Department of Agriculture, New Orleans, La.). The plates were developed with chloroform-methanol (97:3, v/v) and then were dried and examined under ultraviolet (UV) irradiation. The fluorescent zones corresponding to the RF values of aflatoxin standards were eluted from the plates and re-chromatographed as before. The fluorescent zones, including standard, were removed from the plates, and the aflatoxins were dissolved in methanol. The concentrations were de-termined spectrophotometrically (Bausch & Lomb model 600 spectrophotometer) using the molar extinction coefficient e of 21,800 at 362 nm as reported for aflatoxin B, (1,2). The presence of aflatoxins in the same fluorescent zones were verified by chicken embryo assay (7). The aflatoxins were dissolved in 70%Yo ethanol and injected into the embryo via the air sac. Mortality was determined after 21 days. RESULTS AND DISCUSSION Although 24 field samples were collected, 7 were discarded including 3 samples each of Callard and Cutler because they contained less than 1% moldy grain which was an insufficient amount for extraction of aflatoxins. The percentage of moldy grain in each sample, the amount of aflatoxins present, the occurrence of Aspergillus spp., and whether or not these strains were aflatoxin producers are presented in Table 1. Results of the study on soybean samples to be used in poultry feeds are summarized in Table 2, and in Table 3 are the results of the chicken embryo test. The percentage of moldy grain ranged from less than 1% to a maximum of 72%. The highest percentages of moldy grain occurred in experimental lines; the 3 cultivars Wayne, Callard, and Cutler all had between 2 to 7% moldy grain. The highest amounts of aflatoxins recorded were samples from the cultivar Wayne, even though the percentage of moldy moldy grain yet contained only an average amount of aflatoxins, indicating that there was little correlation between percentage of moldy grain and the amount of aflatoxin. Samples 18 and 19, both of cultivar Wayne, were from two different locations and differed in aflatoxin level, whereas samples 10 and 15 (aflatoxin and no aflatoxin, respectively) were from replications of the same experimental line in the same location so that great differences in aflatoxin levels can occur according to cultivar, location, or even rows of the same cultivar in the same field, or perhaps even to pods on a plant. Aspergillus spp. were also isolated from moldy grain, and again there was little correlation between the occurrence of Aspergillus spp. with either amount of moldy grain or level of aflatoxin present. Furthermore, some isolates produced aflatoxins whereas others did not, indicating the difficulty in detecting potential aflatoxin problems by only isolating Aspergillus spp. We also found that even though aflatoxinproducing isolates of Aspergillus spp. could be identified in the soybean samples to be used in poultry feed, detectable amounts of aflatoxins did not always occur ( Table 2). The nonmoldy soybean sample was devoid of Aspergillus spp. and aflatoxins, as expected, whereas the two samples designated moldy mix yielded both aflatoxin-producing isolates of Aspergillus spp. and relatively high levels of aflatoxins. According to Hintz et al. (4) corn and soybeans having up to 50 ug of aflatoxin/kg would not be injurious to swine. The detection of aflatoxins using the chicken embryo test is presented in Table 3. The aflatoxins used in this test were extracted directly from the sample by chloroform, reduced in volume, dissolved in ethanol, and after being quantitated spectrophotometrically were injected directly into the eggs without first being purified by thin-layer chromatography. This may explain the high mortality in embryos injected with extracts from sample 11 and the low level of correlation between aflatoxin level and mortality. Other chloroform-soluble mycotoxins may have been present in this sample which could have increased the percentage mortality of embryos. Our studies indicate that soybeans can be a suitable substrate for growth of aflatoxin-producing strains of Aspergillus spp. especially if heavy rainfall occurs during maturation. Future routine examination of soybeans for the presence of aflatoxins is justifiable especially in areas of high rainfall or where soybeans will be used in poultry or livestock feeds.

v3-fos

2020-12-10T09:04:12.893Z

1972-12-01T00:00:00.000Z

237229055

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Procedure and Analysis of a Useful Method in Determining Mycelial Dry Weights from Agar Plates The evaluation of growth by dry weight determination of fungus mycelium for agar plates was examined. The data obtained were statistically analyzed. This method was shown to be sufficiently accurate to be used as an investigative tool. The evaluation of growth by dry weight determination of fungus mycelium for agar plates was examined. The data obtained were statistically analyzed. This method was shown to be sufficiently accurate to be used as an investigative tool. The more widely used methods (1,6) for monitoring the growth rate of fungi have been either mycelial dry weights obtained from liquid culture or the measurement of increased colony diameter. These methods have been particularly favored because they require neither elaborate equipment nor exhaustive assay techniques. However, there are numerous experiments in which the effect of certain parameters such as light do not lend themselves to this type of assay. One could not use liquid cultures in a shaker, as light spectra through flasks and moving liquid would be difficult to measure, and light effects on a spherical mycelial mass would not be uniform. Use of an increase in colony diameter on an agar plate would measure only lateral growth and neglect any differences in aerial growth. Day and Hervey (3) tried to solve this problem by placing the fungus mycelium-agar content of a petri dish in hot water to achieve a separation and then filtered this through cheesecloth followed by different types of washes. They concluded that washing with hot water freed the mycelium of more agar than did cold water washing and that it most probably dissolved substances from the mycelium as well. Little statistical work has subsequently been done to date to provide a clear picture of the potentiality of this method in determining fungus dry weights. This paper includes an analysis of the feasibility as well as a detailed description of an agar assay method of determining mycelial dry weights which can be used as a criterion for evaluating fungal growth. Based on past experiences with the dissolution of agar by boiling water, as well as the filtration rates and integrity of numerous filter papers, the following separation method was employed. The agar-mycelial contents of each petri dish tested in this experiment were removed and placed in a beaker of rapidly boiling distilled water for 4 min. The contents of the beaker were then filtered through a Buchner funnel by using predried (1 day at 70 C) and preweighed 9-cm Whatman no. 114 filter paper. Two 250-ml samples of rapidly boiling, distilled water were used to rinse the initial boiling flask and the resultant mycelial mat on the filter paper. A constant vacuum of 15 psi was maintained throughout the working procedure. After the final wash the sample was aspirated for 30 sec. Dry weights were obtained by placing the samples in a 70 C forced-air drying oven (Lab-Line) for 24 hr. The dry samples were then placed in a vacuum desiccator charged with Drierite desiccant (Drierite Co., Xenia, Ohio) for 6 days. A single-pan, rapid-reading balance (Mettler model H-10) equipped with desiccant was used to make all weighings. All accumulated data were subjected to a statistical analysis by computer employing the 07V program of the Biomedical Computer Programs (4). The program output included an analysis of variance (including the F ratio test for homologous variances) and the results of Duncan's multiple range test (5). The test fungus used in this experiment was Diplodia zeae (Schw.) Lev., one pathogen which causes stalk rot of corn in Illinois and other states (2). Previously dried and weighed Whatman no. 114 filter paper was subjected to four treatments to establish the amount of standard deviation associated with the assay method. Five filter papers each were (i) redried and directly reweighed, (ii) handled by the assay method but not subjected to any filtration stress, (iii) handled by the assay method and subjected to filtration stress with distilled water, and (iv) handled by the assay method with the filtration stress of a boiling agar solution. By including these four treatments, the sources of variation which might contribute to the resultant mycelial dry weights could be resolved. The amount of deviation attributed to the assay procedure is given in Tables 1 and 2. The analysis of variance shows that the F ratio is not significant, thus, the sample means are all taken from the same population (there was no difference between the results of the four treatments). The cumulative effect of weighing and handling and the stress of water filtration was demonstrated by the third treatment. The variability found in this treatment may be considered to be the same as that found in the broth assay method because the procedures are the same. The last treatment shows the amount of variability inherent in the agar assay method. There is no statistical difference between the effects of the water and the agar assay methods on the resultant dry weights of the filter paper. Both assay methods may be expected to increase the apparent dry weight of a sample due to the retention of media components in the filter paper at a constant rate with a predictable amount of variability. The effect of hot water extraction on the mycelium would also be constant across an experimental design. Therefore, the mechanics of the agar plate-dry weight method are considered sufficiently sound to include its use as a tool for evaluating mycelial growth.

v3-fos

2019-03-20T13:02:34.185Z

1972-01-01T00:00:00.000Z

237234900

s2

Unusual Organism Which Gives a Positive Elevated Temperature Test for Fecal Coliforms Organisms apparently not of fecal origin isolated from a sulfur hot spring gave a positive elevated temperature test for fecal coliforms. At the present time, a distinction is commonly made between coliforms of "fecal" and "nonfecal" origin on the basis of the elevated temperature test (4). This test is based upon the ability of fecal coliforms to produce gas from lactose within 24 hr at 44.5 C. This test may be run by determination of most probable numbers (MPN) in EC broth or by the membrane filter technique on M-FC broth-soaked pads. Nonfecal or soil coliforms do not give a positive reaction under these conditions. Geldreich (2) discussed the significance of fecal coliforms and suggested that these organisms be used as indicators of fecal pollution of recreational waters. Hendricks (3) found that 24% of the Enterobacter (Aerobacter) group (nonfecal in origin) which he isolated from a "relatively unpolluted river" were positive in the elevated temperature test for fecal coliforms. Data presented here describe an organism from a sulfur hot spring which gives a positive elevated temperature test for fecal coliforms. Cultures were isolated and Gram-stained from eosin methylene blue (EMB) agar plates (Difco) inoculated from positive Brilliant Green lactose bile (BGB) broth tubes (Difco), from EC broth tubes (Difco), or from colonies on membrane filters (Millipore Corp., Bedford, Mass.) incubated on M-FC broth (BBL)soaked pads at 44.5 C. All cultures were retested in EC broth, and these inoculations were made by means of a needle. Indole production, hydrogen sulfide production, and motility were performed in SIM medium (Difco studied on nutrient agar supplemented with 0.4% gelatin (5). Cytochrome oxidase tests were performed with test strips (Pathotec, Warner Chilcott Laboratories). Flagellation was determined by use of a flagella stain. Samples were obtained from a sulfur hot spring, from the Laramie river, and from an aerated sewage lagoon. The data in Table 1 show results of studies conducted in April 1970. The finding that 17 of 19 isolates of Escherichia coli produced gas at 44.5 C in EC broth supports the idea that "typical" E. coli isolates give a positive elevated temperature test. In contrast, the fact that 25% (5 of 20) of the Enterobacter aerogenes isolates from the lagoon and river also gave a positive elevated temperature test, a result similar to the value found by Hendricks (4), suggests that a single or few samples are insufficient to judge water quality, since nonfecal coliforms might be falsely confused with coliforms of fecal origin. This could be particularly important in situations in which great distances might limit the number of samples taken. In marked contrast to the results obtained from other sources, 9 of 10 apparent isolates of E. aerogenes from the hot spring drainage produced gas in EC broth at 44.5 C. This result indicates that the incidence of apparent E. aerogenes capable of giving a positive elevated temperature test may depend upon the environment. The isolates that produced gas in EC broth at 44.5 C were obtained throughout the hot spring drainage as soon as the temperature of the water had dropped to 46 C. This along with the visual observation of distance from the spring suggested that these organisms were not of fecal origin. To determine if this was a fortuitous result, another series of samples was taken in November 1970. Data in Table 2 show that 28 of 58 isolates in November were posi- tive in the elevated temperature test, confirming the April result that an unusually high percentage of falsely positive fecal coliforms were found in the hot spring drainage. Four "typical" E. coli cultures were recovered in November, and all were positive in the elevated temperature test. Approximately half of the November isolates were lightly golden in color when grown on nutrient agar at 35 C, and, upon storage at room temperature, these cultures became bright yellow. This finding was not observed in April. The yellow cultures exhibited typical Enterobacter colonies on EMB agar; gave typical indole, methyl red, Voges-Proskauer, citrate (IMViC) reactions; produced gas in BGB broth; were motile; and were gram-negative rods. However, they failed to produce hydrogen sulfide or to liquefy gelatin and were cytochrome oxidase-negative. Flagellation was typical of the Aeromonas group (1). Old cells had predominantly a single polar or subpolar flagellum, whereas young (6to 8-hr) cells showed additional lateral flagella. The exact taxonomic position of these organisms is thus unclear. Data in Table 2 also show that these organisms displayed a positive elevated temperature test when the membrane filter procedure was employed. Moreover, they may be nega- tive (yellow colony) by the membrane filter method and positive by the EC broth method. It should be noted, however, that the color of these organisms by the membrane filter method was not as bright blue as typical E. coli colonies. Regardless of the taxonomic position of the yellow organisms, it is clear that, except for color, they are not easily distinguished from E. aerogenes, particularly in a routine bacteriological water analysis. It, therefore, appears that the use of multiple criteria in determining water quality is desirable.

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2020-12-10T09:04:12.850Z

1972-08-01T00:00:00.000Z

237234296

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Stomaching: a New Concept in Bacteriological Sample Preparation An entirely new mixing device, particularly suitable for preparing bacterial suspensions from foods, fabrics, swabs, and other fairly soft materials, has been developed. With this technique the sample and diluent are put into an inexpensive, sterile plastic bag which is vigorously pounded on its outer surfaces by paddles when placed inside the machine. The resulting compression and shearing forces effectively remove even deep-seated bacteria. After samples are taken for analysis the bag and its remaining contents are thrown away. Labor involved in cleaning and sterilizing reusable homogenizer cups or probes is eliminated, and the device is immediately ready for reuse. Running costs are thus drastically reduced, compared with conventional homogenizers. Additional advantages of this device, which is simple and inexpensive to manufacture, are low noise level, negligible temperature rise, and the small storage space required for bags. Preparation of bacterial suspensions from foods and other materials which cannot immediately be dispersed in water is normally brought about by shaking the sample with diluent in a bottle or homogenizing it in one of a variety of bench-top or hand-held blendors. Considerable labor is then needed to clean or resterilize the homogenizer cup or probe before it can be reused. Such homogenizers frequently possess other disadvantages: for example, high capital outlay for adequate numbers of cups or probes, high noise level, dangerous temperature rises during sampling, and high maintenance costs. To avoid some of these problems, several alternative methods of sampling have been proposed: for example, ultrasound treatment (4,6), vortex stirring (6), surface scraping (9), water spraying (1, 2), vacuunm probe (3), and electrophoresis (8). In certain applications these methods have distinct advantages over homogenization methods, but there is no evidence that they have become popular outside the authors' laboratories. Our experience is that many bacteriologists are suspicious of sampling methods that do not obviously disintegrate the specimen, even though the resulting suspension may, in many cases, be much easier to handle. Since it is axiomatic that, during mixing, some or all of the mixer surfaces contact the specimen and become contaminated, we looked at ways in which the labor needed for resterilizing those surfaces could be avoided. One way to do this was to construct a mixer which would rapidly and automatically carry out a sterilizing routine using, for example, bactericidal solutions, ultrasound, or microwave or steam heating. An alternative and neater way appeared to be to prepare the sample in a disposable mixer. For routine use in a busy laboratory, however, the only containers likely to be available at reasonable cost were plastic bags, and the possibility of applying adequate mixing forces to the sample through the flexible walls of bags was investigated. Results were so encouraging that a series of devices (which have been given the generic name "Stomacher," after the action employed) were designed and constructed so that the potential of the method could be fully evaluated. The stomaching principle is the subject of patent application (Sharpe and Jackson, British Patent Application no. 41395/71). Operating principle of Stomachers. All of the devices feature a means of temporarily sealing the sample and diluent inside the bag and of applying forces to the outside of the bag by means of paddles, wheels, or rollers. A particularly simple and successful design, supplied to us by A. J. Seward and Co. Ltd., (P.O. Box 1,6 Stamford St., London S.E.1), is shown in Fig. 1 The bag is temporarily sealed when the door is tightened. and the bevelled edge of the case as the Stomacher door is tightened. The large knob on top of the machine serves to pull the door firmly into contact with the bevel. When the machine is switched on, two paddles, side by side, alternately pound the bag and compress its contents against the door. A capacitor-type constant-speed motor in the two larger Stomachers, and a series-wound motor in the smaller, provides power to the paddles via two eccentrics, and a degree of resilience is built into the paddle system by the use of rubber connecting rods. Excessive pressure build-up or stalling, when incompressible samples are encountered, is thus avoided. A variety of speeds, paddle conformations (e.g., vertically above one another), flexible coverings for paddles and doors, and tread patterns in these coverings have been employed in experimental Stomachers. However, the smooth metal surfaces of the models shown perform excellently under most circumstances. A variety of flexible containers have been used, for example, polyethylene, polyester, cellulose-polyethylene-polyvinylidene chloride (PVDC) laminates, and rubber. A standard thin-walled polyethylene bag, preferably basewelded, is entirely adequate, however, and preferable from the point of view of cost, handleability, and freedom from toxic effects. Removal of bacteria is probably brought about partly by violent shearing forces as the liquid is swept from side to side, and partly by the series of rapid compressions the sample experiences as it is trapped under the paddles. This repeated "sponging" action may be responsible for the effective removal of bacteria present in fissures or crevices, for example, in the veins and capillaries of meat. Incorrect design can cause rapid bag failure through the production of high hydrodynamic pressures. Failure may then occur at a weld, or in the fabric of the bag, depending on its type. High pressures can be avoided by correct design, however, and catastrophic failure does not occur in the models shown. Sharp objects such as bone splinters may make pinholes through which the suspension will leak, but such damage is infrequent and leaks occur less frequently than with our normal homogenizer cups. The wooden sticks of swabs do not damage bags. Addition of a soft rubber curtain allows even soil samples containing grit and small pebbles to be processed safely. MATERIALS AND METHODS Samples. Food samples were bought locally or were experimental samples obtained from other parts of the laboratory. Frozen foods were thawed, and dried products were resuscitated by soaking for 40 min as 20% suspensions in 0.1% peptone solution before use. Samples were divided and homogenized or stomached separately. Soiled woven fabrics were cut into thin strips and divided. Swabs were added to bags, complete with their sticks. Stomaching. All bacteriological analyses were made using the middle-sized Stomacher, at 230 rev/min. Food (10 g) or fabric samples (4 g) were weighed into sterile polyethylene bags (7 by 12 inches, A. J. Seward and Co. Ltd., or Sterilin Ltd., Richmond, Surrey, England), and 90 ml of 0.1% peptone solution was added. (The procedure was adjusted accordingly where soaking was required.) For swabs, 10 or 50 ml of peptone solution was added. Stomaching times of 30 sec were generally used except where release rates were being studied. With many products (e.g., fruits and comminuted meats) complete dispersal occurred within 5 sec. Homogenization. The same sample and diluent quantities were homogenized in sterile 200-ml stainless-steel cups on an Ato-Mix blendor (MSE Ltd., Spenser Street, London S.W.1., England) for 2 min NEW SAMPLING METHOD (15 sec at 6,000 rev/min, 90 sec at 12,000 rev/min, and 15 sec at 6,000 rev/min). This device is similar to the Waring Blendor. The program is used by many of our laboratories. Count procedure. Three types of counting method were used during the study. Serial 10-fold dilutions in 0.1% peptone solution were inoculated into plate count agar or violet red bile agar (Oxoid Ltd., London S.E.l., England). Total viable aerobic counts (on plate count agar) were made after 48 hr at 30 C, and coliform counts (on violet red bile agar) after 24 hr at 37 C. On other occasions, counts were made by using a rapid technique employing agar droplets (7). Other total viable aerobic and coliform counts were made by using an automatic diluting, inoculating, and pour plate preparing machine (5). Coliform counts were made on only about 20% of samples, and no distinction has been made between the two types of count in analyzing the data. RESULTS About 550 comparisons of samples treated by Stomacher and Ato-Mix blendor showed an excellent recovery of bacteria by the new method (Table 1). Release rate studies indicated that for most foods 15-sec stomaching is adequate, but for routine work 30 sec is preferred. The beef group contained some rather fatty forequarter samples, and it was noticed that, for these, recovery decreased with increasing fat concentration. For samples containing about 95% fat, the recovery was less than half that of the Ato-Mix samples at 30 sec, but improved with increasing stomaching time. Lower recovery was also noted for shortcrust pastry and dairy cream. For high-fat meats etc., therefore, 1 min or more may be preferable. A rough guide is obtained from the appearance of the sample, which is easily visible through the transparent walls of the bag. Recovery efficiencies for coliforms were as good as for total viable aerobic organisms, and it is unlikely that selective retention of any organism would be experienced, except perhaps in high-fat materials. No difference was noted between Stomachers with paddles side by side, or one above the other, and no decrease in count was noted during stomaching times of more than 5 min. Temperature rise during stomaching was negligible (approximately 0.8 C/min for liquids at ambient temperature). This contrasts markedly with certain mechanical homogenizers, in which a most probable rise of 17 C (i.e., most samples warming to 37 C) during the 2 min has been found. Temperatures up to 59 C have, in fact, been recorded in our laboratories in homogenizers having bearings in poor condition. Noise level from the Stomacher is low; its i, Stomacher dull squelch is, in any case, far less irritating 0.05 level). DISCUSSION than the whine of bladed homogenizers. The small temperature rise is desirable when sensitive organisms are likely to be encountered. The whole device is light and easily portable, with a supply of bags. In this respect a Stomacher combined with the rapid droplet technique (7) forms an excellent portable testing method. As an additional benefit, two or three samples may be placed in the Stomacher together and processed simultaneously, thereby effecting even greater economies in time. Only for products of very high fat content is recovery significantly lower than by conventional blending. On no occasion has the difference been greater than 50% after 1 min of stomaching. For these products, slightly warmed diluent is a useful aid, assisting dispersal of fat. This effect seems to indicate that the temperature rise generated by conventional blendors may be a significant factor in their better performance on fatty products. Bag failures are remarkably infrequent. The machine is not particularly demanding in its requirements of bags. The use of a good quality bag is recommended of course, but flexibility is more important than toughness. For this reason, the thinner grades of polyethylene bag (e.g., 200-gauge) are preferable to heavier or laminated bags, which are usually considered to be more durable. The Stomachers shown in the figures have complete protection for the motor and all other electrical components in the event of a bag bursting catastrophically. This has not occurred with these machines; only a small number of pinholes (< 1%) have been obtained with many thousands of samples. Leaks are more likely to occur near a weld than in the fabric of a bag, and, for this reason also, a bag formed from flat polyethylene tubing is preferred since the length of weld is minimal. If samples containing pathogenic organisms are to be handled it is quite possible to place the sample bag inside another, for example a PVDC laminated or rubber bag, to obtain complete protection should the sample bag begin to leak. If no leakage has occurred the outer bag may be reused.

v3-fos

2018-04-03T00:50:38.892Z

1972-12-01T00:00:00.000Z

27004486

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-APPL. MICROBIOL. 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

2020-12-10T09:04:12.439Z

1972-07-01T00:00:00.000Z

237231585

s2

Lactose-Hydrolyzing Enzymes of Lactobacillus Species β-Galactosidase (β-gal, EC 3.2.1.23) and β-D-phosphogalactoside galactohydrolase (β-Pgal) activities were observed in all of 13 Lactobacillus species studied except L. casei and L. buchneri. Only the latter enzyme was detected in nine strains of L. casei. The β-gal from L. thermophilus and the β-Pgal from L. casei were purified and characterized. In comparison with β-gal, the β-Pal was slightly less active (Vmax values were 28.9 and 50.0 μmoles per mg per min, respectively), but the substrate affinitives were similar (Km values were 1.69 × 10-3 M and 1.59 × 10-3 M, respectively). Although the two enzymes had similar amino acid compositions, the molecular weight of β-gal was 5.4 × 105 and that of β-Pgal was 1.3 × 105. The β-gal from L. thermophilus and the β-Pgal from L. casei had optimal temperature and pH activity values of 55 C at pH 6.2 and 37 C at pH 5.0, respectively. The complete absence of β-gal from a homofermentative Lactobacillus species of industrial importance is further evidence of the heterogeneity of this genus. Group N streptococci, with the exception of one strain (2,(17)(18)(19)(20)(21)(22), utilize lactose by the phosphoenolpyruvate (PEP) phosphotransferase system. Kundig et al. (12) first described this enzyme system in Escherichia coli, and it also has been shown (4, 7-9, 11, 13, 14) to be involved in lactose utilization by Staphylococcus aureus. Recently, Romano et al. (26) examined the distribution of this pathway among bacteria for glucose metabolism, pointing out that it allows for conservation of energy among organisms carrying out anaerobic glycolysis. A key enzyme in the PEP lactose phospho- transferase system is f-D-phosphogalactoside galactohydrolyase (j3-Pgal), as first named by Laue and MacDonald (13,14). This enzyme hydrolyzes lactose phosphate, the phosphorylation of which is mediated by PEP, to liberate glucose and galactose-6-phosphate. Because no studies of this enzyme have been reported for Lactobacillus species, the present investigation was undertaken. Results of a survey of lactobacilli for f3-galactosidase (l-gal) and fl-Pgal are presented, as well as the comparative properties of partially purified preparations of these enzymes from L. thermophilus and L. casei, ' Media and growth conditions. Cultures were maintained by weekly transfers in sterile nonfat milk and incubated at their optimal temperatures for 12 to 14 hr; between transfers, they were held at 2 C. Cultures for enzyme studies were transferred (1.0%, v/v) from milk into lactic broth (5) containing lactose as the only energy source, and were incubated for 10 to 12 hr. The L. thermophilus Farr strain was incubated at 45 C (it grew well at 55 C but not at 60 C); the L. casei strains were incubated at 30 C. Harvesting cells. Cells grown in the lactic broth were harvested by use of an RC-2 Sorvall refrigerated centrifuge (1 C) at 3,000 x g. The cells were washed three times with 0.1 M sodium phosphate buffer at pH 7.0. Extraction and purification of ,8-gal from L. thermophilus Farr. Cells harvested from 7 liters of broth were broken by sonic treatment (Raytheon, 10kc) for 30 min and were centrifuged for 20 min at 15,000 x g; the sediment was discarded. A 3-ml amount of 50% streptomycin sulfate (Sigma Chemical Co.) was then added to 50 ml of supernatant 51 fluid which was stirred for 30 min at 5 C. After centrifugation at 15,000 x g for 30 min, the enzyme extract was 65% saturated with reagent-grade (Mallinckrodt Chemical Works) ammonium sulfate. The precipitated protein was dissolved in 5 to 10 ml of 0.01 M tris(hydroxymethyl)aminomethane (Tris)hydrochloride buffer (pH 7.7) containing 5 x 10-' M MgSO4 and 0.002 M mercaptoethanol. The preparation then was dialyzed against three 2-liter changes of Tris buffer for successive 10-hr periods. The dialyzed enzyme was placed in 150 ml of diethylaminoethyl (DEAE) A25 Sephadex (Pharmacia Chemical Co.) slurry and washed with 500-ml volumes of sodium chloride at 0, 1.0, 1.5, 2.5, and 4.0% salt concentrations. The most active fractions were combined, and the enzyme was precipitated by the addition of 40% ammonium sulfate (w/v). The protein was recovered by centrifugation (15,000 x g for 25 min) and was resuspended in 1.0 ml of Tris buffer. The solution was dialyzed as before and then chromatographed on a column (2.7 by 46 cm) of DEAE A25 Sephadex with linear gradient; 0.5 liter at 1.0% NaCl and 0.5 liter at 3.0% NaCl were used in the two mixing vessels. The active fractions (10.0 ml) were combined, brought to 65% ammonium sulfate saturation, and centrifuged. The resuspended protein (5 to 10 ml in Tris buffer) was dialyzed for 36 hr, layered on a 5 to 20% sucrose gradient, and centrifuged at 42,000 x g for 25 hr. Fractions (1 ml) were collected and assayed for activity. Protein was measured by the method of Lowry et al. (16). Extraction and purification of fl-Pgal from L. casei. The procedures followed in purification of this enzyme were the same as for ,B-gal except that Sephadex G-200 chromatography was used in place of sucrose gradient centrifugation after the DEAE Sephadex treatment. Also, the G-200 column was buffered to pH 5.0, the optimal pH for the enzyme. Enzyme assays. The assay used for ,B-gal was described earlier (2). fl-Pgal assays were performed at pH 5.0 as indicated by McKay et al. (20). The onitrophenyl-fl-D-galactopyranoside (ONPG) and onitrophenyl-f,-D-galactopyranoside-6-phosphate (ONPG-6-P) were obtained from Sigma Chemical Co., St. Louis, Mo., and NK Laboratories, Jersey City, N.J., respectively. A unit of enzyme was defined as the number of micromoles of o-nitrophenol (ONP) liberated per milligram of enzyme protein per minute under the assay conditions used. Enzyme properties. An aluminum temperature gradient block with a range from 25 to 65 C was used to determine the optimal temperature; ONP release was measured after 1 min of incubation. The influence of pH on enzyme activity was determined by adjusting 0.1 M NaH2PO4 to pH values from 4.0 to 8.0 by addition of 0.1 M Na2HPO4 and measuring enzyme activity at different pH values at 37 C. To determine stability to heat, purified enzyme in the liquid state was held at different temperatures and sampled at intervals for activity determination. The effect of ethylenediaminetetraacetic acid (EDTA) was determined by dialyzing enzyme preparations for 12 hr at 5 C against 1 liter of EDTA ranging in concentration from 0.005 to 0.5 mg/ml. An estimation of the molecular weight was made by use of the procedure of Andrews (1) as confirmed by Dasgupta et al. (3). To determine amino acid composition, purified enzyme was hydrolyzed for 24 hr at 110 C in concentrated hydrochloric acid. Samples were analyzed on a Beckman Spinco model 170B amino acid analyzer. A Gilford model 2000 multiple-channel recording spectrophotometer was used to follow the kinetics of hydrolysis of ONPG and ONPG-6-P. A Hakke temperature control unit was attached to the jacket of the cuvette chamber to maintain the temperature at 37 C. A wavelength of 420 nm was used. When lactose (23) indicated that this organism may not be L. acidophilus. was used as the substrate, the rate of glucose liberated was measured by use of Glucostat (Worthington Biochemical Corp.). The Michaelis constants were determined from plots of the data made according to Lineweaver and Burk (15). RESULTS Enzyme distribution studies. Results of a survey of Lactobacillw species for #-gal and fl-Pgal appear in Table 1. There were negligible phosphatase activities in the cell-free extracts with p-nitrophenylphosphate as substrate under the conditions used for the a-gal and ,-Pgal assays. Thus, the fl-Pgal activities recorded in Table 1 (Table 3). Kinetic data. Figure 1 shows a reciprocal plot of the enzyme velocity data at different substrate concentrations; typical first-order kinetics were observed, and the Vmax and Km values for both enzymes are recorded in Table 4. The two enzymes had similar Km values or affinity for substrate. Neither ONPG nor lactose was a substrate for f3-Pgal from either L. thermophilus or L. casei. Enzyme properties. The influence of pH and temperature on activity of the enzymes is shown in Fig. 2 and 3, respectively. Enzyme from L. thermophilus, with the higher optimal growth temperature, revealed an expected higher temperature requirement for maximal enzyme activity. Figure 4 shows the effects of storage and heating on the stability of the enzymes; the f3-Pgal from L. casei was less stable to heat than the #-gal from L. thermophilus. EDTA had no effect on the stability of f3-Pgal from L. casei but caused an immediate decrease in activity by 50% when added (50 Aig/ml) to a-gal of L. thermophilus. Storage did not increase the 50% inactivation. 53 VOL. 24,1972 The amino acid compositions of the enzymes from L. thermophilus and from L. casei appear in Table 5. They contain similar amino acids with similar quantitative relative orders for each amino acid; cystine was absent. Figure 5 shows the molecular-weight data; ,-gal from L. thermophilus had a molecular weight of 5.4 x 105, and 6-Pgal from L. casei revealed a value of 1.3 x 105. DISCUSSION Organisms of the Lactobacillus genus are widely distributed in nature, and both homofermentative and heterofermentative species can be isolated from plants (25). Those important in milk fermentations are infrequently isolated from green plants (25) and presumably occur in the intestinal tract, where lactose may be available; however, no recent definitive study on species present in the human intestine has been made. The species used in this study included the homofermenters used in starter cultures in the manufacture of fermented dairy products. Among these are L. acidophilus, L. bulgaricus, L. helveticus, and L. lactis. Other homofermentative species included those such as L. casei and L. plantarum, which are important in bringing about further changes in products such as Cheddar cheese during curing by virtue of their lower optimal growth temperatures. Although it was not surprising to find p3-gal and fi-Pgal missing in L. buchneri, a nonfermenter of lactose, the complete absence of ,Bgal from eight different L. casei strains was surprising, particularly because all other lactose-fermenting strains revealed both ,-gal and ,B-Pgal activities. Whether or not this is of evolutionary significance remains to be seen. It also was noteworthy that the range of specific activities for ,B-gal (300 to 7,014) was considerably greater than for ,B-Pgal (40 to 575). One of the first studies on a lactose-hydrolyzing enzyme from a Lactobacillus organism was made by Monod and Cohen (24), who found that the ,B-gal of L. bulgaricus was activated by monovalent cations as well as divalent metal ions such as Mg2+, Mn2+, and Fe2+. Recently, Iwasaki et al. (10) purified 34-fold the a-gal from L. bifidus (Bifidobacterium bifidum); the enzyme had an optimal pH of 7.0 and maximal activity at 50 C. It also was activated by Mn2+ and Fe2 . To our knowledge, no reports on ,B-Pgal in lactobacilli have been made. Molecular weights of 540,000 and 130,000 have been reported (17) for ,B-gal in Streptococcus lactis; these were the approximate molecular weights determined in the present study for p3-gal and fl-Pgal, respectively. Also, McFeters et al. (17) observed a molecular weight of 500,000 for the monomer form of ,B-gal from S. lactis 7962. McFeters (Ph.D. thesis, Oregon State University, Corvallis, 1967) also noted a quantitative similarity in amino acid content between Escherichia coli and S. lactis 7962 /3-gal, and the same trend was noted in this study (Table 5) for a-gal and O-Pgal. Enzyme purification data (Tables 2 and 3) indicated that O-Pgal was less easily purified to as high a specific activity as O-gal. Storage and kinetic data also suggested that the former enzyme was less stable (Fig. 4) and less active, with a lower Vmax value but the same affinity for substrate. Also, ONPG was preferred over lactose as a substrate for #-gal, (Fig. 1), but purification of this enzyme with ONPG as the substrate may have influenced these results. In acid (>0.75%) also will be studied.

v3-fos

2020-12-10T09:04:12.450Z

1972-01-01T00:00:00.000Z

237234633

s2

Twenty-Four-Hour Immunofluorescence Technique for the Detection of Salmonellae in Nonfat Dry Milk A detection procedure was developed in which a newly devised lysine-iron medium was used as a one-step selective and enrichment medium for detection of salmonellae by the fluorescent-antibody technique. Incubation was conducted in two steps: initially at 30 C for 5 hr to resuscitate sublethally stressed cells, followed by incubation at 39 C for 17 hr. Twenty-seven strains of salmonellae from groups A-I were utilized in the development of this procedure which was sensitive enough to detect one Salmonella bacterium in 100 g of nonfat dry milk. A detection procedure was developed in which a newly devised lysine-iron medium was used as a one-step selective and enrichment medium for detection of salmonellae by the fluorescent-antibody technique. Incubation was conducted in two steps: initially at 30 C for 5 hr to resuscitate sublethally stressed cells, followed by incubation at 39 C for 17 hr. Twenty-seven strains of salmonellae from groups A-I were utilized in the development of this procedure which was sensitive enough to detect one Salmonella bacterium in 100 g of nonfat dry milk. Rapid and reliable means are urgently needed to detect contamination of food by salmonellae. This is especially true of foods like dairy products where wide use and a significant number of very susceptible consumers (infants and aged) require the most careful quality control. Because of the very low incidence of contamination in dairy products, particularly nonfat dry milk (NDM), a rapid screening procedure would minimize the holding period required to assure a safe product for shipment. Numerous methods utilizing the principal biochemical characteristics of salmonellae such as inability to split lactose, production of hydrogen sulfide, and possession of a lysine decarboxylase (1,4,5), as well as modification of media plus serology (12), have been proposed for speeding up salmonellae detection. Specialized glassware utilizing carbohydrate fermentation and the motility of the organism also have been recommended (2). The fluorescentantibody technique also has been proposed in many procedures for detecting organisms in meat (6), eggs (11), miscellaneous foods (8), and NDM (10). The purpose of this work was to determine whether a newly proposed presumptive type selective broth medium (7) for salmonellae could be used as a combination pre-enrichment and selective medium for a fluorescentantibody procedure to determine the presence of salmonellae in NDM within 24 hr. MATERIALS AND METHODS Salmonella serotypes. Serotypes from groups A through I were utilized including those serotypes most frequently isolated from dairy products, viz. S. anatum, S. blockley, S. bredeney, S. cubana, S. derby, S. enteritidis, S. meleagridis, S. Montevideo, S. new brunswick, S. newington, S. oranienburg, S. senftenberg, S. tennessee, S. thompson, S. typhimurium, and S. Worthington. Other Enterobacteriaceae. Representative strains of Enterobacter, Escherichia, Shigella, Citrobacter, and Proteus were included to determine specificity of the method for salmonellae. Medium. The lysine-iron-neutral red-broth presumptive medium as proposed by Hargrove et al. (7) was used to differentiate pure cultures and to detect salmonellae in NDM. The bases of this presumptive medium were pH response to an indicator dye and formation of H2S with novobiocin added as a selective agent. Salmonella antiserum. The Salmonella antiserum used was a commercially available globulin fraction of type 0 polyvalent antiserum (Sylvania Co., Milburn, N.J.). It had been absorbed with strains of Escherichia coli and Citrobacter freundii and conjugated with fluorescein isothiocyanate and contained antibodies against groups A through I. This antiserum stained both somatic and flagellar sites. Fluorescent-antibody microbiology. An advanced laboratory microscope was used throughout this work (American Optical Co., Buffalo, N.Y.; model LIOTU-FDW). Brighter fluorescence was observed with two oil-immersion objectives (x50 and 100) which contained iris diaphragms. The x 100 objective was used for the final evaluation of the 78 slide. Differentiation of pure cultures. Pure cultures were inoculated into tubes of medium and were incubated at 37 C for 18 hr, at which time color was recorded and samples were taken for the fluorescentantibody procedure. Test samples. Since no significant number of commercially contaminated samples was available, samples of NDM powders were prepared by adding known numbers of salmonellae to them. Twentyfour-hour broth cultures were diluted as necessary and blended with NDM in a mortar and pestal; additional NDM was added to obtain the approximate final concentration of salmonellae desired. Thorough mixing was carried out by mixing with large, sterile spoons and subsequent extensive shaking of powder in polyethylene bags. Powders were stored at 4 C. These were then assayed for approximate number of viable salmonellae by addition of trypsin to NDM reconstituted in lactose broth and assayed by conventional procedures. Assay of NDM. Generally, NDM is assayed on a 1:10 (weight of sample/volume of medium) basis; however, when 100-g samples were assayed to detect low levels of contamination, it was found that a 1:15 ratio gave better dispersion of the samples. Trypsin (30 ml of 1% solution) and novobiocin (5 ,g/ml) were added as previously described (7). Based on studies of sublethally stressed cells (9) and optimum temperatures for salmonellae growth (3), the incubation was conducted in two steps: initially at 30 C for 5 hr, followed by 39 C for 17 hr. The stepwise procedure for assaying NDM samples was as follows. (i) Lysine-iron broth medium was prepared by the formula of Hargrove et al: (7), dispensed in 1,500-ml portions in large-mouth Erlenmeyer flasks, and autoclaved 121 C for 15 min. (ii) One hundred grams of NDM powder was mixed aseptically in 1,500 ml of medium in a Waring Blendor. (iii) The broth and milk mixture was returned to the medium flask, and stock solutions of trypsin and novobiocin were added (30 and 7.5 ml, respectively) and mixed. (iv) Flasks were incubated at 30 C for 5 hr and then incubated at 39 C for 17 hr. (v) Flasks were examined for medium indicator-color changes and blackening after a total of 22 hr of incubation. The incubated samples were gently mixed, and 0.05 ml of each sample was placed onto an etched circle of a nonfluorescent glass slide (Aloe Scientific, St. Louis, Mo.). The slides were air dried, fixed in Haglund's solution (a 60:30:10 absolute alcoholchloroform-Formalin mixture) for 30 sec, touched off on absorbent paper, and transferred to absolute alcohol for 30 sec before air drying. Each smear was stained with the polyvalent antiserum at a 1:2 dilution. The slides were incubated for 30 min under inverted 150-mm glass petri dishes containing moist filter paper. This incubation period was followed by a rinse in normal saline, 10 min in phosphate-buffered saline, and a rinse in distilled water. The air-dried slides were mounted in a buffered glycerol fluorescent-antibody mounting fluid (Difco) and covered with a no. 0 Coming cover glass. Final evaluation of these slides was based on bacterial morphology, size, and degree of fluorescence by using x 100 objective with iris diaphragm. Although in these experiments each field usually contained numerous fluorescing cells, a single fluorescent cell in 25 fields examined would be considered as positive for salmonellae. Approximately 2 hr was required for completion of the fluorescent-antibody procedure. RESULTS AND DISCUSSION The 27 salmonellae serotypes from groups A through I that were used in our previous study (7) were tested to determine the specificity of the conjugated antiserum, and all gave a bright fluorescence of somatic and flagellar sites. Additionally, strains of Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paracolobactrum, Proteus, Providencia, Pseudomonas, and Shigella were tested. None of these organisms was positive with this antiserum. Our previous work established (10) that a salmonellae which fluoresced when grown in pure culture would fluoresce when grown in reconstituted NDM. The data in Table 1 indicate that the prepared samples which had been shown to contain approximately one viable Salmonella cell per 50 to 100 g with trypsin-treated NDM assayed by conventional methods were also positive by this method. This is further amplified in Table 2 which also shows that with salmonellae known to be H2S-negative the medium was still a good enrichment, and the fluorescent-antibody test would give positive identification. An explanation of the lack of blackening by S. paratyphi A (WMN), a known H2S producer, in this medium within that time is shown in Table 3. Novobiocin (5 gg/ml) restricted the growth of the two strains of S. paratyphi A as evidenced by the few cells observed on the fluorescent-antibody slides. Thus, the limited growth in 22 hr was probably insufficient to produce enough H2S to show a blackening even though it was sufficient to give a positive fluorescent-antibody test. The variability on response among strains of S. pullorum may have been caused by temperature-sensitive enzymes since Tittsler (13) showed that 30 C is optimum for H2S production of this strain. Neither S. paratyphi A nor S. pullorum has been reported in NDM. The medium provides a very satisfactory one-step enrichment procedure for subsequent fluorescent-antibody examination by providing Black + a Each sample was tested in four separate assays; the level of contamination in these samples ranged from 1 in 50 to 1 in 100 g. 'These samples were negative in 100-g amounts when examined by selenite enrichment without trypsinization. Our previous work (10) indicated that one Salmonella cell in as little as 10 g of nonfat dry milk may not be picked up because of entrapment in coagulated casein. cIn all of these positive slides, the number per field was too numerous to count. "next day" results on NDM samples. Its routine application as a quality control procedure on the production from NDM plants should be valuable in avoiding long holding periods while awaiting test results. Too numerous to count.

v3-fos

2020-12-10T09:04:12.273Z

1972-03-01T00:00:00.000Z

237232452

s2

Comparison of the Antigenicity of Phenol and Ethylenediaminetetraacetate Complexes Isolated from Cell Walls of Salmonella enteritidis Cell walls of Salmonella enteritidis were extracted with phenol and with ethylenediaminetetraacetate (EDTA). Phenol extracts were consistently more antigenic in chickens than EDTA extracts. Differences in the action of EDTA and phenol upon bacterial cell walls were demonstrated by the use of radioisotopes. topes. Ethylenediaminetetraacetate (EDTA) has been reported by several workers to remove portions of bacterial cell envelopes containing lipopolysaccharide (3,4,6). Prior to the use of EDTA, phenol was employed to extract this material (9). The antigenicity of phenol and EDTA extracts from Escherichia coli were compared, and the EDTA extract was more antigenic in mice (4). The present report will show that, in White Leghorn chickens, material isolated by phenol extraction of cell walls of Salmonella enteritidis is more antigenic than that isolated by EDTA. Through the use of radioisotopes, it is shown that differences in antigenicity are possibly related to differences in the modes of action of EDTA and phenol upon bacterial cell walls. Cells of S. enteritidis were inoculated in Trypticase soy broth and incubated for 24 hr at 37 C. They were then harvested by centrifugation and washed twice with physiological saline and distilled water. Two-gram (wet weight) samples of washed cells were suspended in 50 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.0, and sonically treated (Branson sonifier) for 3 min at 5 C. Sonic extracts were centrifuged at 3,640 x g for 20 min at 2 C to separate whole cells. Supernatant solutions were decanted and recentrifuged at 27 ,000 x g for 15 min, and the liquid was discarded. Cell wall pellets were suspended in 50 mg each of deoxyribonuclease and ribonuclease, incubated overnight at 10 C, and then centrifuged. This procedure is a modification of one described previously (2). The absence of significant amounts of cytoplasmic contamination was established by the absence of electrondense material in electron micrographs (1) and the failure to detect ribose in acid hydrolysates of cell walls (3). Cell walls (500 mg dry weight) were resuspended in 50 ml of 1.0 mm EDTA (tetrasodium dihydrate) at 22 C for 15 min. The suspension was then centrifuged, and the supernatant solution was decanted, dialyzed against several changes of distilled water at 4 C for 3 days, and lyophilized. An identical procedure was used to prepare the phenol extract, except that cell walls were resuspended in 50 ml of 90% (v/v) phenol at 65 C for 15 min (9). The isolated extracts were each dissolved in physiological saline (10 mg/ml). Two milliliters of the phenol extract was injected intravenously into each of six adult female White Leghorn chickens. A different group of six chickens was inoculated in an identical manner with the EDTA extract. Procedures for collection of blood and measurement of antibody titer by tube agglutination have been outlined previously (8). Antigen concentrations used in titrations were 200 pg (dry weight) of either the EDTA or phenol extract per ml of distilled water. Basically, the procedure for titration was as follows. Antigen (0.5 ml) was added to tubes containing 0.5 ml of physiological saline plus serial dilutions of serum. The contents of each tube were thoroughly mixed and incubated for 24 hr at 37 C. Positive reactions were indicated by a granular-type agglutination. Zinc is one of the mineral constituents (1) in gram-negative bacteria, and aspartic acid is one of the major amino acids (7) in cell walls of Salmonella. Extracting isolated cell walls labeled with radioisotopic markers of these components should provide insight into the modes of action of EDTA and phenol and help to explain observed differences in the antigenicity of these extracts. Accordingly, cells were inoculated; 4 hr later, 3 ml of either 65ZnCl2 or aspartic-1-14C acid (150 ,gCi/ml, New England Nuclear Corp.) was added to each 250 ml of broth, and the cultures were incubated. The cells were then harvested, and labeled cell walls were isolated and extracted with either phenol or EDTA. Radioactivity was measured for duplicate 1-min intervals, and the percentage lost was calculated by subtraction from controls. Table 1 indicates that in chickens the phenol extract produced a consistently higher antibody titer than did the EDTA extract. The differences in titer remained significant throughout the 8-week period of testing. Similar results have been reported in a previous study (5). When cell walls of Pseudomonas aeruginosa were treated with EDTA, the antigenicity of the isolated lipopolysaccharide was lowered when injected in mice. Perhaps the basis for differences in antigenicity of phenol and EDTA extracts lies in their modes of action upon bacterial cell walls. Approximately two-thirds of the 6"Zn2+ and '4C-aspartic acid was removed from labeled cell walls by extraction with EDTA (Table 2). However, less than 10% of the radioactivity was removed by phenol extraction. In both extractions, the radioactivity was nondialyzable. In a previous study (4), differences were found in the composition of lipopolysaccharide fractions isolated by phenol and EDTA from cells of E. coli. It was reported in that study that EDTA extracts were more antigenic and at least as lethal in mice. In the present study, it appears that in chickens differences in antigenicity of phenol and EDTA extracts may be related to the mode of action of these compounds upon bacterial cell walls, as demonstrated by differences in their ability to extract radioactive components from the walls.

v3-fos

2018-12-06T01:56:52.471Z

1972-11-01T00:00:00.000Z

55828456

s2

Influence of Food Distribution and Human Disturbance on the Reproductive Success of Herring Gulls . A three year study of Herring Gull (Larus argentatus) reproductive success on four i~lands in Maine indicate.d that production of young was controlled by different factors operating on the eggs and chicks. Hatching success was inversely related to the disturbance of co.Ionics by pic~i~kers, which apparently caused the adults to leave their eggs exposed 10 sufficient solar rad1a11on to addle the eggs. The survival of chicks was lower on islands distant ~rom sources of edible r~~use (ou.ter. islands) than o.n. islands close to sources of waste (inner islands), regardless of visits by picnickers. The nutrition and growth rates of chicks on inner and outer islands were similar. The attendance of parents on the territories was found to be less o~ an out~r islan~ than on an inner island. It is concluded that differences in parental behavior associated with greater foraging effort were responsible for a higher loss of chicks to predation on the outer islands. I NTRODUCTION Populations of several species of gulls (Larus spp.) in different parts of the world have increased greatly during the twentieth century (Fordham 1967, Kadlec & Drury 1968, Harris 1970, Spaans 1970). These increases have been attributed to a combination of two factors: 1) passage of laws that limit hunting and egg collecting; and 2) increased food resources in the form of edible wastes supplied by a prosperous and expanding human population. Several studies have sought to demonstrate a relationship between the availability of edible refuse and increases in the gull populations (Spark 1950, Drury 1963aand b, Harris 1965, Ingolfson 1967, Spaans 1970. These authors have shown that gulls gather a large percentage of their food from man throughout the year. However, without data on the carrying-capacity of the natural feeding areas, it is difficult to show that the use of man's waste is required for either the support of the large gull populations or their present rate of increase. The early work of Drury (pers. com.) and of Kadlec and Drury ( 1968 ) suggested that the influence of edible refuse on the reproductive success of H erring Gulls, Larus argentatus, could be demonstrated by comparing the productivity of colonies located at various distances from sources of waste. In this study I have examined this relationship by comparing four breeding colonies of Herring Gulls in Maine. Data were gathered on the productivity of the colonies, the foods brought to the chicks, the growth rates of the young and the parental care given by the adults. STUDY AREA The study was conducted in Penobscot Bay, Knox County, Maine (F ig. 1) during the summers of 1968, 1969, and 1970. The area contained a small number of localized sources of man's waste and a number of gull colonies on islands. Three species of gulls nested in the area. The Herring Gull, Larus argentatus, was the commonest species and comprised 85-90% of the gull population. The Great Black-backed Gull, L. marinus, made up about 8-13 % . The Laughing Gull, L. atricil/a, once common, represented less than two per cent of the gulls in the area at the time of my study. Colonies of Herring Gulls were studied on Goose Rock, Little Green Island, Flat Island and Sloop Island (which was not available for study in 1968). Examination of these colonies provided information about two of the possible factors affecting the reproductive success of gulls, disturbance and distance from foraging areas. Disturbance was measured subjectively on the basis of the number of old fireplaces, beer cans and picnic groups I encountered. Flat Island was a favorite picnicking area for local people, and several groups were encountered during my visits to the island. Sloop was also used by picnickers, but less freq uently than Flat. Goose Rock and Little Green Island were not suitable picnic areas and neither island was visited by picnickers during my study. The distances to sources of man's waste were greatest from Sloop Island and Little Green Island (outer islands), while Flat Island and Goose Rock (inner islands) were relatively close to prime foraging areas (Table 1). Short descriptions of the islands are presented in H unt ( 1970). Natural foraging areas were abundant throughout the study area. Dumps in Camden and Belfast, fish processing plants in Rockland, and chicken processing plants in Belfast (F ig. l) were important sources of food because large quantities of refuse were consistently available to the gulls. Lobster fishing also provided food for the gulls, particularly those nesting on the outer islands. Whenever traps were hauled, the old bait was thrown overboard. Throughout this paper the terms " refuse" and " waste" are used interchangeably. They refer to all foods derived from man, including garbage, sewage and all fish made available to gulls by man's activities. Productivity Nests in each colony studied were marked with a numbered stake. The eggs present in each nest were counted after the clutches were complete. Newly hatched chicks were banded, weighed, and their nest of origin recorded. A chick was banded if it was found on an unnumbered nest and the number of eggs and siblings on the nest was recorded. In these cases the nest was assigned a number and was included in the general statistics of the study. After the initial banding, chicks were recaptured at intervals of two days to more than a week, depending on weather conditions governing travel to the islands and on my success in finding the birds. On each oc- casion the chick was weighed before and after the removal of any food in the foregut. T his procedure was repeated until the chicks could no longer be captured. The number of young surviving per nest was used as a measure of relative reproductive success, hereafter called "productivity." Since my disturbance of the colonies probably caused a lowering of productivity, my measures only provide estimates of the relative breeding success on the islands studied. Because the exact age of many chicks was not known, survivorship curves and productivity were calculated on the basis of the birds living to a given weight. Herring Gull chicks, which weigh approximately 60-70 g at hatching, were considered to have survived the chick stage when they attained a weight of 500 g. In my experience, weight is a better indicator than age of a chick's physical condition and ability to defend itself. It was not practical to follow the survivorship of chicks weighing more than 500 g. Chicks of 600 grams or more wander widely from their nests and become difficult to find. In addition, in two of the three years it was impossible to visit several of the islands late enough in the season to capture all birds attaining a greater weight. Foods Two methods were used to determine the food resources utilized by the different colonies. The first involved marking adult gulls with identifying colors at their nests and then making periodic surveys of feeding areas (see Weaver andKadlec 1970, andHunt 1970, for methods of trapping and marking). The second method was an analysis of the foods brought to the chicks. Stomach contents of young gulls were obtained in a manner which allowed repeated sampling of the same chick. By inserting my index finger down the throat of the chick and hooking it behind the contents of the proventriculus, it was possible to remove all the food in the gull's foregut. Voluntary regurgitations appeared to give a biased sample. Not all the birds regurgitated when handled, often only part of the crop contents were released when regurgitation occurred, and certain soft items such ·as fish or earthworms were given up more readily than others. T he identification of most food items was straightforward. Whether fish in a sample had been obtained from man or from natural foraging areas was surmised by its size, species and condition. Fish of a species and size taken by commercial fishermen which were cut up, decomposed or lacking scales, were judged to have originated from man (see Hunt 1970, for details). Food usage was determined by gravimetric and numeric measures of samples collected at different times of day and heights of tide. Both measures were affected by the presence of long-lasting hard parts which may have been obtained long before the sample was collected. These create a bias in the numeric analysis. Hard parts pose less of a problem to the gravimetric determination as they usually represented only trace amounts. T he nutritive value of the foods was investigated to determine whether the diets on the different islands were equivalent. Jn order to compare the caloric values of the diets in the different colonies, the average number of calories per 100 grams of food was calculated. T his comparison is crude at best, but it is based on the known caloric values of 78-96% of the chicks' diets on the different islands. A comparison of the relative protein content of the diets was made by a similar method. T he caloric and protein values were obtained from the H andbook of Biological Data (Spector 1956), the Agricultural Handbook (Watt and Merrill 1950), and through analysis of samples obtained in the field, .performed by Herbert V. Schuster, Inc., of Boston, Massachusetts, using th!! met· hods of the Association of Official Agricultural Chemists ( 1965). Growth rates Data for the comparison of growth rates were gathered in 1968 and 1969 by weighing chicks after all food in the upper digestive tract had been removed. Growth rates were computed using all chicks of known age from which at least two weights between 125 and 600 g had been obtained. When possible the age of a chick was determined from the observation of hatching. In some cases where hatching was not observed, it was necessary to assume that a chick weighing less than or equal to 70 g was in its first day after hatching. Age was computed from the date of this initial weighing. In order to facilitate statistical comparison of the growth rates, the slope of the straight-line portion of the growth curve between age 5 days and age 25 days (Spaans, 1970) was determined by using a simple linear regression program. GEORGE L. HUNT, J R. Ecology,Vol. 53,No. 6 Parental care Jn 1969 I observed nest attendance by adult gulls on Little Green Island and Goose Rock. On each island about fifteen nests were selected which were visible from a suitable observation point. One adult at each nest was trapped and color-marked with a five per cent solution of silver nitrate. T his colormarking made it easy to distinguish between members of a pair, and therefore a relatively simple matter to record attendance of each at the territory. Observation periods ranged from two to thirteen hours 1 per day on 17 days. Data for comparison of parental care on the two islands were taken at 15 minute intervals, when each bird in a pair was scored as either present or absent. Times of arrival or departure, fights, and the feeding of chicks were recorded when observed between the fixed periods of observation. The length and number of absences of a foraging parent, the percentage of time that a nest or territory had no parent guarding it, and the percentage of time that both parents were present were compiled from these data. Productivity Jn 1970 field studies were stopped by 17 July. Although all living chicks on Sloop and all but one on Flat Island had attained a weight of 500 g by my last visit, 21 of 60 living chicks on Little Green and 15 of 62 chicks on Goose Rock weighed less than 500 g. While the existence of the underweight chicks on Goose Rock creates bias against the hypothesis that proximity of refuse dumps increases reproductive success, those on Little Green Island bias the results in favor of this hypothesis. In order to eliminate bias in favor of this hypothesis, I have assumed, in all calculations involving chick survival and productivity for 1970, that chicks weighing 400 or more g on Little Green Island during my last visit survived to a weight of 500 g. Jn order to avoid any chance of biasing the results in favor of the above hypothesis, the criterion for survival remained at 500 g for chicks on Goose Rock. In all three years Herring Gulls nesting on Goose Rock raised significantly (X.2 test; p < .05) more chicks per pair than those on the other islands (Table 2). However, there were no statistically significant differences in productivity between the other three islands. Throughout the study Herring Gulls nesting on Goose Rock and Little Green Island enjoyed a significantly greater hatching success (X.2 test; p ~ .05) than those on either Flat Island or Sloop Island ( Table 2). The data indicate that hatching success was consistently greater on Goose Rock than on Little Green Island, but only in 1968 was this differ- Although the differences between colonies in the ability of parents to raise chicks (Table 2 and Figure 2) were not always statistically significant within each year, the differences between inner and outer colonies were consistent over the three years they were studied (p < .001 , by Sign Test, Siegel 1956 The differences in rates of mortality between chicks 1>n inner and outer islands were distributed over the entire growth period in all years (Fig. 3). These were statistically significant in 1969 and 1970 (using the Wilcoxin Matched-Pairs test, Siegel, 1956;p > .05, 1968;p < .005, 1969;p < .005, 1970). Chick mortality was greatest on all islands during the first few days after hatching (Figs. 2 and 3). This initial mortality was consistently greater on the outer islands. Foods The distribution of color-marked gulls, compiled from public reports and my own observations, showed that gulls from all colonies used refuse and that they generally restricted their foraging activities to s0urces of waste closest to their colonies (Table 1). Birds from Goose Rock fed primarily at the Camden dump, those from Flat Island at the Belfast dump and chicken processing plants, and those from little Green in Rockland harbor. Very few colored gulls from Sloop Island were seen foraging, although observations suggest that they used the same sources of refuse as the gulls on the other colonies. Although birds from several islands used the Camden dump and Rockland harbor, in general the birds from different colonies appeared not to overlap greatly in the foraging areas they used. Foods sampled from Herring Gull chicks are summarized in Fig. 4. Table 3 shows that there were no clear-cut differences in the total amounts of refuse fed to chicks on inner and outer islands, although the kinds of waste used on the islands differed (Fig. 4). No consistent variation in food types taken with change in date was apparent. This was true for individual colonies as well as for the study area as a whole. Qualitative aspects of the diets of chicks in each of the colonies are presented in Tables 4 and 5. The nutritive value of the diets was similar on. all islands. Indices of the quantity of food provided to the chicks were obtained from the percentage of times that sampled chicks were found empty and the average weight of the samples obtained relative to the weight of the chicks sampled (T able 6). Within any given year there was no indication that chicks on outer islands were either consistently empty more often , or produced lighter food samp!es than chicks on the inner islands. Growth rates When the growth rates of surviving and non-surviving chicks were considered together, the only significant difference between colonies occurred in 1969 when chicks on Sloop Island showed lower growth rates than those on other islands (Table 7). Growth rates of surviving chicks were significantly greater than those of non-surviving chicks (Fig. 5) on all but Sloop Island where the difference between the two groups was not significant. Growth rates of survi ving chicks on Goose Rock were significantly higher than those of surviving chicks on all other islands in l 968 Parental care Parents on Little Green Island left their mates on the territory alone for longer periods than did birds on Goose Rock during both the egg and chick stages. The percentage distribution of absences of a given minimum length is shown in gulls which were incubating eggs than for parents with chicks (Table 8). It appeared that the length of absence during the incubation phase was less critical than it was during the period after the chicks had hatched. Even on Little Green Island, where absences of the foraging mate were longest, seldom did the incubating parent leave the nest completely unattended (Table 9). Absences of the guarding partner would result in the eggs or the chicks being undefended. The percentage of time in which eggs were left undefended was small on both islands and the differences between the behavior of the parents during the incubation period on the two islands is not significant (0.5 > p > 0.25). However, the difference in behavior between birds with chicks is significant (using a 2 x 2 contingency test, "f. 2 = 56.36, p < .0025). The longer absences of the foraging parents with chicks on Little Green as contrasted with those on Goose Rock were important. As the length of absence increased, the adult left to guard the young became restless. Frequent solicitation of food by the young appeared to exhaust the guarding parent's supply. Further begging of food appeared to annoy the parent and it would often seek to avoid the chick. During prolonged absences, the guarding parent often left the territory, thereby leaving the chicks undefended. As can be seen from Figure 6. this became increasingly common on Little Green Island as the chicks grew larger, while the percentage of time that chicks were unguarded on Goose Rock remained more or less constant. On Goose Rock, I was not able to see where the guarding parents which left Another measure of parental attendance is the average number of parents present on the territory at any one time. During incubation the average number of parents present was greater on Goose Rock ( 1.44) than on Little Green Island ( 1.18). This difference held throughout the period when chicks were present ( 1.3 3 vs. 0.91). Using a 2 X 2 contingency test the difference in the average numbers of parents present was found to be statistically significant (p < .01). Figure 7 shows that as the season progressed, the average · number of parents guarding their chicks declined to less than one on Little Green while remaining relatively constant on Goose Rock. On several occasions the presence of the second parent seemed to be important. I once observed a chick taken from its territory by an intruding adult gull while the chick's parent was defending a second chick elsewhere in the territory. On several occasions I observed one parent defending the young while its mate was fighting with another gull. DISCUSSION When a colony is disturbed, the adults leave the eggs unprotected. Gull eggs are sensitive to overheating (Drent 1967) and even in the relatively cool Maine spring, incident radiation from the sun is capable of overheating them. Furthermore, the eggs are exposed to predation by other gulls flying in the vicinity. Hatching success was greatly affected by disturbance of the colony, while it was unaffected by the distance of the colony from major sources of food. Thus, studies attempting to relate reproductive success of gulls to the availability of food must take into account the effect of disturbance on hatching success. The higher survival rates of chicks on the inner islands does not appear to be related to such features as nest-spacing, vegetation and cover, or the roughness of the terrain, which have been studied by others (Patterson 1965;Tinbergen et al. 1967;Brown 1967;I. Nisbet, pers. comm.). Coulson et al. (1969) and Parsons (1970Parsons ( , 1971) have identified egg size as an important factor in chick survival. No measures of egg size were made in the present study, and while it is unlikely that egg size varied between inner and outer islands, this requires further investigation. Likewise, differential exposure to harsh weather is not an adequate explanation for the difference in chick survival. Within the geographic distances with which I was working, exposure of chicks to occasional severe storms was determined more by the cover available in the colony than by the location of the island. On Goose Rock, with its lack of either vegetation or a drift zone, chicks were more exposed to rain and harsh weather than on the outer islands. Furthermore, severe storms were too infrequent to account for the higher mortality (Figure 3) on the outer islands throughout the growth period in 1969 and 1970. The date of egg laying or chick hatching has been found useful in several studies in predicting the survival of young gulls (Paynter 1949, Brown 1967, Harris 1969a, Parsons 1971. Perrins 1970 gave a general discussion). In the present study the timing of reproductive effort was not an important variable. Chicks hatched within any given time interval on the outer islands had lower survival rates than those on the inner islands. The concentration of foraging activity by gulls in the vicinity of their own colony was clear not only from the sightings of color-marked gulls, but also on the basis of the types of waste fed to their chicks. This result agrees with that of Drury and Nisbet (in press) who found that the foraging areas of Herring Gull colonies in Massachusetts were segregated on the basis of the proximity of a resource to a colony. On the average, food samples containing was-te were heavier than those containing natural foods (compare per cent waste by weight with per cent waste by occurrence, Table 3). This relationship was particularly evident on Little Green Island where the major sources of refuse were the fish processing plants in Rockland. In order to bring back larger amounts of food to their chicks, it was apparently more efficient for gulls to fly greater distances to sources of waste than it was to forage in natural areas closer to the colony. There were no consistent changes in the use of man's waste as the summer progressed. This result contrasts with that of Spaans (1970), who found in Holland that as Herring Gull chicks grew larger and demanded greate~ quantities of food, the adults depended more heavily on sources of refuse for feeding their young. The similarity in the quality of the diets and the quantity of food provided on the various islands suggests that nutrition was not responsible for the differences in survival rates between colonies (Tables 5 and 6). Since the diet of the chicks on each of the islands was varied and contained a number of different natural foods as well as waste (Figure 4) , it is doubtful that any of the young birds were lacking an important vitamin or mineral. The comparison of the combined growth rates of all chicks in each colony reflected the similar nutrition of chicks on all islands. Although surviving chicks on Goose Rock grew more rapidly than chicks on other islands, there was no correlation between growth rates in a colony and the percentage of chicks on that island which survived. The difference in growth rates between chicks which survived and those which did not most likely reflects an increased vulnerability of underweight chicks to disease, predation or chilling (Kadlec et al. 1969). The length of absence of a foraging parent appeared to be more important as a factor exposing the chicks to predation than as a factor affecting the nutrition of the young. Although it might be implied from the longer absences and less frequent returns of foraging parents from Little Green Island (Table 8) that those gulls brought food to their chicks less often than parents on Goose Rock, young gulls on Little Green were just as well fed as Goose Rock chicks (Table 6). This appears to be a paradox unless one assumes that not all of the periods of absence of parent gulls from Goose Rock were devoted to foraging, and that their chicks were fed no more often than those on Little Green Island. The fact that chicks on Goose Rock occasionally exhausted the food available from a parent suggests that parents may not have done as good a job of feeding their young as might have been expected from their many brief absences from the colony. The parent gulls must provide not only sufficient food, but also adequate protection for their young, both from bad weather (Vermeer 1963, Harris 1964, Harris and Plumb 1965 and from predation. There is a trade-off between the amount of time adults remain at the colony guarding their chicks and the time they spend foraging (see Perrins, cited in Brown 1967). The time difference between the average lengths of absence of parent gulls on Little Green and Goose Rock was about one hour (Table 8), which approximates the commuting time expected of a gull flying at 40 km per hour (Pennycuick, 1969) over the additional distance that gulls from Little Green must cover to reach mainland food sources. The minor differences in nest attendance on the two islands during the incubation period were not important since hatching success was similar on both islands in 1969 (Table 2). However, the effectiveness of parental protection as measured by attendance on the territory was very different on the two islands once the chicks had hatched. Predation in the colonies I studied appeared to be an important source of chick mortality. Although a few very small chicks were found dead in or next to Ecology,Vol. 53,No. 6 their nests, indicating a failure of their parents to make proper behavioral adjustments, many chicks disappeared without a trace after a few days of growth and are presumed to have been eaten by adult gulls in the colony (see also Harris 1964, Brown 1967, Kadlec et al. 1969. These findings agree with those of Paynter ( 1949), Fordham ( 1964) , Kadlec and Drury ( 1968) 1 a nd Parsons ( 1971). During this period of maximum vulnerability the presence of only one parent guarding the small chicks left broods on Little Green Island more exposed to predation than if two parents had been present. It was observed on Little Green that parents whose food reserves had been depleted by the begging of their growing chicks would leave their territories to forage in the intertidal zone. Casual inspection of the littoral zone of Little Green Island revealed that few food items remained in areas exposed to foraging gulls. This suggests that chicks there were more exposed to predation not only because parents may have guarded them less well, but also because there were more adults seeking food on or near the island. Although [ have only once observed a successful attempt at cannibalism, on many occasions [ have observed chicks attacked by other gulls. Vermeer (1963), H arris (1964), Brown (1967) and Parsons ( 1971) have all concluded that predation and cannibalism are major causes of chick mortality. Lack ( 1968) has suggested that the inshore feeding seabirds will have many small colonies so as to minimize the distance between nest site and feeding area. The mechanism whereby this becomes important is clear from the results of this study. However, it is less clear why the Herring Gulls remain colonial when island nest sites are plentiful and the only predators of consequence appear to be the gulls in the colony. Although it has been shown that the reproductive success of many species of seabirds is limited by the amount of food parents are able to provide their young (Murphy 1936, Ashmole 1963, Harris 1969b), gulls appear to be an exception. Studies of gull productivity in single colonies (Paynter 1949, Vermeer 1963, Harris 1964, Harris and Plumb 1965, Brown 1967, and Spaans (1970) provided no evidence for food limitation of reproductive success. Furthermore, both Vermeer (1963) andH arris andPlumb (1965) have demonstrated that two species of gulls were capable of raising broods of more than three chicks. The differences in the survival of chicks on inner and outer islands, and the year to year fluctuations in reproductive success found by Fordham (1970), demonstrate that the availability of refuse may affect the reproductive output of gulls. The evidence that reproductive success is sensitive to the availability of food, and the conclusion that gull reproduction is not currently limited by the ability of the adults to provide their young with sufficient food, are not nee-essarily mutually exclusive. If predation and the lack of parental care were the proximate cause of the differentia l mortality between colonies on inner and outer islands, then the availability of food must be seen as the ultimate cause of this difference in chick survival. When the parents have to spend large amounts of time seeking food, their ability to provide protection to their young is reduced.

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Enterococci in Insects Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 103 to 3 � 107/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution. by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, fcllowing the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution. The literature provides evidence that the enterococci occur randomly among some genera and species of insects, and with some degree of constancy among others. In some studies, the enterococci are clearly identifiable as Streptococcus faecalis or as Streptococcus faecium, intermediate forms (23), and possibly as Streptococcus faecium var. casseliflavus (10; Cosenza, B., and A. E. Girard, Bacteriol. Proc., p. 45, 1970). They may be recovered from various parts of the insect body (16). Geldreich et al. (18) have obtained them in high numbers from members of the orders Diptera and Coleoptera. They are abundant in the intestinal tracts of the wax moth and the hornworm (5,6), and transmissible to succeeding generations via the eggs (4,8). They have been recovered from ichneumonid parasitoids (7), larvae and pupae of moths and pine sawflies (9,34), flies and roaches (19), muscoid flies (30), desert locusts (35), stable flies (20), from Anopholes but not two other genera of mosquitoes (21), and yellow mealworms (22,37). They have been incriminated as possible agents of insect diseases (12, 14, 31, 32; Cosenza and Gerard, see above). Their use as an insecticidal aerosol has been proposed (13). Some studies have been concerned with the insects in intimate association with human and warm-blooded animal life (19,20,30), and others have dealt with insects which are primarily plant feeders (9,22,35,37). It has been suggested (25) that insects play a role in the seasonal introduction of enterococci to wild and cultivated plants. This study was undertaken to determine more precisely, through enumeration, speciation, and studies of distribution among insects, their role both in dissemination and as an overwintering agent for the bacteria. MATERIALS AND METHODS Collection of insects. All insects were taken from agronomic and wild areas to avoid fields harboring domesticated animal life and human habitation. Some insects were in association with particular habitats, as Anthonomus grandis on cotton plants and Epicauta sp. on soy bean plants. The majority of insect specimens were taken in any convenient manner, including the use of nets, bottles, and forceps, and they were placed immediately into sterile glass containers. All specimens either were cultured promptly upon return to the laboratory or were frozen at -21 C for periods of 2 to 7 days. A total of 403 individuals representing 36 genera of insects was cultured. Culture. Each of the larger insects was homogenized singly in a 15-ml tissue homogenizer in a volume of water equal to 10 times the weight of the insect. Several individuals of the smaller insects were homogenized as one sample to obtain the necessary volume for quantitation of fluid for culture. From the homogenates, serial dilutions were made in azidedextrose (AD) broth and plated on KF agar by the -surface spreading technique. Plates of MMRS agar (36) were also streaked from tubes of AD broth with growth at the highest dilution. Colonies were selected from both plate media to the medium of Mundt and Johnson (27), and cultures were maintained in this medium for routine culture and storage. Identification of cultures. Cultural conditions and criteria employed for identification of the cultures have been described earlier (26,27). All sugar fermentations and ability to grow at 10 C and at 45 C, in 6.5% NaCl broth, and at pH 9.6 were determined in phenol-red meat-extract broth base (PR). Additional media were bile-esculin agar (BE, reference 18) upon which enterococci and but few other bacteria produce growth with blackening (16), and malic acid medium (36), in which S. faecalis and its variants are usually anaerogenic in the presence of glucose, but S. faecium and its variants are usually not. The ability to decarboxylate tyrosine was determined with the basal medium of Deibel, Lake, and Niven (11) to which brom-cresol-purple and 3% gelatin were added; the medium was heated and then tubed in 6-ml quantities in screw-capped tubes. Incubation of the medium through 6 days was practiced because some enterococci decarboxylate tyrosine very slowly with the late restoration of the alkaline color of the indicator. S. faecium var. casseliflavus was recognized by pigment formation on streaked plates of 4% sucrose-Trypticase soy agar (Baltimore Biological Laboratory) after incubation for 4 days. Except as noted, all media were incubated at 37 C. RESULTS Distribution of enterococci. Enterococci were recovered from 53% of 403 cultural assays ( Table 1). The greater number of individuals, representing the orders Coleoptera, Hymenoptera, and Lepidoptera, as compared with numbers in the remaining orders, indicate the relative frequency in occurrence of members of the various orders in the environments at the time of sampling. Enterococci were present in all specimens of the adults and pupae of Camponotus spp. taken from forest litter, of Carabidae taken from forest soils, and of Chrysobothris, Epicauta, and adult and larval forms of Phyllophaga taken from flowering agronomic plants. They were present in 40 to 50% of the specimens of Apis and Bombus spp. taken from flowers, Monophadnoides geniculatus taken from raspberry canes, members of the taxa Diabrotica and Coccinellidae, and the majority of Chauliognathus sp. The weevils, Anthonomus grandis and Odontopus calceatus, and the wasp, Polistes, were devoid of enterococci. Except for the leaf-feeding Hyphantria cunea, 85 and 100% of the larval forms of Lepidoptera yielded enterococci. These were taken from green and also overwintering corn plants. The succulent floral structures of the corn plant frequently bear enterococci in high numbers (23). Enterococci were recovered from adults of Papilio and Pieris, but not from three specimens of related Nymphalidae. Members of the families Acridiidae and Gryllidae of the order Orthoptera associated with grassy areas yielded enterococci, but the few specimens of Microcentrum and Stagmomantis did not. Two of the four specimens of the order Hemiptera and none of the 75 specimens in the order Diptera, Homoptera, and Isoptera yielded the bacteria on culture. Seasonal fluctuation in populations and persistence in dormancy. Representatives of 11 of the 37 taxa of insects listed in Table 1 were encountered during more than one collection period: these are shown in Table 2, together with the month of sampling and other pertinent data. Total numbers of enterococci per gram of insect weight increased with increasing seasonal temperature. This is consonant with earlier observations made with plants. The value of the two retrogressions may be questioned, because one specimen of each was cultured. Enterococci were present in 90% of specimens of insects taken during the dormant season in January, in numbers to 3.9 x 107/g of body weight. Attempts to reproduce survival during dormancy by frozen storage of insects taken during the late summer were unsuccessful; specimens of M. geniculatus and Z. grandiosella taken in the field during January harbored enterococci, whereas those placed into storage did not. Distribution of enterococcal species. Little consistency is seen in the occurrence of S. faecalis, S. faecium, and S. faecium var. casseliflavus within any species of insect (Table 3). Each taxon may occur singly or together with either of the other two or with other spherical bacteria as Aerococcus, Leuconostoc, and Pediococcus, and presence of any taxon may vary with the time of sampling. Of the 37 taxa of insects from which enterococci were obtained, S. faecalis was recovered from Properties of the enterococci. S. faecalis and S. faecium var. casseliflavus exhibit a high percent conformity to the properties established for the enterococci by Sherman et al. (33), to growth in ethyl violet (EV) broth, and in typical pigmentation on BE and tellurite agars and agar containing 1,3, 5-triphenyltetrazolium chloride agar (TTC, reference 1). The small percent nonconformity may be within the limits of deviation for a species noted by Dei-bel (10). The heterogeneity noted earlier among S. faecium obtained from plants (10,28) exists also among cultures obtained from insects. The colonies of the pigmented variant on TTC agar are neither red nor white as described by Barnes (1), but pink, and are recorded as negative. Many cultures of S. faecium failing to grow in EV broth are sensitive to the sodium azide as determined by inoculation of PRglucose broth with and without sodium azide. All S. faecalis taken from insects are anaerogenic in malate-glucose broth, as are 20% of S. faecium and 9% of cultures of the pigmented variant. S. faecium is variable and S. faecium var. casseliflavus were usually negative in the ability to decarboxylate tyrosine, but the majority of isolates of S. faecalis responded typically. Either a transient reduction of litmus in VOL. 24,1972 Sept. Oct. April Sept. June July Jan. April Sept. Sept. Jan. Among the 314 cultures of S. faecalis, the general pattern is the fermentation of melezitose but not of melibiose. Those cultures fermenting melibiose also ferment melezitose. Among 176 cultures of S. faecium, 88 or 50% ferment both sugars, 41% ferment melibiose but not melezitose, and the remainder ferment neither sugar. More than 99% of 148 cultures of the pigmented variant tested ferment melibiose, 3% ferment both sugars, and a few cultures ferment neither. The differences from results obtained by Orla-Jensen (29) and Deibel et al. (11) are a probable reflection in differences in origins of the cultures studied. DISCUSSION The results of this study suggest that insects play a role in the distribution of enterococci within the plant world during the growing seasons of the year. Absent from plants during winter and early spring (25), enterococci appear upon these with new outgrowth and flowering, and are recoverable with increasing frequency with the advance of the season. The cycle corresponds with the extent of insect activity in nature. The apparent randomness in the distribution of the taxa of the enterococci also bears a similarity to that on plants and animals in a wild environment (23)(24)(25). Insects have the potential for being overwintering agents of the enterococci. The enterococci, however, also are constant residents of wild, nonlactating as well as lactating animals, including Mammalia, Amphibia, and Reptilia (24). In selected environments a logical visualization is the reseeding of plants via insects which are both coprophagous and floriphagous and further spreading by insects which are primarily nectarophagous, with reproduction of the enterococci on plants to the extent that environmental conditions permit. Even among insects the life pattern may determine the nature of the intestinal flora. Except for Reticulotermes sp., insects feeding on nectarine foods or dwelling in the humid environment of soil or in deep ground cover harbored enterococci in this study, whereas members of Odontopus, Hyphantria, Aphididae, and Cicadellidae, which feed and dwell on less succulent leaves and stems did not. The prominent feature of S. faecalis taken from agronomic and wild plants and from wild animals (24,25) during earlier studies and from insects is the RSCD reaction in litmus milk. The curd is rennet-like, soft, and flowing, except for a very few cultures not fermenting lactose which produce a grainy curd prior to digestion. The pH of the milk during 1 to 4 days of incubation with several hundred cultures was approximately pH 4.6 to 6.6. Digestion proceeds in stratiform fashion from the top downward terminating in a small, plastic residue reminiscent of sweet-curdling digestion. The reaction is unknown or rare among cultures of human origin (26) and cultures from human sources which we have studied. Therefore, cultures of S. faecalis exhibiting this reaction appear to be part of the natural microflora of nature. The reaction in litmus milk then becomes an excellent marker in analytical laboratories to distinguish between S. faecalis introduced to materials in the remote past by methods characteristic to nature and S. faecalis of recent and human origin.

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1972-06-01T00:00:00.000Z

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Loss of Lactose Metabolism in Lactic Streptococci Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C2F. 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 C2F 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 C2F 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 illustrates the effect of AF treatment on other strains of lactic streptococci. The appearance of lac mutants was increased 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, 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.

v3-fos

2020-12-10T09:02:49.770Z

1972-10-01T00:00:00.000Z

237234940

s2

2,2-Dibromo-3-Nitrilopropionamide, a Compound with Slimicidal Activity Laboratory and field tests demonstrated that 2,2-dibromo-3-nitrilopropionamide was an effective slimicide for use in papermaking systems and cooling towers. It was also effective as a bactericide for soluble oil emulsions. Acute toxicity tests showed that its hydrolysis at pH 9 and 23 C yielded products that were relatively nonhazardous to fathead minnows. MATERIALS AND METHODS DBNPA was prepared by the method described by Hesse (3). The white product which resulted from bromination in an aqueous medium was recrystallized from benzene to give a compound with a melting point of 125 C. Purity was checked by elemental analysis, infrared analysis [IR (Nujol mull), 1,710 cm'l (C=O) l, and by nuclear magnetic resonance spectroscopy [NMR (dimethyl sulfoxided ), 8.36 6 (doublet) J. Physical properties. Table 1 summarizes the solubility of DBNPA in solvents commonly used. Although this compound is relatively soluble in water, its rate of solution can be increased by wetting it with acetone or N, N-dimethyl formamide before addition to water. Chemical properties. The white, crystalline DBNPA has been stable for at least 4 years under laboratory storage conditions. This conclusion is based upon no detectable change in appearance or biological activity during this storage period. DBNPA dissolves in water to give a relatively stable solution in an acid pH range. Its unusual solubility and stability in polyethylene glycol (average molecular weight, 200) make this glycol a preferred solvent. Aqueous solutions hydrolyze under alkaline conditions, with the rate of decomposition increasing with the alkalinity. However, the rate of hydrolysis is not fast enough to interfere with the antimicrobial activity of fresh, alkaline (pH 7 to 9.5) solutions. Heat and ultraviolet and fluorescent light also cause aqueous solutions of DBNPA to degrade, as evidenced by the change of the antimicrobial end point as a given solution ages. This decomposition has also been substantiated by chemical analysis. Toxicological properties. Animal tests, carried out in our toxicological laboratory, have indicated that DBNPA is moderately toxic. The 50% lethal dose (LD5) value ranges from 118 mg/kg of body weight for female guinea pigs to 235 mg/kg for male Sherman rats. Eye-contact tests on laboratory animals indicate that DBNPA damaged the eye seriously enough to cause possible impairment of vision. A single, short skin exposure to DBNPA should result in no significant irritation. A single prolonged or frequently repeated skin exposure, however, may result in irritation, even a burn, depending on the severity of the exposure. Based on animal tests, this material is not likely to be absorbed through the skin in acutely toxic amounts (5). The acute fish toxicity of fresh and aged solutions of DBNPA has been determined for fathead minnows, Pimephales promelas Rafinesque, using dechlorinated Lake Huron water at 50 F and 72 hr of exposure (2). A fresh solution killed the minnows above 1 ug/ml, but parallel studies of solutions aged at pH 9 did not kill at the highest concentration tested, 100 ug/ml. In vitro microbial inhibition tests. Appropriate volumes of stock acetone solutions of the test compounds were added to tubes of melted, sterile nutrient agar (for bacteria) and malt-yeast extract-agar (for fungi) to give the desired final concentration. After being mixed into the media, individual samples were poured into sterile, disposable, polystyrene plates and allowed to harden. Duplicate plates were inoculated with bacteria and fungi in separate operations with an Accu-Drop (The Sylvania Co., Orange, N.J.) dispensing apparatus. Approximately 0.02 ml of each culture was simultaneously dispensed in uniform droplets on the agar surface. Broth cultures (48 hr) of bacteria were used. Fungal inocula were prepared by harvesting the spores from mature agar slants by washing with sterile water, followed by filtration through sterile gauze. Inoculated bacterial plates were incubated for a minimum of 72 hr at 30 C, whereas the fungal plates were incubated for at least 5 days at 30 C. (The plates were observed after 2 days for growth of Rhizopus nigricans, and, if growth had occurred, that portion of the agar was excised with a sterile spatula to prevent overgrowth of the entire plate.) Lack of visible growth at the end of these periods was recorded as inhibition of the organism at the concentration of compound under test. The organisms tested are listed in Table 2 and represent a spectrum of interest in industrial preservation. Inhibition of sulfate-reducing bacteria. Inhibition of Desulfovibrio desulfuricans was determined by the procedure recommended by the American Petroleum Institute (1). Growth of sulfate reducers in the bottles was indicated by an intense blackening of the medium. Slimicidal activity in simulated pulp suspensions. A test substrate of 0.5% ground wood pulp at pH 5.5 and at pH 8 was used to determine the slimicidal activity. The inoculum consisted of a pooled mixture of organisms implicated in causing paper mill slime. One-day-old broth cultures of Candida pelliculosa and Enterobacter aerogenes were used. In the case of Bacillus subtilis, the broth culture was 5 days old to allow for spore formation. Spores of the two fungi, Aspergillus terreus and Penicillium chrysogenum, were harvested with cotton swabs from well sporulated malt-yeast-agar slants, by using 10 ml of sterile saline per slant. The final inoculum was prepared by adding 1.0 ml from each of the above sources to 95 ml of physiological saline. A 1.0-ml inoculum of this mixture was added to 100 ml of pulp suspension to which the test compound had been added. After exposure periods of 3, 24, and 48 hr, portions were subcultured into suitable media to determine the biocidal concentration of the test compound. The subcultures were incubated at 30 C for at least 5 days. Slimicidal activity in headbox pulp suspensions. DBNPA was tested as the solid material (98% active) and as a solution with the solvent, polyethylene glycol, with average molecular weight of 200. Headbox stocks from two mills were used. One mill was producing a wide variety of paper used in a The pH of bacterial medium was 7.0 to 7.2, and that of the fungal medium was 5.0 to 5.5. printing, publishing, and other graphic arts. These stocks contained sizable percentages of ground wood fiber and additives, such as starch, that provided comparatively high nutrient levels accompanied by slime control problems. The second mill was producing mainly heavy paper and board grades on cylinder machines, but also was operating one small fourdrinier machine to manufacture corrugating board from kraft clippings. The kraft furnish gave an alkaline paper stock and was purposely selected to determine whether pH modified the results with the test compound. The test method used was similar to that described by King (4). Essentially, the test involves the use of in-mill headbox stock samples (minus any customary mill treatment) to determine the efficacy of experimental slimicidal additions. Volumes (100 ml) of treated stock in shake flasks, along with proper solvent controls, were kept at the mill headbox temperature peculiar to the sample. Efficacy of treatments was evaluated by appropriate bacterial counts after 3 and 24 hr of exposure. A 1-ml inoculum (or a suitable dilution) was spread over tryptone glucose agar (Difco) contained in a petri plate. The colonies were counted after incubation for 48 hr at 35 C. All samples were run in duplicate. Pertinent properties (Table 3) of the headbox stock samples were determined to make the performance of the test compounds more meaningful. In addition, after exposure, the samples were examined for odor, color, foaming, or pH changes that might have been caused by the slimicide. Slimicidal activity in paper mill trials. A solution of DBNPA was added at several points in the paper-making operation, as recommended by personnel at the particular mill. Slime control was determined by periodic plate counts and by inspection of the critical surfaces of the paper-making equipment, combined with the judgment of experienced operators. Slimicidal activity in cooling water trials. DBNPA was evaluated as a slimicide in a slug treatment for cooling tower water. The water circulated from a large, concrete holding pond with a capacity of 882,000 liteis. The makeup water (95 liters/min) came from a nearby river. Control bacterial counts were established for the pond water, and DBNPA was then added as a solid powder near the circulating pump. Subsequent bacterial counts gave an indication of the effect of DBNPA in the system. In a second field trial, DBNPA was tested with both continuous and intermittent addition. The cooling tower system had a capacity of 727,000 liters with a water makeup rate of 950 liters/min. The cooling tower was located in Louisiana, and the test was started in November, 1970, and continued through November, 1971. A polyglycol solution of DBNPA was added to the cooling tower basin with a metering pump. Slimicidal control was determined by periodic plate counts, by inspection of the cooling tower fill, and by feel for slime formation, as judged by an experienced operator. Soluble oil preservation. DBNPA was added to commercial oil formulations before dilution with water (1 part oil to 40 parts water, as recommended by the manufacturers). An inoculum was prepared by adding 10 ml of 24-hr broth cultures of E. aerogenes and Pseudomonas oleovorans (two organisms commonly cited in contaminated soluble oils) to 80 ml of the diluted oil emulsion under test. Control bacterial counts were in the order of 108 organisms per ml. The oil emulsions were inoculated (5.0 ml of inoculum per 95 ml of emulsion) and tested for viability after 24 hr of exposure by swabbing samples onto brain heart infusion agar with a cotton applicator. RESULTS In vitro inhibition tests. Table 2 summarizes the results of inhibitory tests using micro- Table 3 for control bacterial counts. organisms commonly encountered in industrial practice. DBNPA inhibited the microorganisms tested between 10 and 100 jtg/ml. Slimicidal activity in the presence of pulp. Table 4 shows that DBNPA had activity in the same range as two commercial slimicides that also contain bromine. Slimicidal activity in headbox pulp suspensions. Table 5 shows that DBNPA was active in the two paper mill systems tested, at both pH 4.6 and pH 7.8. These results were substantiated by further tests of headbox stocks from mills producing a wide variety of papers. Also, a field trial carried out over a period of a month at a single mill showed that DBNPA (27 g per 907 kg of paper) gave satisfactory slime control. The compound did not cause any mill problems and did not alter the properties of the paper produced during this trial. Slimicidal activity in cooling water trial. A slug treatment caused a dramatic reduction in bacterial count within 1 hr following the addition of DBNPA (Table 6). This effect persisted for at least 40 hr, and there were no problems (foaming or pH change) that arose subsequent to addition of the compound. DBNPA was used successfully in a second field trial for 13 months. During this time the bacterial count of the cooling tower system remained within the limits demanded by the operators who had had previous experience upon which to base their judgment (Fig. 1). The compound was used both on a continuous and intermittent basis. Soluble oil preservation. The effectiveness of DBNPA varied depending upon the nature of the soluble emulsion tested. The oils tested represented samples from five major producers of soluble or cutting oils. DBNPA killed the natural inoculum in these emulsions in a range of 25 to 100 Ag/ml within a period of 24 hr. Since most soluble oil formulations have an alkaline pH, the hydrolysis of DBNPA limits its extended activity in these systems. DISCUSSION Studies of the rate of kill show that DBNPA is bactericidal and fungicidal over a moderate range of concentrations in 1 to 3 hr. This rate is adequate for controlling bacterial and fungal growth in paper mill and cooling tower systems. However, the rate is too slow for DBNPA to be used as a disinfectant. The breakdown of DBNPA in the pH range from 7 to 9.5 may limit its use in single-dose applications where extended antimicrobial activity is mandatory. For example, a single addition can be used to effect bacterial reduction in soluble oil emulsions. However, these emulsions usually have an alkaline pH, and DBNPA may fail to protect against repeated bacterial insults within a period of 3 or 4 days.

v3-fos

2017-07-29T04:24:53.512Z

1972-04-15T00:00:00.000Z

13860859

s2

Effects of the genes for dwarfism (dw) and naked neck (Na) on chick growth and lipid metabolism SUMMARY In two experiments full sister pairs of chicks, one dwarf (dw) and one non-dwarf (Dw) were reared in individual cage to five weeks of age. Chicks carrying the sex-linked recessive dw gene were identified at hatching by the closely linked fast-feathering gene (k). Approximately half of the pairs carried the autosomal dominant gene for naked-neck (Na). Presence of the dw gene resulted in about a 30 per cent reduction in weight gain, a reduced body temperature, increased carcass content of lipid and of lipid "C activity from injected laC labelled acetate. These latter may be a result of either increased lipogenesis or decreased energy expenditure or, more likely, a combination of the two. Presence of the Na gene appeared to cause increased lipogenesis. The naked-neck bird showed increased energy expenditure in a cool environment and perhaps a greater flexibility of body temperature regulation. An interaction between the dw and Na genes was apparent under cooler environmental condi- tions and seemed to arise from the suppression of the thermoregulatory mechanism of the Na a gene by the dw gene, possibly through inhibition of lipid degradation. 14C-labelled Individual body weights and feed consumption data were recorded weekly. Body tempera- ture was measured cloacally toward the end of the 4 hour fast period at 5 weeks of age. Approximately i.5 hours after they had been re-fed on the final day, each was injected intraperito- neally with io !,Ci of sodium acetate-2 14C (specific activity 20. [jtCi per !,M) in i ml of o.9 percent sodium chloride solution. In experiment 2, sodium acetate-i 1’C was used with a specific activity of 22.7 [tCi per !,M. In Experiment I, five pairs were sacrificed by bleeding at intervals of 0.5, 1, 2 and 3 hours post injection. In Experiment 2, they were all sacrificed 2 hours after radioisotope injection. The livers were removed, frozen and stored for another study. Thecarcasseswereweighed and frozen at - 20°C, then later ground, and freeze died. Total lipids were extracted from dupli- cate samples of the lyophilyzed carcasses by the method of FOLCH et al. The 1’C activities of duplicate aliquots of carcass lipid were measured in a liquid scintillation counter after addition of scintillation fluid (toluene plus PPO plus POPOP). The quantities of lipid and the 1’C content were related to the fresh killed body weight. The data were analyzed using Student’s t test : the method of paired comparisons for the effect of the dw gene (sister pairs), the simple t test for the Na gene (comparison of two means). The likleihood of obtaining significant differences in the latter comparisons was poor because of the limited numbers of animals and of the variability among families due to the heterogeneity of the sires. Under these conditions probability levels of o. i and 0.2 were taken as indicating tendencies, particularly when all of the traits were considered in relation to each other. INTRODUCTION A sex-linked recessive gene for dwarfism in chickens was first discovered by Hun (i 953 ). A similar gene which occured spontaneously in the genetically closed experimental flock at Jouy-en-Josas, France, was reported by MlExA2 (ig6g). This gene caused a reduction in body weight of 30 per cent in females and 40 per cent in males. It also shortened the long bones and reduced the number and weight of eggs produced by io per cent. J AAP and M OHAMMADIAN (ig6g) reported that the dw gene reduced the rate of yolk deposition but did not reduce the rate of egg production. It did lower the per cent of defective eggs produced. These observations pertained to pullets weighing 2 .6 kg at 3 6 weeks of age whereas the mean hen weight of the experimental flock of MÉ RAT was 2 . 2 kg. MA RAT and G UILLAUME (ig6g) provided evidence suggesting that the dwarf birds were slightly hypothyroid, supporting the earlier report by V AN T W ·rxov>~rr et al. (rg66). This hypothyroidism, however, appeared to be secondary and not the primary cause of the dwarf characteristics. LECi!ERCQ ! al. ( 1970 ) reported that feed consumption was practically independent of the energy level of the diet and that the dwarf chicks could withstand severe rationing of energy intake without any ill effects. G UILLAUME (ig6g) observed that the feed consumption of dwarfs was only slightly less than that of normal birds while energy expenditure was considerably reduced. The efficiency of protein utilization was significantly inferior and that of energy utilization significantly superior in dwarf chicks which stored much more fat in their bodies than did normal chicks. The work to be reported here represents a part of the broader goal of studying the metabolic mechanisms involved in this excess lipid accumulation in the dwarf. The naked neck gene which entered the experimental design inadvertently, introduced the aspect of temperature control mechanisms and their relation to lipid metabolism. The information concerning the first reports and description of this autosomal dominant gene were summarized by H U TT ( 1949 ). EXPERIMENTAL METHODS To provide the experimental chicks and in order to identify dwarf and non-dwarf chicks at hatching, special matings were made using cocks from the experimental flock of M>; R n2. These sires were heterozygous for dwarfism (Dwdw) and for early feathering rate (Kk), with these sexlinked genes disposed so that either the dominant or the recessive genes were transmitted together. By chance, 3 of the 4 males obtained also carried the dominant autosomal gene for naked neck (Na). The cocks were mated by artificial insemination with females of a commercial dwarfed broiler strain, JA 57, of the Institut National de la Recherche agronomique (I. N. R. A.) which carried the recessive genes dw and k. At time of hatching the chicks were sexed by the vent method and the females were chosen for these studies. They were separated by rate of early feathering as indicated by relative lengths of primary and covert feathers. Classification by feathering rate was verified at 10 days of age ; the chicks classified as slow-feathering being non-dwarfs, those classified as fast-feathering being dwarfs. They were further segregated for the normal neck or naked-neck condition. Full sister pairs, one dwarf and one non-dwarf were placed in adjacent individual wire-floored cages in electricallyheated battery brooders in a heated room. The first experiment comprised 20 pairs, i of which were normal-neck and 8, naked-neck pairs. The second experiment included 4 normal-neck and 4 nacked-neck pairs. The diet fed was based on corn, soybean meal and fishmeal and provided approximately 3 . 02 kilocalories of metabolizable energy per gram. The analyzed crude protein levels of the diets were i 8.6 per cent in Experiment i and i 9 . 2 per cent in Experiment 2 . These relatively high-energy diets were intended to stimulate lipogenesis. Feed and water were provided ad libitum except that from 2 to 5 weeks of age the chicks were deprived of feed for ¢ hours daily from 10 . 00 to 14 , 00 hours. This was intended to condition the birds so that they would eat at a maximum rate immediately preceding the injection of 14 C-labelled acetate on the final day of the trial. Individual body weights and feed consumption data were recorded weekly. Body temperature was measured cloacally toward the end of the 4 hour fast period at 5 weeks of age. Approximately i. 5 hours after they had been re-fed on the final day, each chick was injected intraperitoneally with io !,Ci of sodium acetate-2 14 C (specific activity 20 . [ jtCi per !,M) in i ml of o. 9 percent sodium chloride solution. In experiment 2 , sodium acetate-i 1 'C was used with a specific activity of 22 . 7 [t Ci per !,M. In Experiment I , five pairs were sacrificed by bleeding at intervals of 0 . 5 , 1 , 2 and 3 hours post injection. In Experiment 2 , they were all sacrificed 2 hours after radioisotope injection. The livers were removed, frozen and stored for another study. Thecarcasseswereweighed and frozen at -20°C, then later ground, and freeze died. Total lipids were extracted from duplicate samples of the lyophilyzed carcasses by the method of F OLCH et al. ( 1957 ). The 1 'C activities of duplicate aliquots of carcass lipid were measured in a liquid scintillation counter after addition of scintillation fluid (toluene plus PPO plus POPOP). The quantities of lipid and the 1 'C content were related to the fresh killed body weight. The data were analyzed using Student's t test : the method of paired comparisons for the effect of the dw gene (sister pairs), the simple t test for the Na gene (comparison of two means). The likleihood of obtaining significant differences in the latter comparisons was poor because of the limited numbers of animals and of the variability among families due to the heterogeneity of the sires. Under these conditions probability levels of o. i and 0 . 2 were taken as indicating tendencies, particularly when all of the traits were considered in relation to each other. RESULTS AND DISCUSSION Out of 19 pairs of dwarf : non-dwarf sisters in Experiment i, one chick exhibiting fast feathering turned out not to be a dwarf according to all other parameters including weight and carcass lipid content. This bird undoubtedly represents a case of crossing-over of the chromosome near the K locus which, according to Hu2!r (1959) occurs at an average frequency of 6.6 per cent. The mean values for body weight gain, feed conversion, body temperature, carcass lipid and lipid &dquo;C content are presented in table I . The experimental results are best understood by examining first the data of Experiment 2 which ended March a 3 , then those Experiment i which ended January 2 i. Although both were conducted in a heated building in heated battery brooders, the seasonal temperature difference appears to have reduced the ambient temperature during Experiment I below a critical point which greatly influenced the results. In Experiment 2 the mean body weight gain to 5 weeks of age in dwarf birds was 73 per cent of that of their non-dwarf sisters. This was similar to the 30 per cent weight reduction resulting from the presence of the dw gene reported by H UTT ( 1953 ) and MA RA T (z 9 6 9 ). Thus, any effect of the k gene of increasing growth rate as reported by G OODMAN and M UIR ( I g65) was minor or insignificant in comparison with that of the dw gene. Feed conversion was very slightly superior for the dwarfs in spite of the fact that their bodies contained rg. 7 per cent of lipid while their nondwarf sisters contained only II . O per cent. These traits were not different between naked-neck and normal-neck birds. Body temperature was reduced by the dw gene and possibly also by the Na gene. The per cent of carcass lipid was significantly increased by the dw gene but not by the Na gene. In contrast, carcass lipid 14 C content was greatly increased by both genes, their effects appearing to be additive. In Experiment I the extreme variability encountered in carcass lipid 1'C content coupled with the limited number of animals precluded observation of differences in relation to time elapsed after injection. Maximum values were obtained, for example, in some cases one half hour post injection. As a consequence, the carcass 1 'C values represent the means over all intervals. Carcass lipid and its 14 C content were again very significantly greater in the dwarf chicks. In the naked-neck dwarfs an even greater augmentation of carcass lipid &dquo;C content confirms the additive effect of the genes dw and Na on the accumulation of labelled precursor in carcass lipids. In the naked-neck non-dwarfs birds, however, the carcass lipid content was significantly diminished whereas the lipid 14 C content was similar to that of the normal-neck non-dwarf birds. In comparison to the latter, these naked-neck non-dwarf birds also exhibited a decreased body weight, poorer feed conversion and increased body temperature. In contrast, their naked-neck dwarf sisters, when compared with the normal-neck dwarfs, showed an increased body weight, improved feed conversion and decreased body temperature. The dze! gene significantly decreased body weight gain and body temperature while it significantly augmented both carcass lipid content and the lipid content of 14 C labelled precursor. This latter effect may be the result of either increased lipogenesis or diminished energy expenditure or, more likely, both. The Na gene appeared to increase both synthesis and degradation of lipid as evidenced by accumulation of labelled precursor on a short term trial without affecting a net accumulation of lipid over the long term 5 -week growth test. This gene also seemed to permit greater flexibility in thermoregulation, increasing body temperature in a cool environment and decreasing it in a warm environment. The dw gene reduced body temperature regardless of the ambient temperature. It also apparently suppressed the thermoregulatory action of the Na gene, perhaps by limiting the degradation of lipid, and this latter effect was responsible for the interaction observed.

v3-fos

2014-10-01T00:00:00.000Z

1971-07-01T00:00:00.000Z

8387179

s2

High density diets for dwarf layers as a potential breakthrough for increasing the feed to food protein conversion of commercial poultry. Evidence from previous experiments at Texas A and M University had suggested that dwarf layers may require a diet with higher nutrient density than their non-dwarf sisters. In an experiment involving non-dwarf birds divided into three weight classes, light, medium and heavy, at 20 weeks of age, the small bodied birds responded more to a higher nutrient density of their diets than the heavier birds. A further suggestion as to the importance of feeding light bodied birds differently from those above a minimum threshold weight, 1300 grams, had been obtained previously from experiments comparing performance of spring versus fall hatched birds in a warm climate and also from the First and Second Texas Gene-Environment Performance Tests. SUMMARY The recent widespread introduction of a simply inherited, sex-linked gene for a dwarf bird into the test programs of a number of commercial breeders has focused attention on body size as a potential breakthrough for increasing the feed to food protein conversion of commercial poultry. Evidence from previous experiments at Texas A and M University had suggested that dwarf layers may require a diet with higher nutrient density than their non-dwarf sisters. In an experiment involving non-dwarf birds divided into three weight classes, light, medium and heavy, at 20 weeks of age, the small bodied birds responded more to a higher nutrient density of their diets than the heavier birds. A further suggestion as to the importance of feeding light bodied birds differently from those above a minimum threshold weight, 1 300 grams, had been obtained previously from experiments comparing performance of spring versus fall hatched birds in a warm climate and also from the First and Second Texas Gene-Environment Performance Tests. Two experiments comparing the laying performance of dwarf and non-dwarf birds have been completed. In the first experiment, two diets ( 1 6 and 17 p. 100 protein) with the same calorie/protein ratio and one, two and three birds per 25 . 4 x 45 . 7 cm cages were compared. The protein level had little effect on body weight and hen-day egg production of the dwarf layers, but egg size, percentage large eggs and feed efficiency were increased by the higher protein level. The non-dwarf birds decreased in egg production as bird numbers per cage increased, whereas the dwarf birds laid more eggs when housed two birds per cage. In the second experiment, protein/calorie (p. ioo/cal kg P. E.) levels of 22/2 145 , y /z o 35 , 1 6/ 1 9 8o and 14/1 925 were compared. A significant improvement in egg production and feed efficiency of dwarfs receiving the 22 p. 100 protein diet was observed. Livability of the dwarf birds decreased as the protein level decreased, but increased for the non-dwarf birds. When all protein levels were combined, the ratio of performance of the dwarfs versus the non-dwarfs was : body weight, 70 p. ioo ; egg production, 8 0 p. 100 ; egg size, 90 p. 100 ; daily food consumption, 75 p. 100 ; feed efficiency, 95 p. 100 and livability, 9 8 p. ioo. These results indicate that the dwarf birds need a higher density diet than currently popular non-dwarf lines. Additional testing must be done to determine the optimum nutrient balance for this dwarf-type bird if it is to be widely adopted for commercial laying operation. INTRODUCTION The nutrient requirements for growth, maintenance and egg production differ significantly. Therefore the optimum laying diet for the most efficient production of eggs differs from time to time and becomes a compromise between these three requirements. To complicate the requirements further, there exist significant interactions between these three variables. Of the 3 6 kilograms of feed consumed by the typical layer per year, 23 are used for growth and maintenance and less than r 3 for the production of eggs. Since feed constitutes from three-fifths to two-thirds of the total cost of egg production, breeders and egg producers are currently giving much attention to minimal body weights required for optimal performance. Breeders have lowered the average body weight of their layers 400 to 5 00 grams during the past five years, reducing the feed for maintenance by approximately 4 .5 kilograms. Their success, in cooperation with the nutritionist, has made the modern laying hen the most efficient converter of feed to food protein the world has ever known. As the adult body weight for laying strains is reduced, new diets of higher nutrient density, more accurate balance and greater palatability must be provided. DWARFS The recent widespread introduction of a simply inherited gene dw for a dwarf bird (H UTT , 1959 ) into the test programs of a number of commercial breeders has focused attention on body size as a potential breakthrough for increasing the feed to food protein conversion of commercial poultry. A growing world population and potential competition with plant protein fortified with synthetic amino acids are only two of many factors that justify exploration of this potential tool for further reduction in the cost of producing poultry meat and eggs. Evidence that small bodied birds responded to a higher nutrient density of their diets than heavier birds of the same strain or cross came from an experiment involving normal, or non-dwarf birds, that were divided into three weight classes, light, medium and heavy, at 20 weeks of age and phase fed different protein levels. Results of this experiment are given in table i. The data in table I suggest that the beneficial effects of phase feeding were mostly limited to the light-weight birds. A further suggestion as to the importance of feeding light bodied birds differently from those above a minimum threshold weight had been obtained previously from experiments comparing performance of spring versus fall-hatched birds in a warm climate and are also contained in table 2 from the First and Second Texas Gene-Environment Performance Tests. The data in table 2 suggest that diets should be formulated for each weight class, particularly the lights, and although not shown in this table each entrant might benefit from specific formulations. This is particularly true for summer grown pullets in warm climates. Flock Replacement Costs for Dwarfs Versus Non-dwarfs On a per bird basis, dwarf birds would require less housing and equipment space than larger or heavier birds. More significant, would be the savings on feed. Differences in feed to 20 weeks of age for one group of birds hatched in early November are given in table 3 . Birds in the latter experiment are currently only slightly more than 12 weeks of age. Time did not permit summarizing the data beyond 8 weeks. Space requirements for dwarf pullets are generally considered to be from one-half to two-thirds that of non-dwarf birds. Body weights of these particular types of dwarfs averaged approximately 6 0 -6 5 p. ioo of the non-dwarfs. For the birds in Experiment i approximately r.8 kilograms of feed were saved to 20 weeks. Perhaps more could have been saved had the dwarfs been raised at a higher density. If the present ratio continues for Experiment 2 , the saving in feed will be 5 to 10 p. 100 more than for Experiment i. Comparison of Laying Performance Dwarfs and Non-dwarfs -Three Experiments Experiment 1. At the time Experiment I was conducted there was a limited number of dwarf birds available and this experiment was conducted to obtain some basic comparative biological data for the dwarf versus non-dwarf birds from which they had been extracted. Two diets ( 1 6 and 17 p. 100 protein) with the same protein/calorie ratio and one, two and three birds per 25 . 4 X 45 . 7 cm cages were compared. Some of the basic biological data collected for the two diets are given in table 4 . Effect of the bird densities, or number of birds per cage, are given in tables 5 and 6. The data given in Tables 5 and 6 are not of too much value in comparing the space needs of dwarfs versus non-dwarfs under the higher densities being used today. The data indicate that the cage space provided even for the non-dwarf birds was adequate or approaching adequacy. Body weight of the dwarfs was approximately 68 p. ioo, egg production 7 6 p. ioo, egg size go p. 100 , daily feed consumption y p. zoo, feed efficiency 9 6 p. 100 and livability 105 p. ioo of the non-dwarf birds. The two traits of most concern to the breeder are egg production and egg size. Improvement in these will, of course, greatly enhance feed efficiency. Experiment 2. For Experiment 2 heterozygous dwarf males were mated to dwarf females of the same breeding line so that both non-dwarf and dwarf progeny would have the same genetic background. The accumulation of data from other experiments had suggested that dwarf birds would respond to higher density diets than the non-dwarfs. For this experiment four protein and four energy levels were used. Performance data for Experiment 2 are given in tables 7 and 8. If averages of all protein and calorie levels are used we do not find much difference from Experiment I in the ratios of performance of the dwarfs versus the nondwarfs. Body weight was 70 p. 100 , egg production 8 0 p. ioo, egg size go p. 100 , daily feed consumption 75 p. IOO , feed efficiency, g 5 p. ioo and livability g8 p. 100 of the non-dwarf birds. However, if tables and 8 are used to compare differences in performance between treatment groups (protein and calorie levels), quite in contrast to Experiment i, a number of differences are noted. The protein/energy ratio of the 22 p. 100 protein diet was considerably lower than the other diets which was probably reflected in a body weight and egg size of the dwarfs that was no greater than the 1 6 and y p. I oo protein diets. On the other hand, for the 22 p. 100 protein group, because part of the protein was no doubt used for energy by the non-dwarf birds there was an increase in body and egg size for these birds (table !). The 14 p. 100 protein diet was too low in protein for both dwarfs and non-dwarfs ; however egg production, egg size, percent large eggs, feed efficiency and livability of the dwarfs, as expected, were more severely depressed than for the non-dwarfs. The two most encouraging observations in Experiment 2 were the significant improvement in egg production of the dwarfs receiving the 22 p. I oo protein diet and the noticeable improvement in feed efficiency. Another interesting contrast was the livability which decreased as the protein level decreased for the dwarf birds but increased for the non-dwarfs. Daily protein consumption of the dwarfs declined from 17 . 1 8 grams per day for the 22 p. 100 protein diet to a low of IL3I grams for the 14 p. 100 diet (Table 8). For the non-dwarfs, the daily protein consumption declined from 22 .66 grams per day to 14 . 95 grams. For the non-dwarfs, protein was severely wasted by those on the 22 p. ioo protein level but fell to less than optimum for those on the 14 p. 100 diet. Experiment 3. In Experiment 3 because of a limited number of available birds of the preferred type, there are two sections to the experiment. In the first section a group of dwarf birds are being compared with non-dwarfs at three densities, one, two and three birds per cage. Performance of these birds will not be reported here. The second section of the experiment consists of dwarf and non-dwarf sibs segregating from the same parent lines but housed one, two and three birds per cage of the same dimensions given for Experiment z. The dwarfs are all receiving 22 p. 100 protein, 2 145 calorie diet and the non-dwarfs are all receiving a 1 6. 4 p. I oo protein, 2 02 6 calorie diet. The calories are given as productive energy per kilogram of diet. This experiment has only progressed through four 2 8-day periods. The dwarf birds laid their first eggs approximately 10 -14 days after their non-dwarf sibs. Thus, with the short experimental period currently completed the dwarfs will tend to show a lower performance score as compared with the non-dwarfs than they would after a full year of lay. Performance data for the first 112 days are given in tables 9 and 10 . Comparison of the basic biological data from Experiment 3 with those of Experiment 2 tends to show some improvement by only one generation of selection. Body weight of the dwarfs averaged 70 p. IOO , egg production of 8 7 p. ioo, egg size 94 p. 100 , daily feed consumption 74 p. ioo, feed efficiency I IO p. 100 and livability I oo p. 100 of their non-dwarf sibs (tables 9 and io). If these improvements continue for the full production period of the birds in this experiment they will certainly be an encouragement to the breeder to continue his selection program with his dwarf lines or crosses. CONCLUSIONS The results of Experiment 3 confirm those of Experiment 2 in that the dwarf birds need a higher density diet than currently popular non-dwarf lines or crosses. They also suggest that much additional testing must be done to find the optimum nutrient balance for this dwarf-type bird if it is to be widely adopted for commercial laying operations. A third conclusion is that for optimum speed of adoption the geneticist and the nutritionist must work in coordination. A close inspection of tables 9 and io reveals a significant improvement in egg production, egg size and feed efficiency for the dwarf birds. Three birds per cage tended to reduce daily feed consumption and rate of lay. In addition to emphasis on the improvement of all other economic traits particular emphasis should be placed on research that will find ways of avoiding this depression in egg production so that higher bird densities can be used to keep down overhead cost as better houses are coming into use all over the poultry world.

v3-fos

2020-12-10T09:04:12.635Z

1972-04-01T00:00:00.000Z

237231795

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 NO3- nitrogen, NH4+ 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 NO3- nitrogen concentrations of less than 1 μg/ml. During the summer months, soil water NO3- nitrogen increased at the 15-cm depth, indicating that nitrification took place at the feedlot surface. However, the low soil water NO3- 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 810 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

2018-04-03T03:52:38.736Z

1972-12-01T00:00:00.000Z

36859450

s2

Effect of Low-Roughage Diets on the Microflora and Lipid Metabolism in the Rumen Changing the diet of five lactating cows and one nonlactating cow from high to low roughage induced milk fat depression in the lactating cows and altered the composition of the rumen microflora. While the numbers of lactic and propionic acid-producing bacteria increased, the numbers of Butyrivibrio spp. decreased. The numbers of lipolytic bacteria and the in vitro lipolytic activity of the rumen fluid were also decreased, as was the extent of hydrogenation of linoleic and linolenic acids combined in soybean oil incubated in vitro with rumen fluid. It is suggested that among the bacterial population in the rumen the vibrios, which were adversely affected by the low-roughage diets, may contribute significantly to both lipolysis and hydrogenation in the rumen. Although most diets of ruminants normally contain high proportions of polyunsaturated acids as phospholipids and galactoglycerides, only small amounts of these acids are directly transferred to adipose tissue or to milk since most of them are metabolized to more saturated derivatives by the rumen microflora (31) before being absorbed from the small intestine. Hydrolysis of the complex dietary lipids is necessary before isomerization and hydrogenation of the fatty acids can take place (15). When cows are fed low-roughage diets, particularly those which depress milk fat secretion, the changes in the fatty acid composition of lipids of milk (25), blood plasma (9,21,27), digesta (29,33), and adipose tissue (30) suggest that hydrogenation in the rumen of dietary unsaturated acids may be reduced. Reductions in the numbers of ciliate protozoa have been associated with reduced hydrogenation (6), and lower numbers of these organisms and of the bacterium Butyrivibrio fibrisolvens, which also actively hydrogenates (23), are known to occur with lowroughage rations (4,18,24). In this investigation the effect of low-roughage diets, including milk fat-depressing diets, on the composition of the microflora and the metabolism of feed lipids in the rumen have been examined more fully. The effect of these diets on the rumen fermentation are reported elsewhere (Latham, Sutton, and Sharpe, in press). MATERIALS AND METHODS Animals and their management. Rumen contents from four lactating Friesian cows with perma-nent rumen fistulas were used for in vitro studies on hydrogenation. Their diet was progressively changed from a control of high roughage (8 kg of hay and 10 kg of concentrate cubes, 20% dry weight as crude protein, daily) to a milk fat-depressing low-roughage diet in which flaked maize was substituted for part of the hay and concentrates until the milk fat content was depressed to less than 2%. The final proportions (in kilograms) of hay, concentrates, and flaked maize varied for each cow and were respectively 1, 1, 9 (cow G10); 1, 6, 4 (cow G9); 1, 6, 4 (cow B20) and 1, 1, 5 (cow G27). Rumen contents from two more fistulated Friesian cows were used for in vitro studies on lipolysis. One (M21) in mid-lactation was treated similarly to the first four cows and received a final milk fat-depressing diet of 1, 2, 8 kg of hay, concentrates, and flaked maize, respectively, per day. The other cow (J), which was nonlactating, received an all-hay diet for 6 weeks and then a low-roughage diet of 20% hay and 80% flaked maize. Sampling. Milk yields were recorded, and daily composite samples of the milk were analyzed for total fat content by the Gerber method (2) and for fatty acid composition by gas-liquid chromatography (26) with either a Perkin Elmer 880 or 900 model chromatograph. For characterizing the microflora, single samples of well mixed rumen digesta were taken from each of the lactating cows when given the high-and the low-roughage milk fat-depressing diets. Characterization of the microflora. The Hungate (16) method of strict anaerobic culture as modified by Latham and Sharpe (17) was used throughout. All media for the enumeration of viable and lipolytic (tributyrin-hydrolyzing) bacteria and for the identification of isolates were based on medium 10 (4) as described by Latham, Sharpe and Sutton (18). Counts were made after 3 days of incubation, and the composition of the flora was determined by picking LATHAM, STORRY, AND SHARPE and subsequently identifying all the colonies (100-150) from suitable roll tubes used for the enumeration of the total viable population. Direct microscopic counts of formalized suspensions of protozoa in glycerol-water (1:1, v/v) were made in a Neubauer counting chamber (Hawksley & Sons Ltd., Lancing, England), and differential counts of bacteria were made by the Breed technique (1) by using Gram-stained preparations. Lipolysis studies. Two methods were used for determining lipolytic activity. (i) An agar diffusion method (19) was used to test the lipolytic activity of the bacteria from cow M21. The rate of hydrolysis of various triglycerides emulsified in buffered agar by test suspensions of bacterial cells was determined by measuring the diameter of the clear zones produced in the opaque gel at intervals during incubation at 30 C. Suitable suspensions of bacterial cells were obtained from 100 ml of rumen fluid which had been filtered through one layer of muslin. The filtered rumen fluid was centrifuged several times at approximately 300 x g for 5 to 10 min to remove the protozoa, and the supernatant fluid was centrifuged at 15,000 x g for 1 hr to sediment the bacteria. The bacteria were resuspended in 2.5 ml of 0.05 M sodium phosphate buffer, pH 7.0, and the amount of protein per milliliter of suspension was determined (20) so that in comparing the lipolytic activity between rations all values could be corrected to a unit value of 25 mg of protein per ml of suspension. (ii) An incubation method was used to determine the lipolytic activity of whole rumen contents from cow M21 and of the protozoal, bacterial, and cell-free rumen fluid fractions of rumen contents from cow J when each cow was given highand low-roughage diets. Sediments obtained by centrifuging at 300 x g (rich in protozoa but contaminated with feed particles and large numbers of bacteria) and 15,000 x g (rich in bacteria) were resuspended in their original volume of cell-free (15,000 x g supernatant fluid) rumen fluid. Duplicate flasks containing each of the rumen fluid fractions were incubated at 39 C in a water bath. Each flask was closed with a rubber bung adapted to gllow continuous stirring and gassing with 02-free nitrogen throughout the incubation. Emulsions of linseed oil or triolein were prepared by sonic oscillation (Dawe Soniprobe type 1130) in 0.05 M phosphate buffer, pH 7.0, containing 0.1% (w/v) gum acacia. The emulsion was purged with O2-free nitrogen, and 10% (v/v, final incubation mixture) added to one (experimental) of each pair of flasks to give 10 mM final concentration of triglyceride. The same volume of phosphate buffer containing only gum acacia was added to the remaining flasks (controls). After thorough mixing, 20-ml subsamples were removed from both the experimental and control flasks (0 hr), the reaction was stopped by acidifying to pH 2.0 with 50% HZSO4, and the sample was shell frozen with CO2-acetone. Additional subsamples were taken at 8 hr and treated similarly. All of the subsamples were then freeze-dried. Total lipid in the freeze-dried material was extracted in 2: 1 (v/v) chloroform-methanol, and the nonesterified fatty acids in the extract were separated by thin-layer chromatography (28) and estimated quantitatively by the colorimetric method of Duncombe (10). Hydrogenation studies. Samples of rumen digesta were taken 4 to 5 hr after feeding from animals on the control diet and again from the same animals after they were established on the low-roughage diet and showing depressed milk fat. These samples were taken directly into each of two sterile graduated flasks and immediately gassed with 02-free nitrogen. Sufficient anaerobic dilution fluid (3) was added to dilute the contents by one-third to give a final volume of 1 liter. At the same time additional rumen samples were taken for microbiological analysis. The flasks were incubated in a similar manner to that used in the lipolysis studies. A 5-ml amount of 10% soybean oil emulsion ("Intralipid": Paines and Byrne, Greenford, England) was injected into the contents of one (experimental) of the flasks. After allowing 2 min for thorough mixing of the emulsion with the digesta, 30-g subsamples were taken from both the experimental and control flasks (0 hr), and again after 1, 2, 4, and 6 hr of incubation. The weight of each subsample was recorded, and the reaction was stopped by immediately transferring the sample into 2:1 (v/v) chloroform-methanol for extraction of total lipids. At each time of sampling, the pH and total numbers of bacteria were also recorded. Extraction of total lipids and total fatty acids and determination of fatty acid composition. Total lipids from whole rumen contents in the hydrogenation studies and from freeze-dried rumen fluid fractions in the lipolysis studies were extracted in 2:1 (v/v) chloroform-methanol and washed with 0.88% (w/v) KCl solution as described by Folch, Lees, and Sloane Stanley (12). Total fatty acids were extracted from the total lipid samples after saponification, and their composition was determined by gas-liquid chromatography (26). RESULTS Milk fat. Although the composition of the low-roughage diets fed to individual animals differed, they -ll gave a marked depression in milk fat secretion. At the same time, the proportions of saturated fatty acids in the milk fat were decreased and the proportions of unsaturated fatty acids were increased (Table 1). Rumen microflora. With the exception of cow G9, the low-roughage diets increased the number of viable bacteria in all cows by 0.6 to 1.6 log units but decreased the number of lipolytic (tributyrin-hydrolyzing) bacteria by up to 3 log units (Table 2). At the same time, these diets reduced by various amounts the numbers of ciliate protozoa. Of the two cows used in the lipolysis studies, M21 lost its ciliates, and there was a reduction in the number of ciliates in cow J which was not quantified. Only one (G10) of the four cows used in the hydrogenation studies lost its ciliates, while in cow G27 they were reduced by 90%. However, the two remaining cows (G9 and B20) lost only 32 and 45% of their ciliates, respectively. The variation in the composition of the bacterial flora between cows given the lowroughage diets was considerable ( Table 3). The proportions of Selenomonas, Peptostreptococcus, Bifidobacterium, and Lactobacillus were particularly variable. Nevertheless, increases in lactogenic and propionogenic bacteria typical of low-roughage diets (4,18) were observed. The most consistent effect of the low-roughage diets on the bacterial flora was the severe reduction in the proportion of Butyrivibrio spp. Borrelia, which like Butyrivibrio is capable of hydrogenation (23,35), was also reduced. Rumen lipolysis. Preparations of mixed rumen bacteria derived from cow M21 when fed the high-roughage diet hydrolyzed buffered emulsions of both tributyrin and tripalmitin, but tributyrin was hydrolyzed almost 2.5-fold as fast as tripalmitin (Fig. 1). On the low-roughage diet the rate of hydrolysis of both triglycerides by similar preparations of mixed rumen bacteria fell to one-fifth of the previously observed rates. Incubations of whole rumen contents with emulsified linseed oil confirmed this apparent reduction in lipolytic activity (Table 4). Progressively less triglyceride was hydrolyzed as the roughage was reduced and the cereal content of the ration increased. In a separate experiment the lipolytic activity of the protozoal, bacterial, and cell-free rumen fluid fractions obtained from cow J given similar high- and low-roughage rations were compared. The bacterial fraction associated with the hay diet had the greatest lipolytic activity, hydrolyzing 87.6% of the added triglyceride (Table 5), whereas the protozoal and cell-free rumen fluid fractions hydrolyzed only 28.6 and 13%, respectively. The same fractions from animals on the low-roughage ration hydrolyzed 5.8, 34.9, and 5.7%, respectively, indicating a considerable decrease in lipolysis by the bacterial fraction offset to some extent by a slight rise in that of the protozc level of unto increased N concentrate Rumen were fed th tions of tot digesta we when they These diff higher diet; age rations of rumen i were appre acids of di incubation any differe C18 fatty controlanm metabolisn The patt similar for mean resu diets are s Over the 6tion of lino form of so, diet, 59 and 63%, respectively, of that obtained on the high-roughage ration. The overall reduced rates of hydrogenation on the low-roughage diet were significant at the 5% and 1% level for linolenic and linoleic acids, respectively. The pattern of hydrogenation also differed between the two diets; with the high-roughage diet the hydrogenation appeared to commence soon after addition of the soybean oil and to progress rapidly up to the end of the fourth hour of incubation, whereas with the low-roughage ration the most rapid hydrogenation occurred between the second and sixth hours. )al fraction. In both experiments the study (18), the composition esterified fatty acid of dietary origin terial flora of nonlactating with increases in the proportion of and low-roughage diets es in the diet. between cows fed the hydrogenation. When the cows present experiment the between-cow Le low-roughage diets, the concentraon the low-roughage ;al lipid and fatty acids in the rumen extended to the protozoa. ore two or three times greater than variability was not clear were fed the high-roughage diets. related to the high food erences can be accounted for by cows. ary intakes of lipid on the low-rough-It has been shown , and by increases in the contribution fats or oils are added microbial lipid (29). Although there rapidly adsorbed on food ,ciable amounts of oleic and linoleic ganisms (13,14); Czerkawski ietary origin present in the in vitro that levels of linseed oil systems, it has been assumed that ml of rumen fluid inhibited nces in the composition of the total Greater concentrations acids between samples from the this, while saturating i experimental incubations reflected rumen fluid (Demeyer, n of the added soybean oil. tion), have not been shown ;emns of hydrogenation obtained were In the present experiment, all four cows examined, and the total lipid and fatty lts for the high-and low-roughage used for the in vitro s hown in Fig. 2 and 3, respectively. lipolytic and hydrogenating *hr incubation period, the hydrogenagreater with the low-roughage lenic and linoleic acids added in the high-roughage diets, ybean oil was, on the low-roughage genation studies the acids in "Intralipid" during incubation with-rumen contents from cows fed a low-roughage diet. was added at the rate of 50 mg/100 ml of digesta, more than 800 mg/100 ml of digesta was added in the lipolysis studies. However, serial samples taken during the various incubation experiments revealed no major change in either the direct count or motility of bacteria and protozoa, indicating that even at the high substrate levels used in the lipolysis studies the added triglyceride had no apparent deleterious effect on the microflora. It is probable that the reduced lipolytic activity of the rumen digesta found with the low-roughage diets was related to the reduced numbers of lipolytic vibrios. These diets greatly reduced the viable counts of tributyrin-hydrolyzing bacteria, and approximately 70% of the tributyrinolytic bacteria isolated on a previous occasion in this laboratory from the same medium were found to resemble butyrivibrios. Five out of 25 of these strains were able to hydrolyze long-chain triglycerides up to triolein. In the present work, the strongly lipolytic vibrio Anaerovibrio lipolytica was not isolated, and subsequent work in this laboratory suggests that this and similar lipolytic organisms constitute no more than 1% of the viable population. This effect of diet on lipolysis is in agreement with recent observations of Demeyer (personal communication) who found that the inclusion of sucrose in the diet ot'a sheep or of glucose in an in vitro incubation of rumen fluid reduced lipolysis. It is not clear why the lipolytic activity of the protozoal fraction should have increased with the low-roughage diet. Clarke and Hawke (7) found that much of the lipolytic activity of this fraction could be transferred to the bacterial fraction after homogenization, and they therefore concluded that many lipolytic bacteria were attached to the particulate matter. Since on the low-roughage diet many starch granules will also sediment at the low "g" values used for separating the protozoal fraction of rumen fluid, they will, in so doing, bring with them even larger numbers of adhering bacteria among which may be actively lipolytic strains. As indicated by the changes in milk fat composition and from the results of the in vitro incubations, the hydrogenating activity of the rumen contents was also reduced by the lowroughage rations. Strains of Butyrivibrio fibrisolvens were the first rumen bacteria to be implicated in the biohydrogenation of polyunsaturated fatty acids and were found to hydrogenate to the monoene (23). Since then laboratory strains of Borrelia (35), a gram-negative micrococcus (22), a strain each of Eubacterium and Ruminococcus (34), and two strains of cellulolytic Clostridium spp. (32) have all been found to biohydrogenate in a similar manner. However, only two strains of bacteria have so far been reported to be capable in pure culture of the complete reductive process to the saturated acid (34). Under normal conditions of high-roughage feeding, Butyrivibrio fibrisolvens is by far the most numerous of all of these biohydrogenating organisms found in the rumen and in the present experiment comprised 25.0% of the viable population in cows fed the high-roughage diets. On the assumption that the isolates of Butyrivibrio and Borrelia included hydrogenating strains, the reduction in their numbers which occurred with the lowroughage diets is consistent with the decrease in hydrogenation observed in vitro. Tove and Matrone (30) reported that, compared with conventional diets, the reduction of the monoenoic to the saturated acid proceeded more slowly with high-concentrate diets. As most workers have not been able to isolate strains which can carry out this final reductive step, such organisms are unlikely to occur in the majority flora, yet, like the butyrivibrios, they would appear to be greatly affected by dietary changes. Although the protozoa-rich fraction of rumen contents has been shown to have biohydrogenating activity (5). the nule (l individual species of ciliate protozoa in this activity remains equivocal (31). However, studies on the plasma lipids of lambs with or without rumen ciliates indicate that dietary C,. unsaturated fatty acids are more effectively hydrogenated in the presence of ciliates (A. K. Lough, Proc. Nutr. Soc., 27:30A, 1968). It is therefore of interest that in the present work the reduction in hydrogenation induced by the low-roughage diets was no greater in the two cows which either lost or suffered a 90% reduction in the numbers of ciliates than in the remaining cows which retained between 55 and 70% of their ciliates. Because Hawke and Silcock (15) have recently shown that only nonesterified fatty acids can be hydrogenated, the present results suggest that, in addition to the adverse effects of low-roughage rations on the numbers of potential biohydrogenating organisms, a reduction in lipolysis may also contribute to the observed reduction in hydrogenation. ACKNOWLEDGMENTS The analysis of total fatty acid composition by A. J. Hall. the care of the animals by V. W. Johnson, and the capable technical assistance of Ann Siddell are gratefully acknowledged.

v3-fos

2018-04-03T02:23:40.293Z

1972-01-01T00:00:00.000Z

33399683

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 manifesta- tions 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 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from 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

2020-12-10T09:04:10.753Z

1972-09-01T00:00:00.000Z

237230259

s2

Radiation Sterilization of Prototype Military Foods: Low-Temperature Irradiation of Codfish Cake, Corned Beef, and Pork Sausage “Screening” packs comprising 10 lots each of codfish cake, corned beef, and pork sausage, each lot containing about 106 spores of a different strain (five type A and five type B) of Clostridium botulinum per can, were irradiated at −30 ± 10 C with a series of increasing doses (20 replicate cans/dose) of 60Co gamma rays. The cans were incubated for 3 months at 30 C and examined for swelling, toxin, and recoverable botulinal cells. Based on the latter criterion of spoilage, median lethal dose (LD50) and D values were estimated for each strain in each food. The most resistant strain in codfish cake, corned beef, and pork sausage was, respectively, 53B, 77A, and 41B. There was no clear-cut trend in the comparative order of resistance between the two antigenic types among the three foods. LD50 values gave essentially the same order of resistances as the D values and may be used interchangeably with the latter for the 10 test organisms. “Clearance” packs consisting of the most resistant strain (about 107 spores/can) with its respective food were irradiated with a variety of doses at −30 ± 10 C, using 100 replicate cans/dose (about 109 spores/dose). These packs were incubated for 6 months at 30 C and assayed for the three types of spoilage. Based on recoverable cells, the experimental sterilizing doses (ESD) for codfish cake, corned beef, and pork sausage were 2.5< ESD ≤ 3.0, 2.0 < ESD ≤ 2.5, and 1.5 < ESD ≤ 2.0 Mrad, in that order. Assuming exponential spore death, the 12D values, or minimal radiation doses (MRD), were 3.24, 2.44, and 2.65 Mrad, respectively. Estimation of the MRD values by a method which assumes that spore death in the cans follows a normal distribution, yielded 3.09, 2.57, and 2.39 Mrad, respectively. Weibull analyis of the pooled 10-strain viable cell spoilage data of the screening packs for codfish cake or corned beef suggested that spore death in the cans follows a normal distribution yielded 3.09, 2.57, pooled data were not amenable to such analysis. Sublethal doses (0.5, 0.75 Mrad) increased the visible spoilage rate of corned beef over that of unirradiated controls. Apparently radiation-injured spores of C. botulinum were sensitized to the presence of food additives such as curing salts, NaCl, and spices. spores/can) with its respective food were irradiated with a variety of doses at -30 10 C, using 100 replicate cans/dose (about 109 spores/dose). These packs were incubated for 6 months at 30 C and assayed for the three types of spoilage. Based on recoverable cells, the experimental sterilizing doses (ESD) for codfish cake, corned beef, and pork sausage were 2.5< ESD < 3.0, 2.0 < ESD < 2.5, and 1.5 < ESD < 2.0 Mrad, in that order. Assuming exponential spore death, the 12D values, or minimal radiation doses (MRD), were 3.24, 2.44, and 2.65 Mrad, respectively. Estimation of the MRD values by a method which assumes that spore death in the cans follows a normal distribution, yielded 3.09, 2.57, and 2.39 Mrad, respectively. Weibull analyis of the pooled 10-strain viable cell spoilage data of the screening packs for codfish cake or corned beef suggested that spore death in the cans follows a normal distribution yielded 3.09, 2.57, pooled data were not amenable to such analysis. Sublethal doses (0.5, 0.75 Mrad) increased the visible spoilage rate of corned beef over that of unirradiated controls. Apparently radiation-injured spores of C. botulinum were sensitized to the presence of food additives such as curing salts, NaCl, and spices. The U.S. Army has been engaged in a longrange research and development program for the preservation of food products by ionizing radiation. Prototype radiation processes have been reported for bacon (5), ham (3), and pork loin (4). All these foods had been irradiated at ambient temperature, i.e., the center of the can contents, which had an initial temperature of 2 to 5 C, were not permitted to rise above 25 C during irradiation. Numerous investigators (8-10, 12, 13, 23-27, 31, 35, 38, 47, 49) had observed that foods irradiated at freezing tem-for study because of the indicative moderate sterilizing doses, even at low temperatures, with relatively high acceptance characteristics. Very little information is available on highdose irradiation of seafood, although doses under 1.0 Mrad have been used extensively to increase their shelf-life (radurization). Data on irradiation of codfish with doses > 1.0 Mrad are even more scarce and of little use for our purpose. Wheaton et al. (48) irradiated codfish inoculated with various strains of C. botulinum spores with only 0.85 and 1.7 Mrad. The fish was initially frozen to -29 C and was kept cold in the spent fuel rod source with cold ethanol during irradiation. Instead of incubating the irradiated pack they made survival counts. Their most resistant strain, 12885A, decreased from 5.8 x 106 spores/g to 8.5 x 103/g at the higher dose. The other investigators of codfish used uninoculated samples. Nickerson et al. (37) reported that they obtained "commercial" sterility with 1.5 Mrep (1.46 Mrad) cathode rays; the irradiation temperature was not indicated. Miyauchi (36) used doses of 0 to 1.86 Mrad and followed survival counts with storage time at 0 to 1.7 C, but he did not give data at doses above 0.7 Mrad. Sinnhuber et al. (41)(42)(43) subjected codfish to 4.5 Mrad at ambient temperature for organoleptic quality studies; the only microbiological test they performed was the verification of the absence of C. botulinum toxin by the mouse neutralization test. The available information on the irradiation of pork sausage is also scanty and involved uninoculated samples. Licciardello et al. (34) found that "commercial" sterility was obtained when the food was irradiated to 3.5 Mrep (3.4 Mrad) with cathode rays at -23 C. In a second study, Licciardello et al. (33) exposed samples to 1.08 Mrad and could not recover any bacteria. Although Coleby et al. (11) irradiated the food to 1, 2, 3, and 5 Mrad at ambient temperature and Kirn et al. (30) The only irradiation data on corned beef which could be found in the scientific literature was reported by Kirn et al. (30). Again, they were not concemed with the microbiological aspects. Consequently, practically nothing is known about the radiation resistance of C. botulinum spores in these three foods. This paper therefore reports on a prototype radiation steriliza-tion process at -30 i 10 C for codfish cake, corned beef, and pork sausage using C. botulinum type A and B spores as the index of microbiological safety. MATERIALS AND METHODS Food preparation. Cod fillet was ground and mixed with 4% white corn meal, 1.5% gelatin, and 0.5% NaCl (noniodized). Approximately 2,268-g (5 lb) lots of the mixture were stuffied into casings (Visking, 3.5 C), clip-sealed, cooked in boiling water to a minimal internal temperature of 71.1 C (about 30 min) to inactivate food enzymes, wrapped in clean wax paper, and chilled overnight at 3 to 5 C. Raw brisket (corned beef) was pumped to 110% green weight with an aqueous solution of 25.10% NaCl (noniodized), 0.35% NaNO3, and 0.13% NaNO2, then was covered with this pickle cure for 24 hr, autoclaved at 104 C to the desired tenderness (about 90 min), wrapped in clean wax paper, and chilled overnight at 3 to 5 C. Pork was ground and mixed with 1.88% NaCl (noniodized), 0.22% black pepper, 0.094% ginger, 0.31% sage (well rubbed), 0.38% sucrose, and 3% chipped ice, stuffed in natural sheep casing to make 28.35-g (1 oz) links, baked at 204.4 C to a minimal internal temperature of 71.1 C, wrapped in clean wax paper, and chilled overnight at 3 to 5 C. The three foods were diced (after removing the casings), packed in 45 i 5 g quantities into 211 by 101.5 C-enamel metal cans, and loosely closed with lids, leaving a 0.6-cm (1/4 inch) headspace per can. Sanitary precautions were followed throughout the handling operations, including the prior autoclaving of the cans, lids, and other food handling equipment for 10 min at 5 psi. Proximate chemical analysis on uninoculated unirradiated random cans of food were performed. Each food product consisted of 10 samples, four cans per sample, and was assayed in duplicate. Average results are tabulated in Table 1. Test organisms, inoculation, can sealing. The 10 strains of C. botulinum spores, the method of inoculating the foods, and the sealing of the cans were previously described (3). The sealed cans were then placed in a -23 C room to await irradiation. Irradiation. A "screening" and a "clearance" inoculated pack were prepared for each food product. The first pack consisted of 20 replicate cans per dose per strain and was irradiated in the range 0. The latter pack provided partial spoilage data from which the "12D", or minimal radiation dose (MRD), was estimated. Irradiation was conducted with 60Co gamma rays at -30 + 10 C. The irradiation temperature was controlled and monitored continuously as described by Jarrett (29). Assay for spoilage. The screening packs were incubated for 3 months and the clearance peaks for 6 months at 30 C and examined for swelling and for the presence of C. botulinum toxin and viable cells as cited earlier (3). Calculation of radiation resistance. The 12D or its equivalent (MRD) was estimated by three independent statistical methods. (i) Assuming exponential spore death in the cans, D values were computed both by the conventional Schmidt-Nank formula The MRD was calculated as D x 12. (ii) Assuming, on the other hand, that the spore death rate follows a normal distribution, the 12D equivalent was found by the Anellis-Werkowski equation (7): (iii) Also, whenev-er spoilage data was adequate, the MRD was computed by the Wiebull expression (6): which makes no assumption about the shape of the death rate curve. RESULTS Comparative radiation resistance of C. botulinum strains. The severest criterion of spoilage of irradiated cans of food is the presence of recoverable C. botulinum cells. Swollen cans always had toxin and viable cells; flat cans frequently contained both toxin and recoverable cells; nonswollen nontoxic cans occasionally harbored dormant survivors, whereas sterile cans were always flat and nontoxic. Hence, Table 2 provides only viable cell spoilage data for the three foods with each of the 10 strains at all radiation levels, as well as Schmidt-Nank D values based upon these data. Interestingly, the D values increased with increasing dosage in each food. This deviation from a nonlinear D value response was also observed with irradiated pork (4) and several other foods (6). This preliminary pack also indicated that the experimental sterilizing dose (ESD) for codfish cake was 2.75 < ESD < 3.0 Mrad, for corned beef was 2.0 < ESD < 2.25 Mrad and for pork sausage was 1.63 < ESD < 1.90 Mrad. Information in Table 2 was used to determine the comparative resistances of the 10 botulinal strains in the three foods. Table 3 lists the LD5, and D values, and their confidence intervals, of each strain estimated by the Spearman-Kiirber (6) and Schmidt (39) expressions, respectively. The Schmidt-Nank formula was not employed due to the dosedependent D values mentioned above. Spores of strain 53B were the most radiation resistant in codfish cake (D = 0.331 = 0.019), 77A in corned beef (D = 0.262 i 0.011), and 41B in pork sausage (D = 0.123 ± 0.012). The order of decreasing resistances of the 10 strains in the three foods is indicated in Table 4. MlD. Clearance pack data reflecting the three types of spoilage for each food are presented in Table 5. For codfish cake, the sterilizing doses were identical for the three According to the most stringent spoilage criterion, the MRD for the three food products was estimated both on the basis of exponential and normal distribution death kinetics ( Effect of sublethal doses on spoilage. Irradiation of the corned beef screening pack to 0.5 Mrad evoked a remarkable increase in the rate of visible spoilage over the unirradiated controls (Fig. 1). The effect manifested itself within 2 weeks of incubation of cans containing strains 62A, 77A, 12885A, 9B, 40B, and 41B. It required 8 to 12 weeks of incubation for the controls to equal the number of spoiled cans produced by 0.5 Mrad; however, control cans with strain 40B never did overtake the 0.5-Mrad cans. A dose of 0.75 Mrad caused an even more notable increase in the spoilage rate of cans inoculated with strains 40B and 41B than when irradiated with 0.5 Mrad; the remainder of the test organisms (33A, 36A, 51B, 53B) were unaffected by these low doses. At doses above 0.75 Mrad, unirradiated cans equaled or exceeded the irradiated spoilage response. Codfish cake and pork sausage did not undergo this phenomenon. DISCUSSION It was observed earlier that a cured (3) and an uncured pork product (4) yielded D values which increased with increasing radiation doses when estimated by the Schmidt-Nank method. Similar findings were made in this investigation with an uncured fish product of low brine content (0.79%), with corned beef of high brine concentration (4.50%) and containing curing salts, and with pork sausage which had a higher level of brine (5.63%) and containing spices instead of curing salts (Table 2). Thus, the types of irradiated foods which gave dose-dependent D values are sufficiently diverse as to make one wary of the Schmidt-Nank statistical procedure. The substitute D value calculation employed herein has no such defect. D values are commonly used to compare resistances between organisms subjected to identical conditions of physical or chemical stress. Table 4 indicates that LD,0 values may be employed as an alternative technique to determine comparative radiation resistances between strains of C. botulinum spores in different foods. The order of decreasing resistances obtained for the 10 strains by the LD,0 and D value estimates are almost identical in the three foods. Since the D value assumes exponential death kinetics, and the Spearman-Karber LD,O does not assume the shape of the dose-response data (6), we find the latter more attractive for ascertaining relative resistances between organisms. Lewis (32) and Vas (46) also evinced a preference for the LD,0 (or ED,,), which they used for evaluating thermal resistance processes. None of the inoculated packs provided sufficient data for analysis by the Weibull distribution. Nevertheless, to determine whether additional useful information may be extracted from the spoilage data at hand, the screening pack viable cell results obtained with each strain in a food were pooled as if every can subjected to a specific dose reflected a mixture of all 10 strains ( Table 7). The resulting data were amenable to statistical treatments by the Weibull, normal and exponential distribution methods (Table 8), and indeed had prediction value for the outcome of the codfish cake and corned beef clearance packs, but not for pork sausage. The ESD of the pooled screening (Table 8) and the clearance ( Table 6) packs for the three foods were remarkably close. The MRD computed by the exponential procedure for the accumulated screening, and clearance, packs did not agree, but the MRD values estimated by the normal distribution were surprisingly close for both codfish cake and corned beef (3.32 and 3.09, and 2.75 and 2.57 Mrad, respectively). The Weibull MRD values for these two foods agreed very closely with the normal calculations. The Weibull technique could not be used with the pork sausage pooled data since the plotted points were all above the 50% sterility level. The Weibull equations for the combined codfish cake and corned beef data are Table 2, footnote a). e Doses increase in 0.25-Mrad levels except for pork sausage, which increase 0.27-Mrad steps. respectively. The shape parameter ,B (3.1, 4.8) indicates that the death rate of the spores in the two foods reflects a normal, or related, type of distribution, and is quite remote from exponential death whose , is near 1.0. Perhaps that is the reason why the predicted exponential MRD values (Table 8) did not agree with the exponential MRD values of the clearance packs (Table 6). Of the three foods, only corned beef exhibited accelerated spoilage with low dose (0.5, 0.75 Mrad) irradiation ( Fig. 1). High brine content did not seem to be the primary cause of this activity, since pork sausage was higher in brine concentration (5.63%) than the corned beef (4.50%). Apparently the curing salts (NaNO2, NaNO This phenomenon also occurred in irradiated cured ham (3). The influence of food substrate on the radiation resistance of bacteria is demonstrated in Table 4. Whereas strain 53B was the most resistant of the 10 organisms tested in codfish cake, it was fifth in the order of decreasing resistance in corned beef and seventh in pork sausage. On the other hand, strain 77A was the most resistant in corned beef and strain 41B was the hardiest in pork sausage. These results emphasize the need to screen a number of test organisms in each food prior to determining a prototype radiation process, rather than use an arbitrarily selected single index organism. Codfish cake, the only food product of the three tested which had no chemical additives of significance (Table 1), had the highest MRD. The two foods which contained additives (curing salts, spices, relatively high brine levels) had notably lower MRD values than the fish (Tables 6, 8). That radiationor heat-injured bacteria are more sensitive to curing agents and NaCl is well documented. That certain spices also enhance the radiation death of C. botulinum spores was observed by Anderson et al. (1), but this phenomenon is not as widely recognized. Although Huber et al. (26) found that spices protected foods against deleterious radiation changes by acting as chemical interceptors of free radicals, they did not investigate the ability of spices to sensitize bacteria. Our data indicate that the proper combination of spices with high brine content were at least as effective, if not more so, in reducing the MRD of pork sausage as were the curing agents with NaCl in lowering the MRD of corned beef (Tables 6,8). Investigations involving spices and NaCl individually, and in combination, would help elucidate the role of these additives on C. botulinum in the radiation process. " Insufficient data for computation. The prototype radiation process, based upon the computation of a 12D equivalent by the normal distribution method (for reasons discussed above), using the clearance pack data (Table 6), is 3.1, 2.6 and 2.4 Mrad for codfish cake, corned beef, and pork sausage, respectively, when irradiated at -30 i 10 C.

v3-fos

2020-12-10T09:04:12.382Z

1972-07-01T00:00:00.000Z

237230508

s2

Machine for Automatic Bacteriological Pour Plate Preparation A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated. A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated. Development of instrumentation for bacteriological analysis has been slow compared with progress in many other branches of science. The plate count, particularly in view of its wide use, has received very little attention, and demands almost the same repetitive manual work that it did at the beginning of the century. Success has been achieved in automation of various bacteriological processes (1, 3-6, 10) but only recently have there been signs that whole, or even usefully large, portions of plate counting can be satisfactorily automated (2,3,(7)(8)(9)(10)(11)(12)(13). Several aids of undoubted value recently have been introduced, for example, the Petri-Scan automatic colony counter (American Instrument Co. Inc., 8030 Georgia Ave., Silver Spring, Md. 20910), the Autofill 600 petri dish filler (Buchler Instruments Div., Nuclear-Chicago Corp., 1327 16th St., Fort Lee, N.J. 07024) and the Petrimat petri dish filler (Bellco Glass Inc., Vineland, N.J. 08360). The last two instruments are intended for use in laboratories where surfaceinoculated plates are used routinely and would be of little value where counts are made routinely with poured plates. The problems encountered in the construction of automatic "classic" bacteriological process equipment are probably unique among the sciences. The most important requirements are for constant provision of sterile containers and media, minimization of hazards and errors caused by carry-over of contamination, dealing with the tremendous range of bacterial concentrations encountered (109 or more), and the difficulties of detecting instrumentally the cues commonly used in recognition or counting (such as colonial morphology or color, or even just distinguishing bacterial colonies from sample debris). In addition, the variety of tests which may be necessary on different samples demand considerable versatility from an instrument system. We have developed an instrument which, we believe, satisfactorily carries out the most time-consuming parts of the preparative and technical work involved in a bacteriological pour-plate count and which can dramatically increase the output of one or more technicians. The instrument, for which patent application has been made (British patent application no. 51377/70), was produced in response to requests from quality control bacteriologists in the food industry and represents one of our approaches to the problems in this area. MATERIALS AND METHODS General description of the bacteriological plating machine. The prototype is shown in Fig. 1-6. From a sample suspension it automatically prepares a stack of inoculated, agar-filled petri dishes, labeled with sample and dilution number, ready for incubation. According to the setting of its controls it will select and prepare up to two dilutions in one agar (e.g., violet red and bile (VRB) for coliform counts) and up to eight dilutions in a second agar (e.g., plate count agar (PCA) for total aerobic counts). The various operations are carried out sequentially and simultaneously. All materials are handled in bulk, and fresh, sterile pipettes for dilutions are produced within the machine as required. Dilutions are made within the petri dish itself, the residual liquid after transferring a sample serving as the inoculum. In its present form, the machine adds 10 ml of diluent to each dish and transfers 1.1-ml samples through the dilution range, thus achieving a set of decimally diluted inocula of 10 ml each. To these are added either 10 ml of double-strength or 5 ml of triple-strength agar. The final content of each petri dish is, therefore, 20 or 15 ml of normalstrength agar. It is convenient to use suitably concentrated nutrient solutions as the diluent and to add blank agar to avoid keeping nutrients for long periods at elevated temperatures; the agar itself is not significantly affected by such holding. Pipettes are produced automatically, as required, from the concentrated agar used to fill the dishes. They are extruded as cylinders from a water-cooled die and discarded after use. Cutting is carried out with a stainless-steel blade, sterilized by electrical heat to 250 C. A constant supply of sterile pipettes is thus assured and carry-over of contamination eliminated. A separate diaphragm pump, connected through the center of the die, controls the volume of liquid taken up and ejected by each pipette. The shape of the agar cylinder used to transfer liquid is unimportant, provided that it is long enough to make contact with the diluent in the petri dish. The machine uses the clover leaf form (Fig. 7) to obtain a large cross-sectional area without tendency to drip. In its present form the machine can prepare one filled, labeled dish every 15 sec, or 2,000 per 8-hr day. Its speed can be increased by up to 30% if necessary. The number of samples for processing is determined by the number of dilutions required. At its lowest value, i.e., if two dilutions in VRB agar and eight in PCA are repeatedly called for, slightly less than 200 samples per day can be processed, allowing for start-up and run-down time. Layout. The agar preparation unit, bulk diluent stores, and metering pumps are located in the lower part of the machine ( Fig. 1 and 2) to avoid the need to lift heavy weights. Diluent bottles of up to 10 liters can be accommodated, allowing up to 4 hr of uninterrupted operation. Agar stores are replenished while the machine is running. All bacteriological operations, i.e., dispensing Agar. For routine work, agar can be prepared in the electrically heated, steam-jacketed lower tank. Agar powder and cold water are added to the tank about 35 min before agar is required. A centrifugal pump circulates the agar suspension during boil-up and is later used to pump the solution up to the storage tank, where it is held at 60 to 80 C until used. An alarm circuit in this tank warns of a low level in time for a fresh mix to be made. Petri dishes. The rotating holder can take up to 200 disposable, plastic petri dishes in eight stacks. As one stack empties, the holder rotates to the next position to bring a fresh stack above the dispenser. Fresh dishes can be added at any time. Electricity and water. A normal-main electric supply is required. The machine consumes about 3.5 kv with all units working, mostly by the agar preparation unit. Cooling water is required for pipette extrusion and for a condenser on the agar steam jacket. Coolant temperatures up to 30 C can be tolerated, but pipette quality decreases rapidly above 25 C. Operation. Start-up operations are completed quickly. These include switching on services and adding agar powder (conveniently by volume) and water to the preparation tank. Next, and before beginning work, occur the following procedures: (i) renewal of diluent bottles and supply tubes; (ii) filling of the petri dish store; (iii) removal of a disinfectant cup from the base of the pipette dispenser (if FIG. 4. Covers are removed from the dilution area. 4, Pipette dispenser; 5, pipette; 6, pipette cutting mechanism; 7, sample container; 8, diluent delivery tubes; 9, dosing pump for emptying and filling pipette. petri dishes, removing their lids, performing dilutions, adding agar, mixing, replacing lids, and labeling dishes are carried out in the covered portion of the machine below the main working surface. Dishes are held in a rotating cross-shaped carrier which rotates from station to station to allow the various operations to be carried out at different positions. Petri dishes and prepared agar are stored above the main working surface ( Fig. 1 and 2). Dishes are added to the rotating store (200-dish capacity) whenever convenient. Agar (10 liters) can be pumped into the storage tank as soon as it is ready, leaving the preparation tank free for rinsing or further preparation. Diluents. These are prepared in bulk, away from the machine, in bottles fitted with supply tubing and delivery nozzles sterilized by autoclaving at the same time as the diluent. Bottles of up to a 10-liter capacity can be accommodated. A length of silicone rubber tubing, included in each supply line, is stretched over the appropriate peristaltic pump head when a new bottle is fitted into the machine. Diluents are prepared as solutions of the media nutrients at 1.5 or 2 times the intended final concentration. Diluents do not, therefore, deteriorate by FIG. 5. A diluent bottle, peristaltic pump, and the being maintained at high temperatures. agar preparation unit. dish is brought into an inclined position. At station 2 the dish engages with a rotating disc and, itself, begins to rotate. (ii) An agar pipette is dispensed. The dispenser moves to the sample container, withdrawing 1.1 ml, and then to the first petri dish where the pipette is emptied. A 10-ml diluent (diluent 1) is pumped into the dish. The contents of the dish mix rapidly as a result of its rotation. The pipette is cut and falls into a waste receptacle. A new pipette removes 1.1 ml of the decimal dilution. A fresh dish (dish 2) is dispensed at station 1. (iii) The carrier indexes around again, taking dish 1 to station 3 (agar addition) where it again rotates while in an inclined position. Agar is pumped into the dish and mixed. At the same time events (i) and (ii) are repeated with dishes 2 and 3, except that the pipette does not move to the sample container. (iv) The carrier indexes again, taking dish 1 to station 4. On the way its lid is replaced. At station 4 the dish is pushed from beneath by the printing mechanism and rides over a set of catches to form the first dish of a stack. Events (i) through (iii) are repeated. (v) At a point determined by the operator when dialing, the pipette dispenser again moves to the sample container, picking up a fresh sample of thẽ suspension. The sequence then continues and uses diluent 2 to obtain a second set of dilutions. The machine will then complete its bacteriological operations while the next sample is prepared. Before, or soon after dialing the next sample, the operator removes the stack of prepared dishes. Only one sample is accepted at a time. Sequence of events after pressing the START button. All operations are carried out with the dishes seated in rings at the ends of a fairly complex cross-shaped carrier mechanism. The carrier indexes around one quarter of a revolution for each operation. The following sequence results when the START button is pressed. (i) A petri dish (dish 1) is dispensed (station 1, Fig. 1) and carried around to the dilution position (station 2). On the way its lid is removed, and the FIG. 7. Used agar pipettes. RESULTS Speed. The machine completes a full dilution-range count preparation from two media in 2.5 to 3 min and smaller count ranges in less time. The operator may use this time to prepare the next sample. With many types of material, sample preparation is then likely to be the rate-determining factor, and it is reasonable to expect that two or more operators can use the machine simultaneously. The potential output (2,000 plates per day or 200 sets of eight PCA and two VRB agar plates) exceeds the capabilities of most bacteriologists. Labor saved on preparative work is marked. All operations concerned with pipettes and small bottles of agar and diluents (uncapping, washing, refilling, recapping, etc.) are eliminated. Agar preparation occupies very little time, equivalent to that spent steaming separate bottles for a normal manual count. Diluent (nutrient) preparation time is equivalent to that required to prepare the same volume prior to dispensation into bottles for the manual count technique. Some laboratories might have difficulty autoclaving the largest and most economical container sizes. Large diluent bottles may be kept in the machine overnight for reuse the next morning, provided the nozzles are removed from their brackets and immersed in Formalin. Total labor saving appears to be about 70% over the whole count in our laboratories when about 150 to 180 samples per day are processed. This represents a cost reduction of about 47% over each manual count. Against this saving, of course, must be weighed the cost of the machine. Accuracy. The machine prepares excellent decimal dilutions, well mixed with agar, showing no sign of "carry-over" and with a maximum temperature of 40 to 42 C after mixing. There is no reason, therefore, to expect the accuracy of the machine to be inferior to that of a properly carried-out manual count. A comparison of approximately 800 sets of data from manual and machine counts, on a variety of foods, including results from the machine at all stages of its increasing operational reliability, showed a significantly lower count (P = 0.01) for the machine. A later set of 144 counts, however, obtained as quadruplicated analyses on 36 specimens (meats, vegetables and swabs) gave the following results (log,0 counts/g): the machine count had a mean of 3.89 (estimate of variance was 0.0027); the manual count had a mean of 3.87 (estimate of variance was 0.082), indicating that the variability of counts made by the machine is significantly less (P < 0.1%) than those done manually. Evaluation is continuing. In the absence of nutrients, and at the high storage temperature, multiplication of bacteria in the boiled agar is most unlikely. However, when prepared by boiling, the agar has a very small but detectable level of residual bacterial contamination. For example, in the worst case during trials in which 250 plates were prepared at a time from boiled agar, 93% of the plates were clear and 6% had one contaminant colony. The corresponding figures for autoclaved agar were 98 and 2%. (This partially may, in fact, be due to aerial contamination, indicating that the addition of a filtered air supply might be desirable.) The convenience of preparing agar within the machine would make a small error in counting acceptable; in fact, for normal quality control work with foods etc., even a 6% chance of finding one contaminant colony on a plate leads to an undetectably small error, since it is unusual for plates containing fewer than 20 colonies to be counted. It is feasible, of course, to prepare agar outside the machine and to sterilize by autoclaving. In most instances, however, the extra labor would be unjustified. Only one plate is prepared from each dilution. Many bacteriologists, however, like to inoculate petri dishes in duplicate from each dilution. The effect of such duplication on the accuracy of the count is questionable; it would be far better if the dilution sequence were also repeated and still better if the whole product were resampled. This is feasible with the machine described here. If the utmost in accuracy is required, therefore, it is more reasonable to take two samples from different parts of the product rather than to duplicate inocula from particular dilutions. DISCUSSION The labor involved in bacterial counting can be conveniently divided into "support" and "technical" work. In large laboratories support work may be carried out by low-level staff and includes preparation and dispensation of media into bottles, cleaning, sterilizing, and distributing homogenizers, bottles, and pipettes, etc., and disposal of contaminated wastes. Technical work includes everything pertaining to the actual count such as sample preparation, dilution and inoculation, counting or estimating numbers of colonies, calculation and reporting of results, and carrying out whatever subsequent special tests may be necessary. Costs can be divided into "direct" costs which include, for example, media, petri dishes, and pipettes consumed during the test (either as disposables or breakages of reusable items) and "indirect" costs which include all labor, steam, gas or other services, and depreciation of equipment. The importance of each item naturally varies greatly among laboratories and according to the types of analyses made. Our main interests were the total viable aerobic and VRB agar coliform counts using poured plates. Average figures for technical time spent on various operations of the viable aerobic count in local laboratories are shown in Table 1. The largest single item was, surprisingly, accurate sample weighing, but diluting plating and (also surprisingly) labeling petri dishes and simply keeping materials at hand were notably time consuming. On the other hand, colony counting occupied a relatively small proportion of the total time. Support labor was notably expensive ( Table 2). A high proportion of this labor, associated with readying pipettes, bottles, etc. could obviously be eliminated if bulk handling were introduced. Pipettes alone accounted for 22.5% of total costs (replacement and labor) and were obviously a particularly important target for automation. In an additional analysis of an individual count which was broken down into individual movements (such as pick up bottle, remove bottle cap, pick up pipette, place pipette in bottle, and so on) over 1,000 operations were noted. This did not include counting itself, for which an operational breakdown was open to many interpretations. The fundamental processes, however, formed less than 10% of these movements. (For example, dilution consists merely of transferring liquid from one container to another; all other operations such as opening bottles are wasted effort but are nevertheless essential to fit the technique to our manual abilities.) It was obviously inefficient, therefore, to design equipment to do single operations (e.g., diluting); at least two stages of the count would have to be automated and integrated (e.g., diluting and plating) to avoid the need for human handling before and after each operation. (This effect was excellently demonstrated by the electronic colony counter tested by Malligo [8]. Although scanning and counting plates in 1 sec, only 40% of counting time was saved, as it did not eliminate the need for human handling before and after operations.) The following criteria were considered most important in the design of a bacteriological plate count machine. (i) The operations carried out by the machine would deviate as little as possible from the accepted manual technique. (ii) Savings in support work were as important as savings in technical work. (iii) Automation of single stages only was not worth considering. (iv) The extent of automation would be related to the capital outlay involved for each stage and the savings resulting from it. (v) Elimination of pipettes and manual pipetting, or both, would be a major objective, partly because of associated costs and partly because of the central position in the count. (vi) The machine would be sufficiently versatile to handle the major portion of routine counting requirements. It was decided that an experimental machine should be made carrying out all parts of the count except sample preparation (on account of the variability encountered), colony counting (because of the likely expense associated with such a small item), and some parts of media preparation. These facilities could be added as peripheral units to the central system if the need later arose. In addition, the machine would have to be able to prepare plates for at least the total viable aerobic and coliform counts, since sequential dilutions were frequently required for these. The ability to prepare plates for counts of Staphylococcus aureus, for example, and yeasts or molds was considered less important, since the levels of these organisms encountered in foods do not normally require sequential dilutions to be made. Work is now underway to redesign certain aspects of the machine to improve accessibility, versatility, and general smoothness of operation. We expect, for example, that increasing the number of media dispensed to at least four and the ability to make up to eight dilutions for each will allow the machine to satisfy any reasonable subsequent demands on performance. At the same time, evaluation work is being done on other types of counts besides those forming our initial interest (total and coliform) to ensure that the pour plate approach embodied in the machine can be used to its fullest in quantitative bacteriology.

v3-fos

2020-12-10T09:04:12.805Z

1972-07-01T00:00:00.000Z

237232311

s2

Gas-Volume Measurement System for Evaluating Effectiveness of Antimicrobial Compounds Yeast spoilage was followed by measuring gas volume produced inside a sealed bag of inoculated fruit. Volume of gas produced correlates with plate counts. Osmophilic fermentative yeasts, the major cause of spoilage in fruit juices and dried fruits, produce gas during the logarithmic growth phase. This phenomenon has been used in the rapid, simple, nondestructive method described below in evaluating the effectiveness of chemical antimicrobial agents for fruit products. Plate-counting the number of organisms present was too cumbersome and was not convenient for analyzing large numbers of samples. Gross examination of the samples for visible signs of spoilage was not satisfactory because it was too insensitive. Our method, though indirect, was found to correlate reasonably well with actual plate counts and to be reliable in determining onset of spoilage. The procedure used is to put the treated, inoculated fruit product (juice or solid) into a polyester bag and heat-seal it after collapsing the bag to remove as much air as possible. In some applications, the inoculated fruit is sealed in the bag and the treatment is added later. For example, a gaseous preservative is added after the bag is sealed by puncturing the bag near the corner with a syringe needle and then resealing it below the puncture. Obviously, more elaborate gas-volume measuring techniques (such as the Warburg respirometer) are available. The advantage of this method is its simplicity. An initial volume measurement is made by submerging the bag in water and measuring the displaced volume (Fig. 1). Further volume measurements are made at appropriate intervals during the period of incubation. The volume data are then plotted (Fig. 2). The logarithmic portion of the curve is determined by extrapolation to the base volume to establish 14( the onset of logarithmic growth. The length of time elapsed until the inflection point was used to gauge the amount of preserving effect of the treatment. This system greatly reduces the necessity of frequent measurements. The important element used in this technique is the bag. We employ Scotchpak heatsealable polyester film bags. These bags have a low permeability to gases and water vapor so that any volume measured is equivalent to the gas produced within the bag. Appropriate controls indicate that the gas is a result of microbial action. Commonly we used 6-by 11inch (ca. 15.2 by 27.9 cm) bags which have a maximum volume of about 1 liter and contain 100 g or 100 ml of product. For smaller samples we used smaller bags. Figure 2 shows the influence of 200 Mg and 500 Mg of potassium sorbate per ml, each added to 100 ml of reconstituted white grape juice inoculated with Saccharomyces cerevisiae, as compared with an untreated control. The potassium sorbate delays the onset of logarithmic growth and thus increases the storage life. Increasing levels of potassium sorbate provide longer preservation (Fig. 2). The viable yeast count of the grape juice in the bags is also shown (Fig. 2). The viable count correlates best with bag volume at the longer incubation times. For the 500 Mg of sorbate/ml treatment the correlation coefficient is 0.99 for seven observations, and for 200 Ag it is 0.92 for six observations. The correlation between bag volume and viable count for all the data in Figure 2 is 0.87. All these correlations are significant statistically (P = <0.01). The correlation for the four observations in the control is 0.75, which is not significant. The lag between viable count and bag volume is due, in part, to the amount of carbon dioxide required to saturate the product. With 100 ml of juice, this calculated volume is about 70 ml of carbon dioxide (1). Another factor is the lag between growth and gas production in microbial growth. The reproducibility of the measurements we have observed is also shown in Figure 2. The first volume measurements of nine bags for the sorbate-treated juice (500 gg/ml), before the volume inflection, ranged from 123 to 130 ml over a period of 73 hr. This is less than 6% variation in measurement when no control was made over pressure or temperature. At higher bag volumes, the repeatability of bag measurements is the same, so the percentage of variation is less. This technique has also been used to examine the spoilage of inoculated and rehydrated dried fruit. In some cases where spoilage is very slow and gas volume is not noticeable for a month or longer, it is possible to observe yeast growth before a definite increase in bag volume is detected. With prolonged incubation, the slow transfer of gases through the plastic bag wall may influence the volume of gas and thus would make this test inappropriate.

v3-fos

2018-04-03T02:59:28.011Z

1972-01-01T00:00:00.000Z

20262138

s2

Unusual Organism Which Gives a Positive Elevated Temperature Test for Fecal Coliforms' Organisms apparently not of fecal origin isolated from a sulfur hot spring gave a positive elevated temperature test for fecal coliforms. between coliforms of and origin the of the temperature This coliforms to produce gas from test may be run by determination of probable (MPN) in or by the filter technique on M-FC broth-soaked Nonfecal or coliforms give a positive reaction under a a elevated temperature test for At the present time, a distinction is commonly made between coliforms of "fecal" and "nonfecal" origin on the basis of the elevated temperature test (4). This test is based upon the ability of fecal coliforms to produce gas from lactose within 24 hr at 44.5 C. This test may be run by determination of most probable numbers (MPN) in EC broth or by the membrane filter technique on M-FC broth-soaked pads. Nonfecal or soil coliforms do not give a positive reaction under these conditions. Geldreich (2) discussed the significance of fecal coliforms and suggested that these organisms be used as indicators of fecal pollution of recreational waters. Hendricks (3) found that 24% of the Enterobacter (Aerobacter) group (nonfecal in origin) which he isolated from a "relatively unpolluted river" were positive in the elevated temperature test for fecal coliforms. Data presented here describe an organism from a sulfur hot spring which gives a positive elevated temperature test for fecal coliforms. Cultures were isolated and Gram-stained from eosin methylene blue (EMB) agar plates (Difco) inoculated from positive Brilliant Green lactose bile (BGB) broth tubes (Difco), from EC broth tubes (Difco), or from colonies on membrane filters (Millipore Corp., Bedford, Mass.) incubated on M-FC broth (BBL)soaked pads at 44.5 C. All cultures were retested in EC broth, and these inoculations were made by means of a needle. Indole production, hydrogen sulfide production, and motility were performed in SIM medium (Difco studied on nutrient agar supplemented with 0.4% gelatin (5). Cytochrome oxidase tests were performed with test strips (Pathotec, Warner Chilcott Laboratories). Flagellation was determined by use of a flagella stain. Samples were obtained from a sulfur hot spring, from the Laramie river, and from an aerated sewage lagoon. The data in Table 1 show results of studies conducted in April 1970. The finding that 17 of 19 isolates of Escherichia coli produced gas at 44.5 C in EC broth supports the idea that "typical" E. coli isolates give a positive elevated temperature test. In contrast, the fact that 25% (5 of 20) of the Enterobacter aerogenes isolates from the lagoon and river also gave a positive elevated temperature test, a result similar to the value found by Hendricks (4), suggests that a single or few samples are insufficient to judge water quality, since nonfecal coliforms might be falsely confused with coliforms of fecal origin. This could be particularly important in situations in which great distances might limit the number of samples taken. In marked contrast to the results obtained from other sources, 9 of 10 apparent isolates of E. aerogenes from the hot spring drainage produced gas in EC broth at 44.5 C. This result indicates that the incidence of apparent E. aerogenes capable of giving a positive elevated temperature test may depend upon the environment. The isolates that produced gas in EC broth at 44.5 C were obtained throughout the hot spring drainage as soon as the temperature of the water had dropped to 46 C. This along with the visual observation of distance from the spring suggested that these organisms were not of fecal origin. To determine if this was a fortuitous result, another series of samples was taken in November 1970. Data in Table 2 show that 28 of 58 isolates in November were posi- tive in the elevated temperature test, confirming the April result that an unusually high percentage of falsely positive fecal coliforms were found in the hot spring drainage. Four "typical" E. coli cultures were recovered in November, and all were positive in the elevated temperature test. Approximately half of the November isolates were lightly golden in color when grown on nutrient agar at 35 C, and, upon storage at room temperature, these cultures became bright yellow. This finding was not observed in April. The yellow cultures exhibited typical Enterobacter colonies on EMB agar; gave typical indole, methyl red, Voges-Proskauer, citrate (IMViC) reactions; produced gas in BGB broth; were motile; and were gram-negative rods. However, they failed to produce hydrogen sulfide or to liquefy gelatin and were cytochrome oxidase-negative. Flagellation was typical of the Aeromonas group (1). Old cells had predominantly a single polar or subpolar flagellum, whereas young (6to 8-hr) cells showed additional lateral flagella. The exact taxonomic position of these organisms is thus unclear. Data in Table 2 also show that these organisms displayed a positive elevated temperature test when the membrane filter procedure was employed. Moreover, they may be nega- tive (yellow colony) by the membrane filter method and positive by the EC broth method. It should be noted, however, that the color of these organisms by the membrane filter method was not as bright blue as typical E. coli colonies. Regardless of the taxonomic position of the yellow organisms, it is clear that, except for color, they are not easily distinguished from E. aerogenes, particularly in a routine bacteriological water analysis. It, therefore, appears that the use of multiple criteria in determining water quality is desirable.

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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-1131 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. 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. 23,1972 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). Differentiation of species. Other characteristics can be used further to differentiate the enterococcal specimens (divisions I and II) from each other. Tolerance to tellurite, the ability to reduce tetrazolium and form acid from sorbitol and glycerol, and the inability to form acid from arabinose indicate that the organism is S. faecalis or one of its varieties; whereas, the opposite reaction on all five tests indicates that the organism does not belong to division L. A clinical laboratory would probably never need to determine the varieties of S. faecalis. The differentiation of these two varieties (liquefaciens and zymogenes) is on very questionable grounds. Updyke (24) showed that 88% of 90 enterococcal isolates were alphahemolytic in sheep blood and beta-hemolytic in rabbit, horse, and human blood-agar pour plates. Deibel (2) has pointed out that S. faecalis strains show frequent loss of hemolytic activity and vary in their proteolytic activity (depending on the type of medium used to test for hydrolysis of gelatin). He thus suggested that the designation of varieties of S. faecalis be discontinued. We are in full agreement with Deibel in this respect. Division II species can be differentiated from species in division I (faecalis) and division III (bovis and equinus) by a number of tests. The formation of acid in arabinose, sucrose, and mannitol broths is used to differentiate S. faecium from S. durans. S. faecium should form acid in all three carbohydrates, but S. durans should not. However, 10 of 13 strains of S. durans formed acid in sucrose broth; thus, it should not be used as a differential characteristic for these organisms. These data also support the contention that S. durans should be considered a variety of S. faecium. Like Duma et al. (5) Additional isolates and characteristics must be studied before conclusions can be made as to the best classification of this organism. These data indicate that the spectrum of tests used here will differentiate about 98% of the group D streptococci isolated from human sources. Emphasis must be placed not on a limited number of tests but on a larger spectrum of tests to recognize varieties within the species. The clinical laboratories probably do not need to worry about species like S. faecium var. casseliflavus, S. avium, or S. equinus; however, they should be aware of the fact that these entities do exist and must be dealt with if correct speciation is attempted. We would like to caution the workers in clinical laboratories against relying on any single test that might be advocated for identifying enterococci. Investigators studying any aspect of species differences should perform at least the number of tests reported here to be of differential value.

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Agar Medium for Differential Enumeration of Lactic Streptococci An agar medium containing arginine and calcium citrate as specific substrates, diffusible (K2HPO4) and undiffusible (CaCO3) buffer systems, and bromocresol purple as the pH indicator was developed to differentiate among lactic streptococci in pure and mixed cultures. Milk was added as the sole source of carbohydrate (lactose) and to provide growth-stimulating factors. Production of acid from lactose caused developing bacterial colonies to seem yellow. Subsequent arginine utilization by Streptococcus lactis and S. diacetilactis liberated ammonia, resulting in a localized pH shift back toward neutrality and a return of the original purple indicator hue. The effects of production of acid from lactose and ammonia were fixed around individual colonies by the buffering capacity of CaCO3. After 36 hr at 32 C in a candle oats jar, colonies of S. cremoris were yellow, whereas colonies of S. lactis and S. diacetilactis were white. S. diacetilactis, on further incubation, utilized suspended calcium citrate, and, after 6 days, the citrate-degrading colonies exhibited clear zoning against a turbid background, making them easily distinguishable from the colonies of the other two species. The medium proved suitable for quantitative differential enumeration when compared with another widely used general agar medium for lactic streptococci. other two species by its ability to produce diacetyl and its reduction products from citrate (13). Reddy et al. (11) exploited the ability of these species to utilize arginine in order to develop a medium for qualitative and quantitative differentiation of S. cremoris and S. lactis in mixtures. On this solid medium, colonies of S. diacetilactis and S. lactis were indistinguishable. The first attempt at devising a differential agar containing citrate for use in identifying the citrate-fermenting lactic streptococci and Leuconostoc species in mixed lactic starters was made by Galesloot et al. (4). They used uniformly suspended, poorly soluble calcium citrate as the specific substrate, thereby imparting pronounced turbidity to the solid medium. Citrate-utilizing colonies developing on this agar exhibited clear zoning through degradation of the relatively insoluble tricarboxylic acid salt; colonies formed by non-citrate-fermenting species failed to show zoning. Nickels and Leesment (8) improved the medium by providing for distinction between slow and fast citrate-fermenting microorganisms. Their technique afforded differential enumeration of not 947 only non-citrate-fermenting lactic streptococci and S. diacetilactis, but also the slow-growing and slow-citrate-fermenting Leuconostoc species. Our medium is an extension of the differential agar developed earlier (11) for the separation of S. lactis and S. cremoris. With this medium, colonies of the three species could be distinguished. This was accomplished by inclusion of relatively insoluble calcium citrate as an additional substrate and by providing suitable pH adjustment of the original S. Iactis-S. cremoris differential agar. MATERIALS AND METHODS Cultures. Fourteen S. cremoris, 10 S. lactis, and 13 S. diacetilactis strains were used in this investigation. The cultures were obtained from the culture collection of the Department of Food Technology, Iowa State University. Cultures were maintained by twice-a-week transfer in reconstituted milk (11% solids). The inoculated cultures were incubated at 21 C for 18 hr. Between transfers, they were stored at 5 C. Agar preparation. The amount of agar required for 1 liter of the medium was suspended in 500 ml of distilled water and steamed until dissolution. In another glass beaker containing 500 ml of distilled water, 10 g of calcium citrate and 15 g of CMC were suspended and heated while being stirred until a homogeneous, white, turbid suspension was formed. The two portions were mixed together in a separate stainless-steel vessel containing the required quantities of tryptone, yeast extract, Casamino Acids, K2HPO4, and arginine. The mixture was covered and steamed for 15 min. The pH of the medium after steaming was adjusted to 5.6 with 6 N HCl. The agar was then dispensed into bottles in 100-ml quantities and sterilized at 121 C for 15 min. Media comparison. Eugonagar (BBL, Bioquest, Cockeysville, Md.) was used to test the efficiency of the differential medium for the quantitative recovery of fastidious streptococci. Plating techniques. The spread plate procedure on previously poured agar surfaces as described by Reddy et al. (11) was followed. At the time of plating, the pH of the medium was 6.35 + 0.05. Wherever more than one species was plated together in any one combination, 1.0 ml from each culture in the combination was mixed together in a sterile screw-cap test tube. Serial dilutions of the mixture were then made, and 0.1 ml of the dilution was spread on the agar surface. Conventional pour platings were made according to Standard Methods for the Examination of Dairy Products (1). The plates were incubated in a candle oats jar at 32 C and were examined after 36 to 40 hr and after day 6 of incubation. Counting. After 36 to 40 hr of incubation at 32 C, the plates were removed from the candle oats jar and counted. First, the total count was taken, and then the counts of yellow S. cremoris colonies were taken. The plates then were returned to the candle oats jar and incubated for an additional 4 days. At the end of incubation, the plates were removed and exposed to the air for 1 hr, and the colonies were counted. Again, the total count was taken first. Then, all colonies showing zones of clearing of the turbid suspension of calcium citrate were counted. The latter figure represented numbers of S. diacetilactis. The sum of S. cremoris (taken after 36 to 40 hr) and S. diacetilactis counts was subtracted from the total count to obtain the S. lactis population in the mixture. An alternate method of counting was used for culture mixtures containing slow-arginine-hydrolyzing or nonhydrolyzing strains of S. diacetilactis. These strains sometimes produced yellow colonies similar to S. cremoris after 36 to 40 hr. In such instances, when the first counts were made, all the yellow colonies were marked with an indelible felt pen (Carter's Marks-A-Lot, Carter's Ink Co., New York). When the final count was taken, if some marked colonies showed clearing, they were counted as S. diacetilactis, and the corresponding number was subtracted from the original yellow colony count to obtain the accurate value for S. cremoris numbers. Test for rates of citrate utilization in a simulated system. To substantiate the need for pH adjustment of the agar to obtain sufficient clearing of the suspended calcium citrate within 6 days at 32 C in a candle oats jar, an experiment in a broth system that would simulate the conditions in the agar was designed. Agar and CMC were deleted, and insoluble calcium citrate was replaced by an equivalent amount of citric acid; other ingredients and their concentrations remained unaltered. Broth pH was adjusted to 6.3 ± 0.05 and 7.00 + 0.05. One-milliliter portions of a 10-5 dilution of fresh 18-hr milk cultures of S. diacetilactis 18-16 and 26-2 were inoculated into broth and incubated at 32 C for 24 hr. Samples of the cultures were aseptically removed at various intervals, and the amount of citrate was determined by the method of Marier and Boulet (6). The pH of the samples also was determined simultaneously. The bottle contents were mixed at intervals to enable the settled CaCO, to neutralize the developing acidity more readily and uniformly. RESULTS AND DISCUSSION Preliminary studies were made to modify the medium described by Reddy et al. (11) to accommodate additional differentiation of S. diacetilactis. When calcium citrate was added to the medium and S. diacetilactis was spread on it, no clearing was observed even after prolonged incubation in a candle oats jar. To increase citrate uptake by S. diacetilactis, the pH was lowered to 6.3. Rapid utilization of calcium citrate by S. diacetilactis then occurred within 2 to 6 days of incubation in a candle oats jar. Lowering of the initial pH in the medium, however, changed the medium color from violet to light yellow and drastically affected the efficiency of differentiation between S. lactis and S. cremoris because the differentiation was based on acid-base reactions of the pH indicator. To offset this loss of efficiency, an increase in arginine level was required. Graphs showing rates of citrate utilization by S. diacetilactis strains 18-16 and 26-2 in broth systems adjusted to pH 6.3 and 7.0 are presented in Fig. 1 and 2. The pH values at the various sampling intervals also are shown. The citrate utilization patterns of these two strains were similar. No appreciable change in pH was VOL. 24,1972 observed within 14 hr of incubation; correspondingly, no utilization of citrate occurred within 14 hr. Beyond this time, a correlation was observed between decrease in pH and citrate uptake. When the broth pH was initially adjusted to 6.3, citrate utilization was complete within 18 hr of incubation, and the pH of the broth registered a rapid decrease after 14 hr. Delayed citrate utilization was noticed in the broth preadjusted to pH 7.0. Similarly, pH depression in the neutral broth system also commenced later, i.e., only after 16 hr of incubation. In both broth systems (pH 6.3 and 7.0), the two strains of S. diacetilactis, after identical incubation periods, exhibited an abrupt shift in pH towards neutrality. In the medium adjusted first to pH 7.0, this phenomenon was noticed after 21 hr, and in the broth preadjusted to pH 6.3 this shift occurred after 17 hr. These results suggest two possible explanations in regard to the relationship of citrate utilization to pH of the medium. It is possible that once citrate utilization is initiated pH does not play a major role in the uptake of the tricarboxylic acid. The other possibility is that a major portion of citrate, if not all, is utilized when the pH is depressed below 6.0. The shift in pH from the acid range toward alkalinity possibly was caused by the liberation of ammonia from arginine. Although these broth systems do not completely simulate the conditions in a differential agar plate, the results obtained showed the need for pH adjustment in the agar medium to obtain rapid clearing of calcium citrate. The requirement for pH adjustment to obtain rapid utilization of citrate by S. diacetilactis was encountered in our earlier investigation in the development of a differential broth for lactic streptococci (10). The optimal pH range for citrate permease probably is involved in this phenomenon. The completed medium gave excellent growth and differentiation for all possible combinations of the 14 S. cremoris, 10 S. lactis, and 13 S. diacetilactis strains. S. cremoris produced yellow colonies with yellow zones (indicative of acid production alone), whereas S. lactis and S. diacetilactis exhibited white colonies (indicative of neutralization of acid by the liberated NH.) after 36 to 40 hr of incubation in a candle oats jar at 32 C. The acid-induced yellow color of the indicator in and around S. cremoris colonies tended to diffuse out rapidly beyond 40 hr of incubation; past this stage, clarity of differentiation was lost. Incubation of plates for 4 additional days in the candle oats jar permitted S. diacetilactis to exhibit clear zones around individual colonies (Fig. 3 and 4). A CO2 incubator could be substituted for the candle oats jar. No clearing was observed, even after prolonged incubation, when the plates were incubated in air at 32 C. The increased rate of citrate utilization in candle oats jar probably is caused by the pH-depressing effect of the high CO2 tension of the enclosed environment. The pH of the medium was lowered from 6.35 + 0.05 to 6.05 i 0.05 in a candle oats jar as compared with 6.1 + 0.05 in a CO2 incubator. The pH of sterile agar in a corresponding poured plate incubated at 32 C in air for the same period was 6.35 + 0.05. By conventional pour plating, distinct differentiation was not achieved. Maximal differentiating efficiency was obtained only when the counts on the individual plates did not exceed 250 colonies and fresh medium was employed. The efficiency of recovery of the fastidious lactic streptococci by the differential medium as compared with Eugonagar is shown in Table 1. The data in Table 1 show that the differential agar provided as good recovery as did Eugonagar. Statistical analysis by the chi-square test FIG. 3. Colonial growth on differential agar of S. cremoris HP-plate I (yellow color, no clearing); S. lactis 7963-plate 2 (white color, no clearing); and S. diacetilactis 26-2-plate 3 (white color, cleared zones). FIG. 4. Differences in appearance of colonial growth of a S. lactis-S. cremoris-S. diacetilactis mixture plated on the differential agar and incubated in a candle oats jar at 32 C. Plate 1 was photographed on the 2nd day; plate 2 was photographed on the 6th day. the count of S. lactis, the second is S. cremoris, and the third is the S. diacetilactis strain in the mixture. c 0.750 < P < 0.900. (15) revealed insignificant differences (see footnote c in Table 1). These results indicate that the proposed medium and technique could be used for both qualitative and quantitative differentiation of mixtures of S. cremoris, S. kactis, and S. diacetilactis strains. In addition to the 37 strains of lactic streptococci used, two strains each of S. faecalis and S. faecium were plated on this medium. Yellowish-white colonies with distinct zones of cleared calcium citrate were formed by S. faecalis when incubated at 32 C in air. S. faecium produced white colonies and did not clear the turbid calcium citrate even after prolonged incubation (10 days). S. faecalis produced larger zones of clearing when incubated aerobically rather than in candle oats jars at 32, 37, and 45 C. These findings raise two points worthy of comment. The medium is not selective and must only be used in pure culture studies; natural products such as cheese or contaminated cultures would give erroneous results. Secondly, incorporation of selective factors in this medium with additional refinement might produce a selective and differential medium for the study of enterococcus or group D streptococci. From a practical standpoint, the triple-species, differential agar described here could be used for studying associative growth relationships in the triple-species, starter mixtures involving strains of S. lactis, S. cremoris, and S. diacetilactis. This agar greatly reduced the tedium of picking colonies from a general-purpose agar into different test media to identify members of lactic group streptococci. S. diacetilactis strains could cause the slit defect of cheddar cheese (16), floating curd in cottage cheese (12), and the green flavor defect in cultured sour cream and butter (5). Therefore, our medium could be used to verify the composition of mixed starter culture in the manufacturing plant. The culture manufacturer also could use our medium for screening single strains for compatibility in mixed cultures.

v3-fos

2016-05-12T22:15:10.714Z

1972-04-01T00:00:00.000Z

15828088

s2

Polychlorinated biphenyl residues in milk of environmentally and experimentally contaminated cows. Polychlorinated biphenyl (PCB) residues have been found in the milk of cows. In some instances the residue levels exceeded the FDA guideline 0.2 ppm in milk (equivalent to 5.0 ppm in milk, fat), and the milk was removed from the market. The major source of PCB residues in milk is Aroclor 1254 that has been used in coatings for concrete silos. Aroclor 1254 is unaltered in silos, and most of the contamination is adjacent to the walls (1, 2). We have observed a number of farms with PCB-treated silos and have fed Aroclor 1254 to cows under controlled conditions. This paper summarizes our major findings. chlorinated the samples to remove possible interferences of DDD and DDT. DDE did not interfere, because those components of Aroclor 1254 with the same retention time as DDE did not occur in significant amounts in milk (Fig. 1). We obtained milk tank samples from six farms with PCB-contaminated silos. The PCB levels in milk fat are summarized in Table 1. In all cases PCB concentration in milk fat exceeded the FDA guideline, at least part of the time, when silage was fed. When the silage was not fed, levels of PCB in milk fat were always lower than the guidelines. The farms represent a variety of feeding and management conditions. The results suggest that any dairyman with a contaminated silo will probably exceed the guideline at least some of the time during the year. While a few higher levels have been reported for herds removed from the market, most were within the range of our observations. The number of farms with PCB-contaminated silos is not known. However, PCB contamination of milk is probably more prevalent than the regulatory record would suggest. Comparison of DDE and PCB We have observed a herd that was simultaneously contaminated with high levels of DDE and Aroclor 1254 from a silo. By a fortuitous set of circumstances, the cows had been removed from the sources of both contaminations at about the same time. A detailed description of this work has been prepared (4). Silage was not being fed when italicized samples were obtained. b Not Sampled. Relationship of the rate constants for DDE and PCBs within individual cows is presented in Fig. 2. The correlation (r = 0.82) between the rate constants of the two compounds was significant (P<0.01). The usual linear regression was calculated, and the intercept did not differ significantly from zero (6). Therefore, the regression in Fig. 2 was recalculated, forcing the intercept through zero. The regression coefficient, 0.974, was essentially unity. In some cases there was considerable variation between the rate constants for DDE and for PCBs within a given cow. The rate constants for the two compounds within a given cow were tested statistically (6), but the differences were not significant. The usefulness of phenobarbital alone, or in combination with activated carbon, in accelerating the reduction in milk concentration of DDE and PCBs was tested using three groups of cows. Neither phenobarbital nor phenobarbital in combination with activated carbon had an effect on the relative reduction in concentration of either compound (Table 3). We have previously reported a small effect of phenobarbital on DDE, but we did not consider it of practical significance (7). The absence of an effect here is consistent with that interpretation. The failure of activated carbon, even in combination with phenobarbital, to affect the reduction of DDE and PCB concentrations is consistent with our previous interpretation of laboratory experiments (8). Aroclor 1254 Feeding Study We have carried out a controlled feeding study using nine Holstein cows. The cows were fed 200 mg per day Aroclor 1254 for 60 days. Milk samples were obtained periodically during the 60-day feeding period and the subsequent 60-day period. Body fat samples were obtained by biopsy at 30-day intervals. The cows were selected to have a range in both the stage of lactation and level of milk production. However, preliminary examination of the results indicates that there was little difference in the residue levels due to these factors. Therefore, only average data for all cows are presented. The average level of PCBs in the milk fat during the feeding period is presented in Fig. 3. The standard deviation of any given point did not exceed :1 10% of the mean. The concentration of PCBs increased rapidly and approached a "steady state" at about 40 to 60 days. As expected from our field studies, the shape of the curve is similar to that for other chlorinated hydrocarbon pesticides, particularly DDE (5). The decline in PCB concentration in the milk for the 60 days after feeding stopped is presented in Fig. 4. This curve is quite typical of previous findings with DDE and was resolved into a twocomponent first-order system (5). The equation for the curve normalized to an initial concentration of 1.0 ,ug/g is: C = 0.52e0°*25I+0.48e-o0 oos Where C is concentration, e is the base of the natural logarithms, and t is days. The constants are similar to our previous observations for DDE (5). While the rate for the second component in this study was less than the values found in the field, it was within the range of values that one might expect. The level of PCB in body fat at various times is presented in Table 4. For reference purposes, milk fat levels at these times are also presented. Environmental Health Perspectives While the PCBs were being fed, the level in milk fat was higher than that in body fat. When feeding stopped, milk fat levels dropped below and appear to reflect body fat levels. We have conducted a number of studies with chlorinated hydrocarbon pesticides under conditions similar to those of this PCBs study. A summary of levels in milk fat at 20 and 60 days and body fat at 60 days with normalized intakes is presented in Table 5. The levels of PCBs are similar to levels of DDE and dieldrin in corresponding samples. In contrast DDD, p,p'-DDT, and o,p'-DDT are transferred to the body fat and milk fat at much lower rates than DDE and PCBs. Conclusions Farms having silos that have been treated with PCB-containing paints will frequently have residues of PCBs in milk which exceed the FDA guidelines. The behavior of this residue in the cows is similar to DDE and other chlorinated hydrocarbon pesticides resistant to metabolic degradation. The only practical countermeasures appear to be to decontaminate the silos sufficiently to remove most of the PCBs or to discontinue the use of these silos completely. Limited field experience and the similarity of PCBs to DDE suggest that there are no practical procedures to minimize the transfer of PCBs from the diet to the milk or to speed up the elimination of PCBs from cows.

v3-fos

2020-12-10T09:04:12.813Z

1972-01-01T00:00:00.000Z

237229508

s2

Effects on Turkey Poults of Rations Containing Corn Invaded by Fusarium tricinctum (Cda.) Sny. & Hans Consumption of an otherwise balanced ration containing 1% of corn invaded by Fusarium tricinctum isolate 2061-C resulted in the death of 13% of turkey poults within 35 days, in decreased feed efficiency and weight gain, and moderate development of bilateral necrotic lesions at angles of the mouth, especially in those that succumbed. Consumption of a ration with 2% of corn invaded by F. tricinctum resulted in death of 60 to 83% of the birds, in greatly reduced growth and feed efficiency in the survivors, and in development of severe mouth lesions. Consumption of rations containing 5, 10, and 20% of corn invaded by the fungus resulted in death of all birds in 5 to 15 days. Wollenweber and Reinking (4) applied the name Fusarium tricinctum to a species within the Sporotrichiella section of the genus Fusarium, the section including, according to their arrangement, F. chlamydosporum, W There are numerous reports of toxin production by members of the Sporotrichiella section as used by Wollenweber and Reinking or of F. tricinctum as applied by Snyder and Hansen. Joffe (1) reported that a toxin produced by F. sporotrichioides growing on overwintered millet in the U.S.S.R. was responsible for a serious outbreak of poisoning in the people who consumed the millet. Isolates of F. tricinctum (Cda.) S. & H. from grains and from other plant parts produce a very potent toxin that has been characterized as a trichothecane and that has been designated T-2 (2). We isolated F. tricinctum (Cda.) S. & H. from numerous samples of corn stored on the cob in cribs. Some of these lots of corn were suspected to have been involved in the illness of cattle, and the isolate used in the present tests (designated 2061-C in our records) was from such a lot. The aim of this work was to determine its effects on turkey poults when consumed in the ration as it might be in practice. Moist autoclaved corn was inoculated with isolate 2061-C of F. tricinctum and was incubated for 2 weeks at 22 to 25 C followed by 2 weeks at 14 C, a schedule resulting in high production of toxin (2). The corn was then dried, milled, and added to the ration as 1 to 20% of the total. The other ingredients of the ration consisted of 30 to 49% food-grade corn plus 50% turkey premix. One-day-old Wrolstad White turkey poults were obtained from a commercial hatchery and were kept for 1 week on a balanced ration in the test pens to detect and eliminate any weaklings and to accustom the others to the environment. They were then divided into groups and fed for 5 weeks. In the first trial, with 0, 2, 5, 10, and 20% of F. tricinctum-invaded corn in the ration, each group, with one exception, consisted of four replicates of five birds each in pens arranged in a Latin square design, the exception being the group of eight birds that received the ration containing 20% of F. tricinctum-invaded corn. In the second trial, with 0, 1, and 2% of F. tricinctum-invaded corn in the ration, each group consisted of three replicates of 10 birds each, and the groups were distributed among the pens according to a Latin square design. The birds were observed by one of us daily during the 5week trial period and were weighed each week. Birds that died during the course of the trial were necropsied, and the survivors at the end of the trial were sacrificed and examined, and the hearts and livers were removed and weighed. Trial 1. The average weight of the birds is shown in Fig. 1. None of the control birds died; all of the birds receiving 5, 10, and 20% of F. tricinctum-invaded corn in the ration died within 5 to 15 days, 12 of 20 (60%) of those receiving 2% of F. tricinctum-invaded corn in the ration died, and the survivors on this ration were stunted and unthrifty. Most of the birds which died from day 10 on and most of the survivors on the ration containing 2% of corn infected with F. tricinctum developed pronounced bilateral necrotic lesions at the angles of the mouth. Trial 2. The average weight of the birds in trial 2 is shown in Fig. 2. Again, none of the controls died, 4 of 30 (13%) of those on a ration containing 1% of F. tricinctum-invaded corn died, and 25 of 30 (83%) of those receiving a ration containing 2% of F. tricinctum died. mal); for those receiving 1% of corn invaded by F. tricinctum it was 2.02, and for those receiving 2% F. tricinctum-invaded corn in the ration it was 3.28. Many of the birds developed severe necrotic mouth lesions before death (Fig. 3). There was no significant difference between the heart weight/body weight or the liver weight/body weight of the controls and the two treatments.

v3-fos

2020-12-10T09:04:12.439Z

1972-07-01T00:00:00.000Z

237235379

s2

Analysis of Tobacco and Smoke Condensate for Penicillic Acid Gas chromatographic analyses of smoke condensate from commercial, unfiltered cigarettes spiked with penicillic acid (500 or 1,000 ppm), a reported carcinogenic substance from certain fungi, indicated approximately 3% of unchanged compound was transported in the smoke. Analysis of tobacco on which either Aspergillus ochraceus or Penicillium cyclopium was grown revealed microgram quantities of the compound. Small amounts of the material were also found in moldy tobacco from commercial storage. The results of these investigations suggest that fungi may be a source of carcinogenic compounds in tobacco and tobacco smoke. Fungi produce numerous biologically important compounds on the substrates they inhabit. Many of these substrates, such as tobacco, are utilized by human beings. Because of its implicated role in the smoking and health problem, tobacco is currently receiving considerable attention. Aspergillus ochraceus Wilhelm and Penicillium cyclopium Westling are two of the many fungi found on tobacco during marketing and storage (11). Like numerous other microorganisms, these fungi produce substances hazardous to some test animals (4,5,6) and therefore are potentially hazardous to human beings. Penicillic acid (PA) was first isolated in 1913 (1) during an investigation of the cause of pellagra. The compound, C8H1004 (2), exists in tautomeric equilibrium with its lactone. The toxic properties of penicillic acid have been studied by several workers (4,5,6); however, only recently has the carcinogenic nature of the material been reported (3). The work reported in this paper is part of a larger study of the fungi and their metabolic relationship to tobacco. MATERIALS AND METHODS Moldy tobacco samples. Samples of stored, moldy (damaged) tobacco were obtained from a commercial tobacco company. After removal of midribs, a total of 3.7 kg (sample 1) of leaf lamina was 1 Paper No. 3670 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. 27607. macerated in chloroform in a large commercial blendor. The chloroform extract was filtered through cheesecloth and Whatman no. 42 filter paper and concentrated to a thick gum. Sample 2 (35.4 kg) of moldy commercial tobacco was air dried at 35 C to 5 to 10% moisture and shattered by hand. Enough chloroform was added to cover the tobacco which was allowed to stand overnight. The chloroform was decanted and the tobacco extracted a second time with the same volume of chloroform. The extracts were combined, filtered through Whatman no. 42 filter paper, and evaporated to a thick gum. Cultures of A. ochraceus and P. cyclopium were isolated from tobacco in 1968, lyophilized, and stored at 3 to 4 C until used for the following experiments. To obtain monocultures of moldy tobacco, 50 g of commercial, shredded tobacco and 50 ml of distilled water were placed in each of 15 flasks. The flasks were plugged with cotton and autoclaved for 20 min at 20 psi. Five flasks were each inoculated with 5 ml of a spore suspension of either A. ochraceus (2.5 x 106 spores/ml) or P. cyclopium (5.0 x 104 spores/ml). The remaining five flasks each received 5 ml of sterile, distilled water and served as controls. The flasks were covered with aluminum foil to prevent desiccation and incubated at 25 C for 4 weeks. After incubation, 300 ml of chloroform was added to each flask and they were allowed to stand overnight. The chloroform extract was filtered through cheesecloth and Whatman no. 42 filter paper. Each flask was flushed with an additional 300 ml of chloroform which was filtered and combined with the original extract. The combined extracts were then evaporated to a thick gum. Smoke samples. PA for the following experiments was isolated from liquid cultures of A. ochraceus. After incubation, the culture medium was filtered and evaporated to 300 ml. Two hundred ml of SMOKE CONDENSATE FOR PENICILLIC ACID xylene was added to form a liquid-liquid interface. After warming and stirring the liquids for 2 hr, the xylene was decanted and evaporated to dryness. The concentrate was dissolved in 50 ml of carbon tetrachloride and stored at -18 C. Crystals of PA began to form in 3 to 5 hr. Verification of identity was made by infrared and mass spectral analyses. Twenty commercially manufactured 82-mm cigarettes (sample 1) were spiked with 10 ppm of PA (0.2 mg/2.0 ml of distilled water), 20 cigarettes (sample 2) were spiked with 100 ppm of PA (2.0 mg/2.0 ml of distilled water), 100 cigarettes (sample 5) were spiked with 200 ppm of PA (20 mg/10 ml of distilled water), 20 cigarettes (sample 3) were spiked with 500 ppm of PA (10 mg/2.0 ml of distilled water, and 40 cigarettes (samples 4 and 6) were spiked with 1,000 ppm of PA (40 mg/4.0 ml of distilled water). The core of each cigarette received 0.1 ml of solution from a microsyringe drawn along the length of the cigarette as the solution was expelled. Control cigarettes were spiked with sterile, distilled water. After air drying overnight, cigarettes at each PA level were mechanically smoked on a Phipps and Bird smoking machine (capacity of 20 vacumm ports). Each port was programmed to draw a 35 4 2 cm3 puff for 10 sec of 60-sec intervals. Smoke was collected in Pyrex condensers, each containing 2 g of Sokafloc (Brown Company, Berlin, N.H.), and partially submerged in a dry ice-acetone bath. Contents of the condensers were combined and twice flushed with reagent grade acetone. After filtering through Whatman no. 42 filter paper, the combined sample was evaporated to dryness. Analysis of samples. Gas chromatographic analyses for PA were carried out by using a Varian Aerograph model 2100 gas chromatograph with a flame ionization detector and an accessory model 480 Varian-Aerograph digital integrator with printout. Derivatization and programming were carried out according to the method reported by Pero et al. (8). RESULTS The results of gas chromatographic analyses for PA in extracts of moldy tobacco are summarized as follows. The smaller sample of commercial moldy tobacco (sample 1) contained approximately 23 jg of PA per 100 g of tobacco. Sample 2 contained approximately 11 jig of PA per 100 g of tobacco, about half as much PA as sample 1. Sample 2, however, was visibly less moldy than sample 1, which might account for some of the difference. Also, sample 1 was macerated in a blendor, whereas sample 2 was shattered into pieces no larger than about 1/4 inch in diameter. More PA was found in samples of tobacco on which A. ochraceus (1,800 ,g/100 g) was grown than in samples on which P. cyclopium (320 gg/100 g) was grown. This suggests that A. ochraceus may produce more compound under storage conditions than does P. cyclopium. No gas chromatographic peaks with the same retention time as PA were found in extracts of nonmoldy tobacco. Table 1 summarizes the results of gas chromatographic analyses of smoke condensates. A trace of PA was detected in condensate from cigarettes spiked with either 10 or 100 ppm (samples 1 and 2), whereas samples 3 and 4 contained 2.4 and 3.9%, respectively, of the amount originally added. For samples 5 and 6, a finer-mesh Sokafloc (SW-60) was used to trap the smoke, and no PA was detected in the condensate. Lack of recovery of PA in these samples may have been due to decreased air flow through the finer-mesh filter. DISCUSSION Several workers (7,9,12) have detected aflatoxin and aflatoxin-like compounds in tobacco. Aflatoxin, like PA, is a carcinogenic compound produced by a fungus, Aspergillus flavus, cultured from flue-cured tobacco. The presence in tobacco of a carcinogenic compound produced by fungi growing on tobacco and its transport in cigarette smoke, although in small amounts, emphasizes the importance of evaluating the possible contribution of microbial contaminants to tobacco in general and to the solids and volatile components of cigarette smoke. A. ochraceus and P. cyclopium are only two of numerous fungi which have been isolated from moldy, commercial tobacco (10). When one considers the myriads of compounds produced by these organisms, the chances of one or more being harmful to man are considerable. If mold- damaged tobacco is blended with undamaged tobacco, PA or other fungal toxins could find their way into commercial products. At very low concentrations, these compounds, acting alone or in combination with carcinogens or co-carcinogens of fungal or other origin, may after many years of exposure produce effects similar to those obtained when the compounds are tested on a short-term high-dose basis. Further studies are needed to determine the effects of long-term exposure to these materials alone and in combinations with other tobacco and smoke components.

v3-fos

2020-12-10T09:04:12.292Z

1972-06-01T00:00:00.000Z

237230269

s2

Factors Affecting Organic Acid Production by Sourdough (San Francisco) Bacteria Previous workers from this laboratory observed considerable variation in the proportions of acetic and lactic acids produced in pure broth culture as compared to consistently high proportions of acetic acid produced in the sourdough and flour suspension systems. In the latter the proportion of acetic acid was always in the range of 20 to 35% of the total, whereas in pure broth culture frequently less than 5% acetic acid was produced. In the natural environment, the sourdough bacteria, tentatively identified as lactobacilli, coexist with a yeast, Saccharomyces exiguus, and this study was undertaken to determine whether this yeast or flour ingredients including glucose or other factors were involved in this variable production of acetic acid. The proportion of acetic acid produced in broth culture on maltose, the preferred carbohydrate source, was found to depend almost entirely on the degree of aeration. Essentially anaerobic conditions, as obtained by thorough evacuation and flushing with CO2 or N2, resulted in very low (5% or less) proportions of acetic acid. Aerobic conditions, achieved by continuous shaking in cotton-plugged flasks, yielded high levels (23 to 39% of the total) of acetic acid. Similar effects of aeration were observed with glucose as the substrate, although growth was considerably slower, or in nonsterile flour suspension systems. It is theorized that, under aerobic conditions, the reduced pyridine nucleotides generated in the dissimilation of carbohydrate are oxidized directly by molecular oxygen, thereby becoming unavailable for the reduction of the acetyl phosphate intermediate to ethyl alcohol, the usual product of anaerobic dissimilation of glucose by heterofermentative lactic acid bacteria. Comparative studies with known strains of homo- and heterofermentative lactobacilli showed similar effects of aeration only on the heterofermentative strains, lending additional support to the tentative grouping by previous workers from this laboratory of the sourdough bacteria with the heterofermentative lactobacilli. Previous workers from this laboratory observed considerable variation in the proportions of acetic and lactic acids produced in pure broth culture as compared to consistently high proportions of acetic acid produced in the sourdough and flour suspension systems. In the latter the proportion of acetic acid was always in the range of 20 to 35% of the total, whereas in pure broth culture frequently less than 5% acetic acid was produced. In the natural environment, the sourdough bacteria, tentatively identified as lactobacilli, coexist with a yeast, Saccharomyces exiguus, and this study was undertaken to determine whether this yeast or flour ingredients including glucose or other factors were involved in this variable production of acetic acid. The proportion of acetic acid produced in broth culture on maltose, the preferred carbohydrate source, was found to depend almost entirely on the degree of aeration. Essentially anaerobic conditions, as obtained by thorough evacuation and flushing with CO2 or N2, resulted in very low (5% or less) proportions of acetic acid. Aerobic conditions, achieved by continuous shaking in cotton-plugged flasks, yielded high levels (23 to 39% of the total) of acetic acid. Similar effects of aeration were observed with glucose as the substrate, although growth was considerably slower, or in nonsterile flour suspension systems. It is theorized that, under aerobic conditions, the reduced pyridine nucleotides generated in the dissimilation of carbohydrate are oxidized directly by molecular oxygen, thereby becoming unavailable for the reduction of the acetyl phosphate intermediate to ethyl alcohol, the usual product of anaerobic dissimilation of glucose by heterofermentative lactic acid bacteria. Comparative studies with known strains of homoand heterofermentative lactobacilli showed similar effects of aeration only on the heterofermentative strains, lending additional support to the tentative grouping by previous workers from this laboratory of the sourdough bacteria with the heterofermentative lactobacilli. The microorganisms responsible for the leavening and souring activities in the dough used in perpetuating the San Francisco sourdough bread process were successfully isolated recently and described by Kline and Sugihara of this laboratory. The leavening function was numerically correlated with the occurrence of a yeast identified as Torulopsis holmii, the asporogenous form of Saccharomyces exiguus (8), and the souring activity was correlated with the presence of a bacterium tentatively identified as a lactobacillus, whose requirement for maltose, at least for isolation purposes, and other differences suggest it to be a previously undescribed species (3). As reported by Kline and Sugihara (3), discrepancies were observed between the propor-tions of acetic and lactic acids produced in doughs or other flour culture systems and that produced in the pure broth cultures. Thus in flour systems, whether a natural "starter" or pure cultures of yeast and bacteria were used as inocula, the acetic acid produced was between 20 and 30% of the total whereas in pure broth cultures inoculated with the bacterium alone the proportion of acetic acid was frequently less than 10% and highly variable. The sourdough yeast per se does not produce significant amounts of acids in the dough (6), but it was not known whether it, or factors contributed by the flour, exerted any influence on the type of acidity produced by the bacterium. Accordingly, the present studies were undertaken to determine the conditions responsible for the variable production of acetic acid by the sourdough bacteria. For reference purposes, comparative studies were also made on known strains of heterofermentative and homofermentative lactobacilli. MATERIALS AND METHODS Pure culture media. The SDB (sourdough bacteria) agar and broth were prepared as described by Kline and Sugihara (3). Although these workers used only maltose as a fermentable carbohydrate source, I found glucose to be utilized also, although more slowly, and the medium was made up to contain either 2% (w/v) maltose or glucose and designated as SDBM or SDBG, respectively. Initial pH of the medium was adjusted to 5.5 with HCl. Fresh yeast extractives (FYE) prepared as described previously (3) were routinely used at a concentration of 0.5% unless stated otherwise. Flour cultures. Preparation and incubation of sour bread doughs were as described by Kline, Sugihara, and McCready (4). Flour slurry cultures were also made by mixing 100 g of flour, 2.2 g of NaCl, and 250 ml of distilled water and adjusting the pH of the slurry to 5.5 with HCl. The slurry was inoculated with a bacterial cell suspension freshly prepared from 20 ml of an overnight culture in SDBM broth by centrifuging at 15,000 rev/min for 10 min in a Sorvall refrigerated centrifuge and suspending the cells in 5 ml of sterile physiological saline. Methods of cultivation. All cultures were incubated at 30 C. For achieving essentially anaerobic conditions, broth cultures (100 ml) in loosely capped 250-ml Erlenmeyer flasks or slant cultures in loosely capped test tubes were placed in vacuum desiccators which were evacuated to about 50 mm Hg followed by refilling with either pure CO2 or N2 to atmospheric pressure; the process was repeated three times. The tubes or flasks were then removed from the desiccators, the screw caps were tightened, and the cultures were incubated without shaking. For aerobic conditions, cultures were incubated in cotton-plugged flasks and shaken in a New Brunswick rotatory shaker-incubator operating at a frequency of about 100 excursions/min. The same methods were applied to flour slurry cultures, except these were shaken in all cases during incubation to prevent settling of the flour. Organisms. The sourdough bacteria used in these experiments were those isolated and described by Kline and Sugihara (3) and designated by them as strains B, C, L, and T according to the source (San Francisco sourdough bakery from which dough was obtained). These were maintained on SDBM slants. For comparative purposes, known species of lactobacilli obtained as lyophilized cultures from the Northern Regional Research Laboratory culture collection (courtesy of W. C. Haynes) were also studied. These included: L. delbrueckii The lyophilized cultures were taken up in SDBM broth, and, after growth at 30 C, loopfuls were streaked out on SDBM agar plates where well isolated colonies were selected and transferred to SDBM agar slants. A lactobacillus strain isolated by T. F. Sugihara of this laboratory from a commercial frozen concentrated sourdough starter culture, designated as Sardo SF, was also evaluated. Organic acid analysis. For determining acids in doughs, the doughs were extracted by blending a weighed piece of dough (about 50 g) with about 50 to 60 ml of distilled water in a 250-ml osterizer jar for about 1 min. Sodium hydroxide (1.0 N) was then added to bring the pH of the extract to about 8, and the mixture was again blended for 1 min. The extract was then centrifuged at 2,000 rev/min for 30 min, and the supernatant fluid was decanted. The pellet was washed with another 20 to 30 ml of water and again centrifuged. The supernatant fluid and washing were combined and made up to a volume of 100 ml. An acidified sample was used for analysis as described below. Flour slurries were extracted in a similar manner. Alkaline extraction was found to result in more complete extraction of the acids than when distilled water was used, but the proportion of acetic to lactic acid was not significantly affected. Acids were analyzed by chromatographic separation on a celite column and titration according to the procedure described by Wiseman and Irvin (10). All extracts or samples to be chromatographed were acidified by addition of H2SO, (1.0 N) to an approximate concentration of 0.1 N, and a 2-ml sample was placed on the column. The eluates were titrated to a cresol red end point by using standardized 0.01 N NaOH and agitation with CO2-free air. Values obtained on culture filtrates were corrected for the blank values in the uninoculated SDB medium which contained average concentrations of 7.3, 4.5, and 0.71 Mmoles/ml, respectively, of acetic, lactic, and formic acids. Diacetyl determination. Diacetyl was determined by the method of Westerfield as described by Neish (5). The reaction time selected was such that equal weights of diacetyl and acetoin would give equal color intensities. Therefore, the "diacetyl" values reported are the sum of these two compounds, and no attempt was made, for the purpose of this report, to distinguish between the two. Diacetyl (2,3-butanedione) obtained from Eastman Chemicals was employed as the standard. RESULTS Organic acids in sour bread dough. Table 1 shows the amount and proportions of acetic and lactic acids produced in fully developed sour bread doughs or in the "starter sponges" which are special pieces of dough serving as inocula for preparing these bread doughs (4). No other acids except for a barely measurable trace of formic acid were detected. Acetic acid is seen to represent 25 to 35% of the total a Made in conventional fashion by using a natural starter sponge as the inoculum (starter sponge is a special piece of dough which is constantly rebuilt with fresh flour and water to perpetuate the yeast and bacterial activities). Incubated at 30 C for 7 hr. bMade with pure cultures of yeast (strain C) and bacteria (strain C). c C and L refer to source (bakery). acidity produced. The higher total acidity figures for the starter sponges, as compared to the bread doughs, are attributed to the fact that these starters are a more concentrated system in terms of flour substrate and initial bacterial counts. Organic acid production in SDBM under anaerobic conditions. Since Kline and Sugihara reported stimulatory effects of CO, on growth (3), their usual procedure was to evacuate and gas with CO2 before incubation. In the present study this evacuation plus gassing was repeated three times providing essentially anaerobic conditions. As shown in Fig. 1, all four strains (B, C, L, and T), growing on SDBM broth, produced only low levels of acetic acid and for three of the strains (B, C and T) amounting to less than 5% of the total throughout the 60-hr period of fermentation. Accordingly, it was decided to evaluate factors contributed by the flour or dough systems 'which might account for their higher content of acetic acid. These included, as described below, effects of the glucose contributed by the flour, degree of aeration, and concentration of fresh yeast extractives, the latter relating to a possible contribution from the sourdough yeast moiety. Effect of glucose and of FYE concentration. Sourdough bacteria, which prefer maltose as a fermentable carbohydrate and appear to require it for isolation (from the dough) purposes, will ferment glucose after a lag of about 24 hr (Ng, unpublished data). Since flour does contain a small amount of glucose and more is released after the dough is formed (6), the ef-E HOURS FIG. 1. Production of organic acids by sourdough bacteria in SDB broth with maltose as carbon source. The cultures were incubated at 30 C under CO2 atmosphere. Samples were removed at the indicated times and acetic (0) and lactic (x) acids determined as described in Materials and Methods. fect of glucose as a substrate on the proportion of acetic acid formed was studied. As shown in Table 2 with strain B, this proportion was not significantly increased by glucose used separately or in combination with maltose under the essentially anaerobic conditions used. The slow release of acids associated with the lag in growth on glucose is also apparent. Two other strains tested, C and L, reacted similarly to growth on glucose. Table 2 also shows that the concentration of FYE used in the medium does not affect the proportion of acetic acid produced, although the total quantity of acids produced is higher, reflecting the heavier growth observed at the higher FYE level. Effects of aeration on organic acid production. In the conventional mixing procedure for preparing a dough, ample opportunity is provided for considerable trapping of air in the structure. Flour also routinely contains small amounts of oxidants such as potassium bromate which could increase the oxidation-reduction potential. Since studies in pure culture under essentially anaerobic conditions consistently resulted in minimal production of Table 3 for maltose as the substrate, and Table 4 for glucose, aeration had a profound effect on increasing the proportion of acetic acid produced by the sourdough bacteria; thus for strain L ( Table 3) the proportion of acetic acid produced was increased to 36% by aeration as compared to only about 3% under CO2. The percentage of acetic acid was also about 3% when incubated under N2, so that the low proportion of acetic acid is very likely a result of anaerobic conditions and not unique to CO2 incubation. That shaking or interchange with the air atmosphere is essential was shown by the results on strains B and T ( Table 3) where use of semi-anaerobic conditions (cotton plug but not shaking) yielded acetic acid values of only 6 to 9% as compared to 23 and 39% when shaking was employed. Thus aeration is shown to increase the proportion of acetic acid produced in pure culture on maltose by the sourdough bacteria to approximately that reported for the dough systems. No diacetyl was produced by the sourdough bacteria under either aerobic or anaerobic conditions. Of the known lactobacilli included for comparative purposes, only the heterofermentative L. brevis showed any similarity to the sourdough bacteria in its response to aeration, and the effect was even more striking with the proportion of acetic acid produced on SDBM broth being increased from 1 to 64%. L. brevis, also like the sourdough bacteria, produced no diacetyl under either condition. Aeration did a Total acids represent the sum of the acetic and lactic acids in micromoles per milliliter of culture supernatant fluid. Percent acetic is calculated by dividing the acetic acid value by the total acids and multiplying by 100. b The amount of acids produced was too low to get an accurate value. have a slight effect on the homofermenters, L. delbrueckii and L. casei, increasing the acetic acid produced on SDBM from 3.4 to 11.6% and from 3.4 to 16.5%, respectively. In addition, aeration markedly increased the production of diacetyl by these homofermenters. The isolate from the commercial "sourdough" starter culture appeared dissimilar to the San Francisco sourdough bacterial isolates in producing a minimal proportion of acetic acid (ca. 5%) even under aerobic conditions. It would appear to be a homofermenter except that it did not produce diacetyl as did the particular known species of homofermenters studied. The results in Table 4, with glucose used as substrate, show that the aeration effect was not unique to maltose as a substrate. Additional known strains of lactobacilli were studied. Thus the two additional heterofermenters, L. buchneri and L. fermenti, responded to aeration in similar fashion to the sourdough bacteria and L. brevis in markedly increasing their production of acetic acid under aeration and in not producing diacetyl under either condition. Results with L. acidophilus, a homofermenter, were similar to those obtained with L. delbruekii, although L. acidophilus did not grow well on the SDBG broth. L. plantarum, a facultative homofermenter, showed proportionately more acetic and less diacetyl increases on aeration than did L. casei. The similar effect of aeration in a flour system was demonstrated in the study shown in Table 5. Here the B strain of sourdough bacteria was inoculated into a flour-water slurry with added salt. After 24 hr, the flour was removed by centrifugation and the acids were determined in the supernatant liquid. The proportion of acetic acid produced by aeration was increased from 9.5 (anaerobic) to 19.9% (aerobic). The magnitude of the effect was not as great as that observed in the broth systems, possibly due to the failure to attain complete anaerobiosis resulting from a difficulty of removing occluded air or the fact that the requirement for shaking to keep the flour suspended could result in incorporating air if the screw caps leaked. However, qualitatively, the effect of aeration was significant and similar. DISCUSSION The foregoing experiments demonstrate that the proportion of acetic acid produced by the sourdough bacteria, and by known strains of heterofermentative lactobacilli, are dependent upon the degree of aeration to which the cultures are exposed. These observations offer a possible explanation for the higher proportions of acetic acid developed in the dough systems as compared to that observed in essentially anaerobic pure culture fermentations. Varia- bility in the earlier results with pure cultures reported by Kline and Sugihara are also likely attributable to variability in the degree of "anaerobiosis" achieved in their culturing techniques. This explanation is further supported by the finding that the degree of aeration also affected the proportion of acetic acid produced in a flour slurry system. The mechanism by which acetic acid is produced by these recently isolated sourdough bacteria has not been studied. However, based on mechanisms which have been postulated for the oxidative metabolism of glucose by heterofermentative lactobacilli, it is presumed that, under aerobic conditions, the reduced pyridine nucleotide generated from the oxidation of glucose to 6-phosphogluconic acid via the hexose monophosphate shunt transfers its electrons directly to molecular oxygen so that the acetyl phosphate intermediate yields acetic acid rather than being reduced to ethanol, the usual product under anaerobic conditions. In this connection, Stamer and Stodola (7) have shown that L. brevis, a heterofermenter, produces increased amounts of acetate under aerobic conditions and at the expense of decreased production of ethanol. White and Sherman (9) also have reported that anaerobically Streptococcus lactis converts 99.4% of the glucose to lactic acid whereas aerobically only 43.2% of glucose is recovered as lactic acid. No effort was made by these workers to account for the missing carbon, but it is very likely that it has been diverted to acetic acid. A similar explanation may also be invoked to account for the increased production of diacetyl by the homofermentative lactobacilli under aerobic conditions. In this case, pyruvate which becomes reduced to lactic acid anaerobically is now diverted to diacetyl aerobically for lack of reduced pyridine nucleotides. This is in general agreement with the data in Tables 3 and 4. This postulated mechanism is also supported by the report of Bruhn and Collins (1) who showed that under aerobic conditions, there is an increase in diacetyl and acetoin production by Streptococcus diacetilactis with a concomitant increase in production of the enzyme nicotinamide adenine dinucleotide oxidase. This enzyme presumably facilitates the transfer of electrons to molecular oxygen instead of reducing pyruvate to lactate. The present findings lend additional support to the preliminary tentative positioning of the sourdough bacteria by Kline and Sugihara as heterofermentative lactobacilli (3). The strong preference of the sourdough bacteria for maltose and other differences suggested to these workers a lack of identity with known strains. Work is under way at another laboratory to compare the genetic composition and hybridization relationships of known lactobacilli and the sourdough bacteria. Thus far the preliminary report by Nelson et al. (Bacteriol. Proc., p. 4,1971) suggests that the guanine plus cytosine content of the sourdough bacteria is about 37% or substantially lower than that of known strains of heterofermentative lactobacilli, lending further support to the view that the sourdough bacteria are previously undescribed strains. Further definitive studies on hybridization, complete analyses of fermentation products, and determination of their enzymatic makeup (2) will help to establish the taxonomic position of these bacteria.

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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. between good and poor plants and between geographical areas were examined by using the within-lot variation as the estimate of random error. 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 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 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 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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2018-04-03T06:25:00.465Z

1972-09-01T00:00:00.000Z

46595157

s2

Identification of a germination system involved in the heat injury of Bacillus subtilis spores. Bacillus subtilis A spores were injured by exposure to heat treatments of 110 to 132 C. Injury was demonstrated by the inability to form colonies on fortified nutrient agar (FNA) unless the medium was supplemented with CaCl(2) and Na(2) dipicolinate (CNA). A preliminary heat treatment fully heat-activated the spores, was not lethal, and did not prevent injury by subsequent secondary heat treatment. Exposure of heat-activated spores to 122 C reduced germination in FNA. The primary germination agents in FNA were identified, and a defined germination medium of glucose, NaCl, l-alanine, and sodium phosphate (GNAP) was developed. Germination of heat-activated spores in GNAP was equivalent to germination in FNA. Injury measured by colony formation on FNA and CNA was correlated to injury measured by reduced germination in both FNA and GNAP. Inactivation of the FNA and GNAP germination systems by secondary treatment exhibited similar kinetics. Therefore, injury expressed as the inability to form colonies on FNA involved alteration of the GNAP germination system. The detection of surviving spores is required for thermal process evaluation, and the inability to accurately predict the survivors quantitatively has both economic and public health significance. Survivors of thermal processes may be injured and unable to grow under cultural conditions that are satisfactory for unheated spores. Little information is available as to whether germination, outgrowth, or vegetative cell growth is affected in heat-induced spore injury. Levinson and Hyatt (11) reported that for Bacillus megaterium at least 94% of the spores heated at 75 to 85 C retained germinability but were unable to form colonies due to damage to the cell-division process. Less than 6% were nonviable because of the lost ability to germinate. Campbell et al. (4) reported the isolation of germination mutants of B. stearothermophilus from spores heated at 121 C. Unlike unheated spores, the injured spores and their progeny were unable to form colonies on a minimal medium unless certain amino acids were added. Colony formation was I Paper no. 3633 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Ra-leigh, N the criterion of injury and survival; therefore, the authors could not distinguish between germination, outgrowth, or vegetative cell growth as the stage affected by the apparent mutations. Cassier and Sebald (5) observed improved recovery of heated Clostridium perfringens spores when egg yolk emulsion or lysozyme was added to the plating medium. Duncan et al. (6) recently reported that lysozyme germinated the injured C. perfringens spores whose normal germination system had been inactivated by heat treatment. Edwards et al. (7,8) reported that B. subtilis A spores were injured by exposure to ultrahigh temperatures (UHT). The injured spores did not form colonies on a fortified nutrient agar unless the medium was supplemented with CaCl2 and Na2 dipicolinate. This suggested that B. subtilis A spore injury involved the germination systems. The objectives of this investigation were to identify the germination system(s) active when B. subtilis A spores were enumerated in FNA and to determine whether this germination system(s) was involved in the UHT injury. Preliminary reports of these findings have been given previously ( (7). The inoculated slants were incubated at 44 C for 24 hr and stored at 2 C. Media. The plating media were fortified nutrient agar (FNA) and FNA supplemented with 44 mM CaCl2 and 40 mm sodium dipicolinate (K & K Laboratories, Inc., Plainview, N.Y.) (CNA). The composition and preparation of the FNA and CNA were described by Edwards et al. (7). Constituents of the germination media were autoclaved separately at 121 C for 15 min and stored at room temperature. Preparation of spore suspensions. Portions (0.5 ml) of a 16-hr nutrient broth (BBL) shake culture grown at 45 C were dispensed into 100 large petri dishes (150 by 22 mm) containing 100 ml of FNA, and were spread over the entire agar surface. The plates were incubated upright for 24 hr at 44 C in a water-jacketed, natural convection incubator, inverted and incubated for an additional 24 hr, and then stored at 4 C for 18 to 24 hr. The growth was harvested with cold, sterile deionized water, and the spores were washed and purified by centrifugation according to the procedure of Edwards et al. (7). To obtain spore crops of less than 1% vegetative cells required 10 to 18 centrifugations and washings. The spore crops were stored at 2 C in deionized water. Heat treatments. The heat activation or primary treatment was 90 C for 60 min. The spores were suspended at a concentration of 107/ml in 25 mm Na phosphate buffer, pH 7, and were heated in a covered water bath. The secondary or damaging heat treatments were at 110 to 132 C. The spores were suspended in 25 mm Na phosphate buffer, pH 7, at a concentration of ca. 106/ml. When germination was measured as a reduction in optical density (OD), the suspension for both the primary and secondary treatments contained ca. 109 spores/ml. This suspension was dispensed in 0.04or 0.05-ml quantities into glass capillary tubes (Kimble 34507; 0.9 to 1.1 by 90 mm) using a syringe equipped with a repeating dispenser (Hamilton Co., Whittier, Calif.), and the capillaries were sealed in a gas flame. The capillaries were placed in a wire mesh basket and immersed in a constanttemperature oil bath (Colora Ultra-Thermostat, Germany). The heat treatment was terminated by plunging the capillaries into an ice bath. Exposure time was measured with an electric timer (Precision Scientific, Chicago, Ill., model 69230) calibrated in 0.1-sec units. The temperature change during comeup was measured with a copper-constantan thermocouple implanted in a sealed, water-filled capillary tube and was recorded on a Speedomax G recorder (Leeds & Northrup Co., Philadelphia, Pa.). The heat contribution during come-up was calculated by the method of Halvorson (10) assuming a ZD of 10 C. Six seconds were required for temperature equilibration, and the contribution during come-up was equivalent to 3 sec at bath temperature. When germination was measured by change in OD, the capillaries containing the heated spores were washed, both ends were broken off, and the contents were blown into a small test tube. The spores were diluted 1 to 10 with deionized-distilled water and used in the germination experiment. When injury was measured by colony counts or germination was measured by loss of heat resistance, two capillaries that each contained 0.05 ml of heated spore suspension were washed, rinsed in sterile deionized water, and crushed in a 99-ml phosphate buffer dilution blank (3). The samples were then dispensed in germination media or were further diluted as described in Standard Methods for the Examination of Dairy Products (3), plated in triplicate on FNA or CNA, and incubated for 18 to 24 hr at 44 C. Germination. Germination was measured as a loss of heat resistance or as a reduction in OD at 625 nm. When the germination medium contained agar, the agar was melted, cooled to 43 C, and added to the germination mixture. Germination was initiated by the addition of spores to the germination mixture after the system had been allowed to equilibrate to germination temperature. When measured as loss of heat resistance, germination was terminated by exposing the entire mixture of substrate and spores to 90 C for 15 min and then diluting the mixture immediately before colony count with CNA. RESULTS AND DISCUSSION Effects of multiple heat treatments on the apparent viability of B. subtilis A spores measured on FNA or CNA are shown in Fig. 1. Treated spores received a primary or heat-activation treatment (90 C for 60 min) prior to exposure at 121 C. Germination in FNA required heat activation and the primary treatment fully activated the spores. The number of primary treated spores enumerated on FNA and CNA was approximately equal to the direct microscopic count (data not presented). The plate counts of untreated spores on CNA were similar to those of primary treated spores plated on FNA. This indicated that the Ca dipicolinate (CaDPA) in CNA medium germi- on March 18, 2020 by guest http://aem.asm.org/ Downloaded from nated the spores directly and without previous heat activation. Riemann (12) also observed the germination of B. subtilis A spores by CaDPA. The primary treatment was not lethal as shown by equivalent plate counts on CNA before and after primary treatment. The injury induced by the secondary treatment was demonstrated by the difference between the apparent number of survivors enumerated on FNA and CNA. After a secondary treatment, a spore was considered dead if it was unable to form a colony on CNA, and a viable spore unable to form a colony on FNA was considered injured. The presence of CaDPA in CNA overcame the damage of injured spores but had no apparent effect on the additional cellular damage in dead spores. This was also observed by Edwards et al. (7,8) for spores heated in skim milk. The action of CaDPA as a germination agent for B. subtilis A spores and the requirement for outgrowth and vegetative cell growth for colony formation by injured spores on CNA strongly indicated that the site of injury was the spore germination system. Heat-activated spores that received a primary treatment appeared to be more resistant to the damaging effects of the secondary treatment. Similar observations were made by Carawan (M.S. thesis, North Carolina State University, Raleigh, 1970) who reported that a sublethal heat treatment protected B. subtilis A spores against thermal inactivation by subsequent UHT treatments. However, up to 90% of the primary treated spores surviving exposure at 121 C were injured. Therefore, the primary or heat activation treatment required for germination in FNA did not interfere with a study of the influence of secondary treatments on spore germination. Data on germination in FNA and CNA by primary treated and primary plus secondary (121 C for 6 sec) treated spores are shown in Fig. 2 and 3, respectively. Germination was measured as the loss of resistance to heat treatment at 90 C which also served to melt the agar when germination was carried out at 30 or 37 C. The controls indicate germination in 2% agar. With primary treatment only (Fig. 2), germination in FNA and CNA at 43 C were similar and greater than 90% germination occurred in 2 to 4 hr. Germination in FNA increased with increasing temperatures. The extent of germination in CNA, however, was much less influenced by temperature. This indicated that germination by CaDPA was either unaffected by temperature or increased with decreasing temperature, and thus com-pensated for the reduced germination in the FNA base of the CNA medium. Riemann (12) reported that the optimum temperature for CaDPA germination of B. subtilis A spores was between 10 and 30 C, and germination at 45 C was greatly reduced because of crystallization of the CaDPA. After secondary treatment (Fig. 3), germination in FNA at 43 C was reduced by ca. 50%. A direct relationship between germination temperature and the extent of germination in FNA also existed for secondary treated spores. In CNA, however, secondary treated spores exhibited an inverse relationship between germination temperature and the extent of germination. This reflects reduced germination of the spores by CaDPA above the 30 C. The injured spores were unable to germinate at 43 C in the FNA base of the CNA medium. About 80% of the spores capable of germination in CNA after primary treatment germinated in CNA at 30 C after primary plus secondary treatment. Complete germination of injured spores for plate counts at 43 C when CNA was used to enumerate survivors (Fig. 1) apparently occurred because of the lower temperatures during and after solidification of the CNA medium in the pour-plate technique. It was apparent, therefore, that the inability of injured B. subtilis A spores to form colonies on FNA was due to the inability of these spores to germinate unless CaDPA was added to the medium. The involvement of germination in heat injury necessitated identification of the germination system(s) that responded to FNA constituents and was suppressed by secondary heat treatment. This required evaluation of the FNA constituents, alone and in combination, for germination of uninjured and injured spores (Table 1). Germination in FNA after primary treatment or primary plus secondary treatment was similar to that shown for 90 min in Fig. 2 and 3. Deletion of minerals, Gelysate, or NaCl had no effect on the germination of spores that had received only a primary treatment. Removal of beef extract slightly reduced germination, and deletion of glucose severely retarded germination. Minerals, Gelysate, and especially glucose appeared to be important for the germination of spores that had also been subjected to a secondary treatment, although only a fraction of the survivors were able to germinate in the total or complex medium. Glucose was the only individual FNA constituent that by itself stimulated germination of heat-activated (primary treated) spores. Other FNA constituents in combination with glucose increased germination, and germination in glucose, NaCl, and beef extract approached the germination observed in FNA for primary treated and primary plus secondary treated spores. Amino acid analysis (Beckman amino acid analyzer) of FNA indicated the presence of 11 amino acids at the following concentrations: arginine, 1.65 mM; lysine, 0.76 mM; alanine, 0.62 mM; leucine, 0.49 mM; glycine, 0.44 mM, methionine, 0.33 mM; phenylalanine, 0.32 mM; tyrosine, 0.24 mM; isoleucine, 0.22 mM; serine, 0.19 mM; glutamic acid, 0.02 mm. For germination of primary treated and primary plus secondary treated spores in defined media, the media constituents were at the concentrations found in FNA; glucose, 0.55 mM; NaCl, 140 mM; Na phosphate, 2 mM; amino acids as given above. In glucose and the full complement of amino acids plus NaCl and phosphate, germination approached that observed in FNA. Maximal germination in L-alanine required the presence of other FNA constituents, especially glucose and NaCl. The defined germination medium (GNAP) used in future experiments therefore contained glucose, 0.55 mM; NaCl, 140 mM; L-alanine, 0.62 mM; and 37 Na Phosphate, 2 mm. After secondary treatment the extent of germination was reduced, as was the influence of the germination medium composition. Data describing the germination of B. sub-8 Fig. 4. The primary treatment increased germination in the defined medium by about threefold as had also been observed for colony formation in FNA (Fig. 1). After a primary and secondary treatment, germination in GNAP was reduced by 55 to 60% from that observed for primary treated spores. This agreed well with the 57% reduction in germination after 90 min in FNA (Fig. 2 and 3). A comparison of the effects of secondary treatment on the germination of heat-activated spores in FNA and GNAP is shown in Table 2. After primary treatment, the spores germinated to about the same extent in FNA and in GNAP. Injury induced by each secondary treatment and measured on FNA and CNA was accompanied by similar but reduced germination in both FNA and GNAP. After a primary treatment and a secondary treatment of 122 C for 6 sec, 82% of the spores retained viability as measured on CNA. Of these survivors, however, only 41% and 38% germinated in FNA and GNAP, respectively. These values correspond to reductions in germination activity (relative to the controls) of 52% and 59% for FNA and GNAP, respectively. These values are in agreement with others obtained when germination was measured as a loss of heat resistance (Fig. 2, 3) FIG. 4. The influence of primary (P) and secondary (S) heat treatments on the germination of Bacillus subtilis A spores in the defined medium. Open circles are control spores that received no heat treatment. P, 90 C for 60 min; S, 122 C for 6 sec.

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2018-04-03T02:12:01.920Z

1972-03-01T00:00:00.000Z

32719214

s2

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 col- lected in 44 plants had aerobic plate counts in the range of 500,000 or fewer/g; 88% contained 100 or fewer E. coliig; 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. 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

2018-04-03T03:03:58.856Z

1972-03-01T00:00:00.000Z

331889

s2

Identification and Aflatoxin Production of Molds Isolated from Country Cured Hams Of 562 molds isolated from country cured hams, 403 isolates were of the genus Penicillium, 121 were Aspergillus, and 36 were Cladosporium, Alternaria, and other genera. Country cured hams (6) are very popular in the southeastern United States. The surface of country cured hams often is covered by the growth of different species of mold, some of which might be considered desirable for improving the flavor; yet undesirable molds could cause spoilage of the product or could be pathogenic or toxigenic for man. Strains of Aspergillus flavus Link ex Fries and A. parasiticus Speare are able to produce aflatoxins (1,14). One aflatoxin-producing strain was recovered from an Italian-type salami (3). Five strains of A. flavus were isolated from a single country cured ham by Strzelecki and coworkers (12). Four of these five strains produced aflatoxin. Experimentally, A. flavus and A. parasiticus were shown to be capable of producing aflatoxin on fresh beef, ham, and bacon (4,5). Since the presence of A. flavus on hams conceivably could constitute a potential health hazard, it was considered of interest to note how often mold isolates capable of producing aflatoxin are found. Samples from country cured hams were taken from different ham processors in the State of Georgia. Four hundred and fifty-five swabs were taken from 356 country cured hams from 11 ham processors. Each swab was inoculated on the following media: Czapek Dox agar, Czapek Dox agar plus 16% NaCl, malt agar, and potato dextrose agar (8). Penicillia were identified to genus only, and all species of aspergilli were identified by the methods outlined by Raper and Fennell (10). Production of aflatoxin by isolated strains of mold was determined by the screening method used by De Vogel et al. (7). Fluorescence under long-wavelength ultraviolet light was checked after 3 and 10 days of incubation at 27 C. Isolates which showed blue or green fluorescence by the screening test were inoculated into 50 ml of YES broth (20% sucrose and 2% yeast extract) and onto sterile moist rice. Inoculated broth and rice were incubated at 27 C for 7 to 12 days. After the mold cultures were extracted twice by shaking for 10 min with 75 ml of chloroform, the chloroform layer was collected with a separatory funnel, filtered, and evaporated to dryness at 40 C with a flash evaporator (12). The residue was dissolved with 5 ml of chloroform, and the mycotoxins were separated by using thin-layer chromatograms (TLC) coated with MN Silica Gel G-HR (Brinkmann Instr., Westburg, N.Y.). Chloroform-acetone (85:15 v/v) was used as the TLC developing solvent for aflatoxin. Production of aflatoxin was determined visually by comparing the sample with aflatoxin standards (Southern Utilization Research and Development Laboratories, New Orleans, La.). From producing stains, the chloroform extract was rechromatographed on a preparative scale, and the suspect spot removed. The aflatoxin was eluted from the silica gel with chloroform and filtered, and the ultraviolet absorption spectra were obtained on a Perkin-Elmer model 202 spectrophotometer. The method of Verrett et al. (13) was used for bioassay. Chloroform extracts of broth were evaporated to dryness and dissolved in 2 ml of sterile propylene glycol. A 0.03-ml amount of this solution was inoculated into the air sac of eggs by sterile syringe. A control was inoculated with the same amount of pure propylene glycol. The development of the embryo was observed after 4, 6, and 8 days. After 8 days, all eggs which failed to develop were discarded. Of the 562 mold isolates taken from 356 country cured hams, 403 Penicillium, 121 Aspergillus, and 36 other mold isolates, mostly members of the genus Cladosporium or Alternaria, were identified ( Table 1). The number of aspergilli depended on the age of the hams and on the amount of moisture in the storage room. All aspergilli were more abundant on the surface of hams aged for 12 months or longer than on 1to 3-month-old hams. Under dry storage conditions, more aspergilli were isolated from 1to 3-month-old hams, whereas under moist conditions these hams yielded more penicillia. The largest number of aspergilli isolated were members of the A. glaucus group ( Table 2). Twenty-one were A. repens, 18 were A. amstelodami, 3 were A. pseudoglaucus, and 2 were A. ruber. The A. versicolor group was the second most common, having 21 of A. versicolor and 13 of A. sydowi. These groups are of fakus. Each of the three possessed distinct morphological and color characteristics. By comparing extracts of these strains with aflatoxin standards on TLC, it was found that only two were able to produce aflatoxin. Only aflatoxin Bl was produced in detectable amounts by either strain. The absorption spectra of the eluted TLC spots for both strains showed the characteristic maxima of aflatoxin B1 at 363 and 265 nm. The embryos of all 48 chick eggs inoculated with extracts of broth cultures of the two aflatoxin-producing strains of A. flavus failed to develop. In the same experiment, 20 of 24 control eggs hatched. These results, together with the screening test, TLC, and ultraviolet spectrophotometry, indicate that two of the three strains of A. flavus isolated from country cured hams produced aflatoxin B. when inoculated on experimental substrates. Of 356 hams examined, only two yielded strains of A. flavus having the ability to produce aflatoxin. Previous work from this laboratory (Strzelecki et al., 1969) has shown that had these two toxigenic strains been provided with appropriate conditions for toxin production, they could have posed a real hazard. There still is no definite evidence to date that such hams contain harmful amounts of aflatoxin.

v3-fos

2020-12-10T09:04:12.895Z

1972-02-01T00:00:00.000Z

237233249

s2

Possible Origin of Clostridium botulinum Contamination of Eskimo Foods in Northwestern Alaska Soils from beaches in northwestern Alaska have been found to contain Clostridium botulinum type E, providing evidence of one environmental source of food contamination. Soils from beaches in northwestern Alaska have been found to contain Clostridium botulinum type E, providing evidence of one environmental source of food contamination. Alaska has the highest rate of morbidity from type E botulism in man in the United States (8). More than one-half of the outbreaks in Alaska have occurred in the northwestern region, and in all but one outbreak the foods implicated have been prepared solely from marine mammals (3). Insanitary conditions, a cultural preference for uncooked foods of marine origin, and the presence of type E Clostridium botulinum spores in the surroundings have been considered to be prerequisites for endemic type E botulism (5). In Alaska, the first two conditions have been met, whereas the third has been suggested by the number of cases of botulism that has occurred. Although the organism has been isolated from, or detected in, fish (9) and crabs (7) taken from Alaskan waters and foods implicated in cases of botulism (3), a source within the physical environment has not been demonstrated. Dubovsky and Meyer (6) were unable to confirm its presence in a soil sample found in south central Alaska. Studies of the marine sediments from the Bering and Chuckchi Seas did not show the presence of C. botulinum (5). Consequently, a search for alternate nidi of the organism was indicated. Isolations from beach shorelines are not uncommon, having been reported from Lake Michigan (1) and Hokkaido, Japan (10); therefore, beaches near recent outbreaks or beaches used in the production of the implicated foods were sought. One set of nine soil samples was collected at Kotzebue, Alaska, and one set of four samples was collected at Point Hope, Alaska, coastal communities where the most recent outbreaks had occurred. An additional set of 10 The soil samples were collected aseptically in screw-cap vials and refrigerated until cultured 12 to 14 months later. Five grams of soil was transferred into 120 ml of freshly prepared TPGY (Trypticase, 5%; peptone, 0.5%; glucose, 0.4%; yeast extract, 2%; sodium thioglycolate, 0.1%) and incubated for as long as 7 days in loosely stoppered water dilution bottles under anaerobic conditions at 28 C. Cultures were examined for the presence of toxin at 4 and 7 days. Toxin detection and typing by intraperitoneal injection of supernatant fluids into mice and the identification of isolates were performed by the procedures of the Center for Disease Control (11). The Center supplied the antitoxins used for typing. Of the 23 samples, 17 produced type E toxin in culture, and the organism was isolated from at least one culture from each locality (Table 1). At the time the samples were collected, marine mammals were being butchered on the beaches at Kotzebue and at Elephant Point; the animals had been killed at sea and towed to the beach for further processing. The skinning and butchering of animals on such beaches provide ample opportunity for contact of the meat and blubber with soils that might contain C. botulinum. Our results do not rule out other means by which the food might have become contaminated. The animals themselves could harbor the organism, providing an endogenous source of contamination for both the foods and beaches. Earlier investigations into sources of botulinum contamination have been reviewed by Dolman and Chang (4). Recent studies of botulinum contamination of soils along the shores of Lake Balkhash in the Kazakh SSR suggest that one source of type E contamination of soils may be fish entrails (2). Despite the unsuccessful attempts by Dolman and Iida (5) to isolate the organism in sediments off Alaskan coasts, the role of sediments near the shore as a reservoir of C. botulinum remains unknown. Our results do, however, identify beach soils as one source of the C. botulinum in foods prepared by Eskimos in northwestern Alaska.

v3-fos

2018-04-03T01:25:57.921Z

1972-03-01T00:00:00.000Z

24514171

s2

Ethylenediaminetetraacetate Complexes Isolated from Cell Walls of Salmonella enteritidis' Ethylenediaminetetraacetate (EDTA) has been reported by several workers to remove portions of bacterial cell envelopes containing lipopolysaccharide (3, 4, 6). Prior to the use of EDTA, phenol was employed to extract this material (9). The antigenicity of phenol and EDTA extracts from Escherichia coli were compared, and the EDTA extract was more antigenic in mice (4). The present report will show that, in White Leghorn chickens, material isolated by phenol extraction of cell walls of Salmonella enteritidis is more antigenic than that isolated by EDTA. Through the use of radioisotopes, it is shown that differences in antigenicity are possibly related to differences in the modes of action of EDTA and phenol upon bacterial cell walls. Cells of S. enteritidis were inoculated in Trypticase soy broth and incubated for 24 hr at 37 C. They were then harvested by centrifugation and washed twice with physiological saline and distilled water. Two-gram (wet weight) samples of washed cells were suspended in 50 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.0, and sonically treated (Branson sonifier) for 3 min at 5 C. Sonic extracts were centrifuged at 3,640 x g for 20 min at 2 C to separate whole cells. Supernatant solutions were decanted and recentrifuged at topes. Ethylenediaminetetraacetate (EDTA) has been reported by several workers to remove portions of bacterial cell envelopes containing lipopolysaccharide (3,4,6). Prior to the use of EDTA, phenol was employed to extract this material (9). The antigenicity of phenol and EDTA extracts from Escherichia coli were compared, and the EDTA extract was more antigenic in mice (4). The present report will show that, in White Leghorn chickens, material isolated by phenol extraction of cell walls of Salmonella enteritidis is more antigenic than that isolated by EDTA. Through the use of radioisotopes, it is shown that differences in antigenicity are possibly related to differences in the modes of action of EDTA and phenol upon bacterial cell walls. Cells of S. enteritidis were inoculated in Trypticase soy broth and incubated for 24 hr at 37 C. They were then harvested by centrifugation and washed twice with physiological saline and distilled water. Two-gram (wet weight) samples of washed cells were suspended in 50 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.0, and sonically treated (Branson sonifier) for 3 min at 5 C. Sonic extracts were centrifuged at 3,640 x g for 20 min at 2 C to separate whole cells. Supernatant solutions were decanted and recentrifuged at 27 ,000 x g for 15 min, and the liquid was discarded. Cell wall pellets were suspended in 50 mg each of deoxyribonuclease and ribonuclease, incubated overnight at 10 C, and then centrifuged. This procedure is a modification of one described previously (2). The absence of significant amounts of cytoplasmic contamination was established by the absence of electrondense material in electron micrographs (1) and the failure to detect ribose in acid hydrolysates of cell walls (3). Cell walls (500 mg dry weight) were resuspended in 50 ml of 1.0 mm EDTA (tetrasodium dihydrate) at 22 C for 15 min. The suspension was then centrifuged, and the supernatant solution was decanted, dialyzed against several changes of distilled water at 4 C for 3 days, and lyophilized. An identical procedure was used to prepare the phenol extract, except that cell walls were resuspended in 50 ml of 90% (v/v) phenol at 65 C for 15 min (9). The isolated extracts were each dissolved in physiological saline (10 mg/ml). Two milliliters of the phenol extract was injected intravenously into each of six adult female White Leghorn chickens. A different group of six chickens was inoculated in an identical manner with the EDTA extract. Procedures for collection of blood and measurement of antibody titer by tube agglutination have been outlined previously (8). Antigen concentrations used in titrations were 200 pg (dry weight) of either the EDTA or phenol extract per ml of distilled water. Basically, the procedure for titration was as follows. Antigen (0.5 ml) was added to tubes containing 0.5 ml of physiological saline plus serial dilutions of serum. The contents of each tube were thoroughly mixed and incubated for 24 hr at 37 C. Positive reactions were indicated by a granular-type agglutination. Zinc is one of the mineral constituents (1) in gram-negative bacteria, and aspartic acid is one of the major amino acids (7) in cell walls of Salmonella. Extracting isolated cell walls labeled with radioisotopic markers of these components should provide insight into the modes of action of EDTA and phenol and help to explain observed differences in the antigenicity of these extracts. Accordingly, cells were inoculated; 4 hr later, 3 ml of either 65ZnCl2 or aspartic-1-14C acid (150 ,gCi/ml, New England Nuclear Corp.) was added to each 250 ml of broth, and the cultures were incubated. The cells were then harvested, and labeled cell walls were isolated and extracted with either phenol or EDTA. Radioactivity was measured for duplicate 1-min intervals, and the percentage lost was calculated by subtraction from controls. Table 1 indicates that in chickens the phenol extract produced a consistently higher antibody titer than did the EDTA extract. The differences in titer remained significant throughout the 8-week period of testing. Similar results have been reported in a previous study (5). When cell walls of Pseudomonas aeruginosa were treated with EDTA, the antigenicity of the isolated lipopolysaccharide was lowered when injected in mice. Perhaps the basis for differences in antigenicity of phenol and EDTA extracts lies in their modes of action upon bacterial cell walls. Approximately two-thirds of the 6"Zn2+ and '4C-aspartic acid was removed from labeled cell walls by extraction with EDTA (Table 2). However, less than 10% of the radioactivity was removed by phenol extraction. In both extractions, the radioactivity was nondialyzable. In a previous study (4), differences were found in the composition of lipopolysaccharide fractions isolated by phenol and EDTA from cells of E. coli. It was reported in that study that EDTA extracts were more antigenic and at least as lethal in mice. In the present study, it appears that in chickens differences in antigenicity of phenol and EDTA extracts may be related to the mode of action of these compounds upon bacterial cell walls, as demonstrated by differences in their ability to extract radioactive components from the walls.

v3-fos

2018-04-03T06:07:23.405Z

1972-05-01T00:00:00.000Z

45591194

s2

Cultivation of leptospires: fatty acid requirements. Both the parasitic and the saprophytic leptospires grow well on a pair of fatty acids (one saturated, the other unsaturated) if they contain at least 15 carbon atoms. The fatty acid requirements of serotypes of Leptospira interrogans were previously investigated utilizing a medium which contained "fatty acid-poor" fraction V bovine albumin (Pentex, Inc., Kankakee, Ill.) (3). We recently became aware that, although this albumin contained only trace amounts of free fatty acids, it was contaminated with 2.25 mg of lipid per g of albumin. These lipids markedly affected the lipid composition of leptospires (4). Accordingly, the fatty acid requirements were reinvestigated using a "lipid-poor" albumin which contained <50 jig of lipid per g of albumin (4). The medium used in this study is the same as that previously described except for the albumin component (3). Leptospires used in this investigation were from cultures in the logarithmic or early stationary phase of growth. Unless stated otherwise, a 1% (v/v) inoculum which yielded approximately 3 x 106 cells per ml was used. The results presented in this report were obtained from the third transfer in the test medium. In the absence of added fatty acids, none of the leptospires could be subcultured in the medium. Growth was measured daily with a Coleman (model 7) photonephelometer calibrated with an arbitrary standard. The relationship between nephelometer reading and number of organisms was verified by periodic counts with a Petroff-Hausser counting chamber. All incubations were conducted at 30 C for 5 to 9 days. In the designation of fatty acids, when two numbers are used the first indicates fatty acid chain length, and the second indicates the number of double bonds. When three numbers are used, the first indicates position of double bond, the second, the fatty acid chain length, and the third, the number of double bonds. Both parasitic (eight serotypes) and saprophytic (eight serotypes) leptospires required fatty acids containing at least 15 carbon atoms. These results are in contrast to our earlier finding which indicated that the saprophytes could grow on fatty acids containing less than 15 carbon atoms (3), whereas the parasites required the longer chain fatty acids. The basis for this discrepancy was found to be the ability of the saprophytes to utilize shortchain fatty acids when very low levels of longchain fatty acids were present. The saprophyte patoc grew on 4 x 10-4 M 12:0 (lauric acid) if as little as 0.5 x 10-5 to 10-5 M 16:0 (palmitic acid) was provided. The concentration of longchain fatty acids associated with contaminating lipid of the fatty acid-poor albumin was calculated to be 10- Other studies carried out in the lipid-poor albumin medium further elucidated fatty acid requirements of the leptospires. The unsaturated fatty acid cis-9-18: 1 (oleic acid) was generally a poor substrate, especially for the parasites (Table 1), whereas the combination of cis-9-18: 1 and 16: 0 or 16: 0 alone were good substrates. The saturated fatty acid 18:0 (stearic acid) supported good growth of the saprophytes, but only a few of the parasites grew on this acid ( Table 1). The two parasites ballum and hardjo required a combination of a saturated and a cis-unsaturated fatty acid (16: 0 + cis-9-18: 1) for growth ( Table 1). The trans form of 9-18:1 (4 x 10-4 M) was found to substitute for the above pair of acids with an equivalent level of growth resulting (40 x 107 leptospires/ml). Similar results with trans-9-18:1 have been reported for the Kazan and Reiter strains of Treponema pallidum (1) and the goat mycoplasma strain -y (5), microorganisms which also require a pair of fatty acids for growth. Fatty acids (2) and fatty alcohols (T. Auran and R. C. Johnson, Bacteriol. Proc., p. 27, 1968) are the only two readily utilizable major carbon and energy sources known for the leptospires. Since the fatty alcohols were previously tested in a medium which was not lipid-poor, they were reinvestigated using the lipid-poor albumin medium. The results obtained with palmityl alcohol and oleyl alcohol were similar to those observed with the corresponding fatty acids (Table 1). In addition, a lipid analysis was conducted on patoc cells cultivated on palmityl alcohol. The fatty acid composition of the phosphatidyl ethanolamine of these cells was found to be the same as that of cells cultivated on palmitic acid (4), indicating that the fatty alcohols and fatty acids are metabolized in a similar manner. This work was supported by Public Health Service grant AI-06589 from the National Institute of Allergy and Infectious Diseases. We thank Brian Livermore for conducting the lipid analyses.

v3-fos

2018-04-03T02:19:29.647Z

1972-03-01T00:00:00.000Z

25864464

s2

Enterovirus Concentration on Cellulose Membranes Cellulose nitrate membranes were used as one of the adsorbents in concentrating viruses from water. For adsorption to occur, salts were required. With increase in valency of salt, less salt was necessary for enhanced virus adsorption to membranes. Trivalent salts were more effective because they could be used at only 1% the concentration required for divalent salts. Thus, 0.5 mm AlCl3 was as effective as 50 mm MgCl2. For testing 500 gal of water, only 0.24 kg of AlCl3 was required in contrast to 20 kg of MgCl2. Virus could then be eluted from such membranes, having an area of 486 cm2, with 250 ml of pH 11.5 buffer. Lowering the pH of the eluate and adding AlCl3 permitted the virus to be quickly readsorbed on a smaller cellulose membrane, i.e., 4 cm2. Virus for assay was eluted from the small membrane in 1 ml. This procedure has provided the basis for concentrating minute amounts of virus from large volumes of water. MATERIALS AND METHODS Monkey kidney (MK) cells. Kidneys obtained from immature vervet monkeys were trypsinized and grown as described (4). Virus and virus assays. A plaque-purified line of poliovirus type 1 (Mahoney strain) was used in all experiments, unless otherwise indicated. Other vi-476 ruses used were attenuated poliovirus type 1 (LSc strain), echoviruses type 1 (Farouk) and type 7 (Wallace), and coxsackieviruses A9 (Grigg) and B3 (Nancy). Stock viruses were grown in MK cells using an input of 1 to 10 plaque-forming units (PFU)/cell and stored at -70 C. Virus assays were made by the PFU method as used in this laboratory (4). Virus adsorbents. Cellulose membranes (Millipore Corp.) with a porosity of 0.45 Am were used throughout this study. The diameter of membranes is 25 mm, having an available surface area of 4.0 cm2, unless otherwise indicated. The method used for concentrating viruses on cellulose membranes has been described (10,11). Virus eluents. Proteins, as in serum (10) and beef extract (1), that adsorb to cellulose nitrate membranes, and wetting agents (10,11) exchange for virus and elute it. The membrane-coating components which are used in the eluent are then found in the eluate, where they interfere with reconcentration of virus on smaller surface membranes. Thus, another eluate had to be found. Protein-free salt solutions at pH levels from 2.0 to 8.5 are required for adsorption of viruses to membranes; above pH 8.5 the efficiency of adsorption is decreased. Enteroviruses and myxoviruses can also be eluted with a nonprotein solution at pH 11.5 without detectable loss of infectivity (8,12 Purified water. Water containing no more than 0.01 Mg of dissolved solids per ml and no detectable organics was obtained by passing tap water (see below) through a water purification system (Carborundum Co., Niagara Falls, N.Y.), and was used in place of distilled water. Tap water. Tap water contained 457 ppm dissolved solids and 350 ppm suspended solids. When used, it was dechlorinated with 1-3 ppm sodium thiosulfate. Clarifying filters. To prevent clogging of cellulose membranes, AP20 fiberglass pads (Millipore Corp.) were sometimes used as clarifiers to remove suspended solids and ferric complexes from tap water. To prevent virus adsorption, the pads were first treated with 1% Tween 80 (100 ml for each 100-cm2 surface); residual Tween was removed by thorough washing of the pad. RESULTS Effects of different pH levels on elution of virus from membranes with glycine buffer. Table 1 shows the results of eluting poliovirus (Mahoney) from cellulose membranes at different pH levels. At pH 11 to 12, virtually 100% of the virus was recovered from the membrane. In these tests, 5 ml of glycine-NaOH buffer was used to elute virus from 25mm cellulose membranes; the membrane was then washed with 5 ml of glycine-HCl buffer at pH 2.0 to 4.4, thus yielding a total volume of 10 ml of neutralized eluate. The minimum volume required for elution of virus was found to be 0.5 ml for each 4-cm2 surface. Thus, virus adsorbed to a 25-mm membrane (4 cm2) could be eluted with 0.5 ml of buffer (pH 11.5), immediately followed by 0.5 ml of pH 2 neutralizing wash, to make a final recovery volume of 1.0 ml. Effects of salts on virus adsorption to cellulose membranes. Several monovalent, divalent, and trivalent salts dissolved in purified water were tested to determine the lowest concentration required for virus adsorption. The experimental procedures and results are shown in Table 2. Trivalent aluminum salts proved best, and AlCl3 and Al2(SO4)3 were just as effective at about 1% the concentration of MgCl2. Thus, the amount of salt which would have to be transported to the field is significantly lower for AlCl, than for MgCl2. Only 0.24 kg of aluminum salts would be required for adsorption of poliovirus to membranes possessing 500 gal of water, whereas 20 kg of MgCl2 would be necessary ( Table 3). Effects of MgCl2 and AICI3 on virus suspended in tap water. Since our goal is to concentrate virus in the field from natural water, Houston tap water was used to determine the effects of Mg and Al ions on virus adsorption. Figure 1 shows the experimental procedures and results. Again, AlCl3 was at least 200 times more efficient than MgCl2 in facilitating poliovirus adsorption. Effects of AICIl on large volumes of tap water. The experiments described above required that only 5-ml samples be passed through the cellulose membrane. When large volumes of tap water were treated with AlCl3, the aluminum hydroxide gel which formed clogged the filter. However, enteroviruses can line, and 10-ml samples were filtered through each of six 25-mm cellulose membranes (0.45 Mm pore size). The membrane filtrates were collected, pooled, and assayed (control virus, membrane filtrate). Each membrane was then washed with 10 ml of saline at pH 6.9, and the membrane washes were pooled and assayed. Each membrane was then treated with 5 ml of the eluent at the indicated pH, and the membrane was washed with 5 ml of buffer made acidic with HCl so that the total 10 ml recovered would be neutral. be adsorbed to cellulose membranes at low pH levels, and the gels are soluble in acid. Therefore, an experiment was performed to determine flow rates of tap water containing 0.0005 M AlCl3 through membranes at different pH levels. Tap water was filtered through 90-mm membranes (0.45 gm pore size) at constant pressure. Tap water at 5 psi, free from Al ions, required 24.4 min for filtration of 10 liters. Tap water containing 0.0005 M AlCl3 at pH 7 clogged the filter before 1 liter passed the membrane. Similarly, at pH 6 and 5, immediate clogging occurred. However, at pH 4.5, 4.0, 3.5, and 3.0, the flow rate was faster (13.6 min/10 liters) than the control sample free from AlCl3. The increased flow rate is attributable to the fact that the acid dissolved some of the natural gels present in tap water, especially ferric hydroxide. All subsequent experiments were carried out by adjusting virus-tap water mixtures to pH 3.0 with HCl and then adding AlCl3 to give a final concentration of Table 1, except that tap water was used as a virus and salt diluent. Average number of PFU/0.1 ml present in the control was 150 (±15). 0.0005 M AlCl3. The water must be made acid before addition of the Al ions. Effect of AICI3 on adsorption of enteroviruses to cellulose membranes. A number of enteroviruses were studied ( Table 4). All viruses tested (polioviruses, echoviruses, and coxsackieviruses) were adsorbed to the membranes with 0.0005 M AlCl3 and were quantitatively eluted with pH 11.5 buffer. a Viruses were diluted in pH 3.5 tap water containing 0.0005 M AlCl3, and 10-ml samples were filtered through 25-mm cellulose membranes (0.45 tm pore size). The membranes were washed with 10 ml of saline and then treated with 5 ml of pH 11.5 eluent followed by 5 ml of pH 2 neutralizing buffer. Reconcentration of poliovirus adsorbed to cellulose membranes. Virus recovered from membranes as outlined above can be readily readsorbed to new membranes, since the eluate does not contain any membrane-coating components (10). This is done merely by adjusting the pH levels and adding AlCl3. Under conditions which require the processing of large volumes of water at high flow rates, 293mm membranes (0.45 um pore size) require 200 to 300 ml for virus elution. The virus can then be readsorbed on a 25-mm membrane (0.45 gm pore size) at acidic pH levels and eluted from this membrane with as little as 0.5 ml of pH 11.5 buffer. An experiment was conducted to show that by pH control and salt addition poliovirus can be recycled on and off cellulose membranes. A total of 1,300 PFU of poliovirus was adsorbed to a 293-mm membrane in the presence of 0.0005 M AlCl3 at pH 4.0. The membrane was washed with saline to remove residual Al ions to avoid subsequent gel formation with the basic eluent and thus to prevent clogging of membranes. The 293-mm membrane was then treated with 300 ml of pH 11.5 eluent. The eluate was collected in 300 ml of pH 2 buffer to yield 600 ml of virus suspension at pH 6.7. [If the suspensions are not acidic (pH 4.0 to 4.5) at this point, they should be made acidic with HCl before addition of AlCl3.] AlCl3 was added to yield a final concentration of 0.0005 M to enhance virus readsorption to a freshly prepared 25-mm membrane (0.45 lim pore size). Virus was then eluted from the membrane with 0.5 ml of pH 11.5 buffer, and the membrane was washed with 0.5-ml volume of pH 2 buffer. The 1 ml of suspension now con-tained 1,250 PFU of poliovirus, virtually all of the virus initially contained in the 5-gal test volume. This type of concentration and elution of poliovirus was carried out many times with 90 to 100% recovery of the virus in the concentrate. Processing of large volumes of tap water. These new findings were then applied to recovery of virus from large volumes of tap water. Five gallons of 0.05 M AlCl3 in 0.5 M HCl acid was placed in a 5-gal pressure vessel. The salt solution was mixed into running tap water (20 psi) by nitrogen pressure (50 psi) and a metering valve so that the mixture was diluted 1: 100 in the water to give a final concentration of 0.0005 M AlCl3 and a pH of 3.0. A pH meter was used to monitor the effluent, and the metering device was adjusted to produce a flow that gave the desired pH of 3.0. Upstream a second vessel with 5 gal of purified water containing poliovirus, sodium thiosulfate, and 0.04% phenol red was connected to the nitrogen pressure. These fluids were also diluted 100-fold in the running tap water to give a final concentration of virus as indicated in Table 5 (i.e., 0.5 PFU/gal when 250 PFU input was used for 500 gal), 2 Mg of thiosulfate per ml, and a faint pink color. Immediately before the downstream pressure vessel, a sight glass was inserted in the tubing to monitor the color of the fluids, and in this way the quantity of virus and thiosulfate being added was controlled. The results of three consecutive tests in which 500-gal batches of water were processed by adsorption on a 293-mm cellulose membrane (0.45 um pore size) and then reconcentrated on a 25-mm membrane are shown in Table 5. Additional experiments have shown that methyl orange can serve as an indicator to determine pH, since the dye changes from light yellow to bright red at pH 3.0. A solution of 0.5 M HCl, 0.05 M AlCl3, and 0.1% methyl orange was prepared in a 5-gal pressure vessel so that 1:100 dilution would yield a pH of 3.0, 0.0005 M AlCl3, and 0.001% methyl orange. The concentrate was forced into running tap water under nitrogen pressure until the color changed from yellow to red. Testing the effluent by a pH meter confirmed that pH 3.0 had been obtained and also assured that the concentrate was being delivered into the running water at a dilution of 1:100. At this dilution, methyl orange was not toxic to the tissue culture cells (thus the effluent containing dye could be assayed without dilution), nor was the dye virucidal to poliovirus. With Houston tap water (pH 8.2), the concentration of HC1 indicated above was required. However, for other areas, the water under test must be titrated to determine the amount of HC1 required to bring the water to pH 3.0. The concentration needed must then be determined so that a 100-fold dilution of the salt-dye-acid stock can be injected into the water to yield the optimal salt-pH concentrations. DISCUSSION The concentration of viruses from large volumes of water in the field is now feasible with the use of aluminum salts for adsorption of viruses to cellulose membranes. Aluminum salts can be used in 1% the concentration of magnesium salts. For virus to adsorb from a 400-gal sample, 45 lb of MgCl2 is needed. On the other hand, with AlCl3, 1,000 gal of water can be processed with only 1 lb of this trivalent salt. In this report, we have also described a method for concentrating enteroviruses by serial adsorption to and elution from cellulose membranes. By manipulating hydrogen ion levels, viruses in 400to 500-gal samples can be adsorbed to 293-mm membrane surfaces at pH 3 to 4 at high flow rates, eluted at pH 11.5 with 250 to 300 ml of protein-free buffer, and readsorbed to a small surface membrane (25 to 47 mm). Viruses can then be removed into a APPL. MICROBIOL. final eluate of 1 to 5 ml, which can be conveniently assayed.

v3-fos

2020-12-10T09:04:12.776Z

1972-10-01T00:00:00.000Z

237230119

s2

Death of Lactobacillus bulgaricus Resulting from Liquid Nitrogen Freezing1 Concentrated cell suspensions of Lactobacillus bulgaricus prepared from cells grown in semisynthetic media were frozen in liquid nitrogen. After storage for 24 hr, the cell suspensions were found to have decreased colony counts and acid-producing capacity in milk. The amount of loss varied among the different strains tested. The addition of known cryoprotective agents to cell suspensions of the most labile strain before freezing provided little or no protection to the cells. However, storage stability of all strains investigated was improved by supplementing the growth medium with Tween 80 (polyoxyethylene sorbitan monooleate). The concentration of Tween 80 necessary for maximal storage stability varied among strains. Concentrated preparations of starter bacteria are being used successfully in the manufacture of various cultured milk products. Lactic streptococci (1,7,10,12) and leuconostocs (6) can be successfully preserved and used after freezing and storage in liquid nitrogen. Optimal performance by the cultures is dependent on the stabilization of their viability and biological activity during freezing and storage. In projecting the use of concentrated starter cultures for additional foods, it is reasonable to expect that concentrated cultures of lactobacilli could find useful applications, particularly in the manufacture of products such as yogurt. Certain lactobacilli are not sufficiently resistant to freezing to permit their use as frozen concentrated starter cultures, especially for the direct inoculation of the final milk without intermediate starter preparation. In this study we report on factors involved in the susceptibility of Lactobacillus bulgaricus to freezing, and on means whereby the organism can be made more resistant to freezing and storage in liquid nitrogen. MATERIALS AND METHODS Cultures. The strains of L. bulgaricus selected for this study are used commercially in yogurt and Italian cheese manufacture. The cultures were routinely propagated in sterile litmus milk. A 1% inoculum was used with incubation at 37 C for 15 hr, and ' Paper no. 3783 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh. cultures were stored in a refrigerator between transfers. Three daily transfers in litmus milk were made before use in an experiment. Growth media. The cultures were grown in lacto-'bacilli MRS broth (Difco) or in a medium containing 2% tryptone (Difco), 1% yeast extract (BBL), and 2% lactose. The latter medium was designated as TYL and was used as the basal medium to study the effects of medium composition. In some experiments, this medium was supplemented with 0.5% (w/v) corn steep solids (TYLCS). Concentrate preparation. Cultures of L. bulgaricus were grown statically in 100 ml of the test media (milk dilution bottles) at 37 C for 12 hr. The cultures were cooled in an ice-water bath for 10 min, and the cells were recovered by centrifugation for 20 min at 6,780 x g and 0 C. The resulting pellet was resuspended in sufficient cold sterile 10% (w/v) nonfat milk solids (NFMS) to produce a concentrated culture having approximately 10' cells per ml. In experiments involving the use of cryoprotective agents, the cell pellets were resuspended in sterile NFMS (20%, w/v) and then diluted 1:1 with aqueous solutions of the protective agents. The concentrated cell suspensions were aseptically weighed (1-g quantities) into 1.5-ml sterile plastic vials. The vials were sealed with a 1: 1 mixture of acetone and chloroform, and were then submerged and held in liquid nitrogen (-196 C) for 24 hr. Colony counts. Colony counts were determined before and after freezing by the pour-plate method. The vials of frozen cells were thawed by immersion SMITTLE, GILLILAND, AND SPECK in 1 liter of tap water at 17 C for 5 min. The initial dilution (1: 100) was made by adding 1 g of cell suspension to 99 ml of cold 10% NFMS (steamed for 30 min on the day of use). Additional dilutions were prepared with sterile 99-ml dilution blanks containing 0.1% NFMS and 0.01% silicone antifoamer (Sigma Chemical Co.). Duplicate plates were prepared from the required dilutions. The plating media and incubation times were selected to maximize the count for each strain. The YEPT plating medium contained 5 g of yeast extract (BBL), 5 g of Phytone (BBL), 5 g of Trypticase (BBL), 5 g of dextrose, 4 g of K2HPO4, 15 g of agar, and 1,000 ml of distilled water. The mixture was adjusted to pH 7.0 before autoclaving at 121 C for 15 min. This medium was used for L. bulgaricus strains NCS1 and NCS2. Lactic agar (3) was employed for strain NCS3. The medium of Rogosa et al. (11), modified by omitting the acetic acid and autoclaving for 15 min at 121 C, was used for strain NCS4. The plates for strain NCS1 were incubated for 48 hr at 37 C; those for the remaining strains were incubated for 72 hr at 37 C. All colonies were counted with the aid of a Quebec colony counter. Acid production. Portions (10 ml) of the initial dilution of the cell suspensions were incubated at 45 C for 4 hr. The acid produced in each sample was measured by titration with 0.1 N NaOH to pH 8.6 by use of a Radiometer automatic titrator. Compensation was made for titratable acid present in uninocu-. lated 10% NFMS. The percent acid production lost was then calculated by the following formula: percent acid production lost acid produced by unfrozen cells acid produced by frozen cells =~~~~~~~~~x 100 acid produced by unfrozen cells RESULTS Effect of liquid nitrogen freezing on L. bulgaricus. Cultures varied in their stability to freezing and storage at -196 C (Table 1). Strain NCS1 was very labile to freezing, whereas NCS4 was essentially unaffected. Since strain NCS1 was the most sensitive to freezing, it was chosen for further study. Freezing menstruum additives. The addition of 2.0% monosodium glutamate, 10% glycerol, or 2.0% polyvinylpyrrolidone to the menstruum provided little or no protection of the cells against freezing ( Table 2). The number of lactobacilli killed was slightly less in the samples supplemented with 4% monosodium glutamate and a mixture of 2.5% glycerol and dimethylsulfoxide. The mixture of glycerol and dimethylsulfoxide afforded slight protection of the acid-producing capacity. However, the apparent protection resulting from these additives was not great enough to be significant in preserving the cultures. Effects of growth medium composition on storage stability. The effects of growth medium composition on stability of cells to subsequent freezing and storing in liquid nitrogen was examined in further attempts to improve the stability of the lactobacilli. Cells of L. bulgaricus NCS1 grown in lactobacilli MRS broth were resistant to freezing, whereas those grown in TYLCS were not ( Table 3). The major differences in these media with respect to the types of ingredients appeared to be the presence of Tween 80, supplementary mineral salts, and buffer in the MRS medium. Based on this information, MRS broth was prepared with these components omitted, and TYLCS was supplemented with the components singly and combined. The results ( Relationship of growth time to storage stability. Since the incubation time for Supplementation of freezing menstruum with Tween 80. Cell suspensions of L. bulgaricus NCS1 grown in the presence of Tween 80 may have contained small amounts of this material; therefore, the effect of incorporating Tween 80 into the freezing menstruum was investigated. For these experiments, the cells were grown in TYL broth and resuspended in sterile 10% NFMS containing various levels of Tween 80. The incorporation of 0.05% Tween 80 into the freezing menstruum did not protect L. bulgaricus NCS1 from damage caused by freezing. Use of the TYLCS medium was discontinued at this point because the variable composition of corn steep did not permit a predictable yield of cells. Effects of Tween 80 in the growth medium on the stability of other strains of L. bulgaricus. To ascertain whether the effects of adding Tween 80 to the growth medium of L. bulgaricus NCS1 was a particular requirement of this culture, other strains of L. bulgaricus were investigated. All cultures that were grown in TYL plus 0.1% Tween 80 survived freezing better than those grown in TYL (Table 4). Of the cultures grown in TYL, strain NCS4 was most stable to freezing. The stability of strains NCS1, NCS2, and NCS3 was markedly im-proved when 0.1% Tween 80 was in the growth medium. The improvement for strains NCS2 and NCS3 was not as great as for strain NCS1. Subsequently, the effect of different concentrations of Tween 80 on L. bulgaricus NCS3 was examined. A much lower concentration (0.025%) of Tween 80 was required for increasing the numbers of organisms that survived freezing (Table 5). This demonstrated variability among the cultures with respect to the optimal level of Tween 80. DISCUSSION The instability of concentrated cultures of L. bulgaricus to freezing in liquid nitrogen is in contrast to characteristics reported for concentrated cultures of lactic streptococci and Leuconostoc citrovorum (1,6,7,10,12). The unsatisfactory storage stability of the lactobacilli imposes severe restrictions on the commercial use of concentrated cultures prepared from them. The use of additives to protect bacterial cells from damage due to freezing has been Raleigh, 1970). Glycerol and dimethylsulfoxide are believed to protect cells by reducing electrolyte concentrations around the cells during freezing (8). The lack of response to 10% glycerol and the poor response to 2.5% glycerol and dimethylsulfoxide indicated that storage instability of lactobacilli is probably not due to an excessive electrolyte concentration during freezing. Morichi et al. (9) found that L. bulgaricus was not adequately protected by glutamic acid during the process of freeze-drying; our results with monosodium glutamate were similar. Although monosodium glutamate and glycerol provided some protection, the concentrated cell suspensions of L. bulgaricus were not sufficiently protected for commercial application. The mechanism whereby Tween 80 imparts freezing stability to the lactobacilli has not been elucidated. Calcott and Postgate (2) reported that Tween 80 incorporated into the freezing menstruum was effective in protecting Aerobacter aerogenes from freezing damage. The results from our study, however, indicate that Tween 80 used in this manner did not protect the lactobacilli during freezing. Storage stability of L. bulgaricus cells was markedly improved only when the cells were grown in broth supplemented with Tween 80, which has long been known to stimulate the growth of the lactobacilli (13). However, little information is available concerning its physiological role. Such material may be directly involved in lipid metabolism or may have a physical effect on the cells. Most of the lipid material in gram-positive microorganisms is associated with the cell membrane; thus, if Tween 80 is metabolized, it could play a role in developing cell membranes whose integrity is maintained during freezing and storing of L. -bulgaricus in liquid nitrogen.

v3-fos

2020-12-10T09:04:17.146Z

1972-11-01T00:00:00.000Z

237233954

s2

Identification of T-2 Toxin in Moldy Corn Associated with a Lethal Toxicosis in Dairy Cattle Over a 5-month period during the winter of 1970-71, 20% of the lactating Holstein cows in a Wisconsin dairy herd died after prolonged ingestion of a diet containing 60% moldy corn infested with Fusarium tricinctum (2 � 105 propagules per g of moldy corn). Ethyl acetate extracts of the ground dried corn induced severe dermal reactions when applied to the skin of shaved 60-g albino rats and killed four of five 100-g rats that were force fed 1 ml in 2 ml of pure corn oil. T-2 toxin (3-hydroxy-4, 15-diacetoxy-8-[3-methylbutyryloxy]-12, 13-epoxy-Δ9-trichothecene) at concentrations of 2 mg per kg of dry corn was identified in purified extracts of the moldy corn by means of gas-liquid chromatography and mass spectrometry. This concentration of T-2 toxin in the moldy feed and the nature of the toxic effects observed strongly suggest a major causal relationship. Moldy corn has frequently been associated with outbreaks of severe toxicity in farm animals (W. F. 0. Marasas, Ph. D. thesis, Univ. of Wisconsin, Madison, 1969; reference 9). Such problems are worldwide in distribution and have been associated with a variety of mycotoxin-producing fungi and their metabolites. Only a few clear-cut instances are recognized, however, in which positive causal relationships have been established between the presence of toxigenic fungi and their metabolites in the suspect product and the animal toxicosis (4). The vast majority of these associations are circumstantial, with the equivalent of "Koch's Postulates" remaining to be completed (7). The particular fungal flora and the resulting toxic metabolic products developing in moldy corn appear to be closely linked, in general, to the prevailing climate of the particular geographic region. In the warmer areas, the development of Aspergillus flavus and production of aflatoxins are common (12). In the northern temperate regions, moldy corn is much less likely to be contaminated with A. flavus (9). Instead, fungi are present which develop at the low temperatures common to the northern winter storage conditions. Thus, toxin-producing strains of Trichothecium roseum, Ni-grospora sp., Epicoccum nigrum, Alternaria tenuis, Fusarium roseum, F. moniliforme, F. tricinctum, various penicillia, and others are commonly isolated. Clearly, however, F. tricinctum is consistently the most toxic of the fungi isolated from moldy corn in low-temperature storage, and its presence in moldy corn samples has been highly correlated with farm outbreaks of moldy corn poisoning (9). Experiments to duplicate in the field conditions leading to the development of toxic moldy corn were unsuccessful in the absence of F. tricinctum contamination (Marasas, Ph.D. thesis). Several toxic metabolites have been identified and characterized from pure cultures of these toxic strains of F. tricinctum. These include a butenolide (4-acetamido-4-hydroxy-2butenoic acid-y-lactone) (13), occasionally the estrogen, zearalenone (6-[10-hydroxy-6-oxo-1undecenyl] beta resorcylic acid-mu-lactone) (3), and members of the 12, 13-epoxytrichothecenes (1). A member of this latter group, T-2 toxin (3-hydroxy-4, 15-diacetoxy-8-[3methylbutyryloxy ] -12, 13 -epoxy -AV-trichothecene), is produced in large quantity when pure cultures are grown at low temperatures (J. R. Bamburg, Ph.D. thesis, Univ. of Wisconsin, Madison, 1968). Although toxins of the tri-684 chothecene group are readily obtained from pure cultures, they have not previously been identified chemically in naturally toxic products such as moldy corn. Thus, the causal relationships of these toxins to moldy corn poisoning has remained in doubt, even though the symptoms of toxicosis in farm animals resemble those produced by artificial administration of pure trichothecenes (8,9). Demonstration of these compounds in toxic natural products at biologically active concentrations is needed to establish causal relationships. We report here the detailed biological and chemical analyses of a case of moldy corn poisoning in dairy cattle which developed over a 5-month period during the winter of 1970-71 on a farm near Hortonville (Outagamie County), Wis. MATERIALS AND METHODS Field history. The virtual absence of moldy corn toxicosis in Wisconsin livestock since the severe outbreak years of 1962, 1964, and 1965 (9) has frustrated attempts to discover practical methods for detecting trichothecenes in naturally toxic moldy feeds. During the winter of 1970-71, however, a number of cases of toxicosis, possibly related to moldy feed, were called to our attention through the Wisconsin Animal Health Laboratories. In the case selected for study, 7 of 35 lactating Holstein cows died over a 5month period after prolonged ingestion of a diet containing 60% ground moldy corn. Postmortem examination revealed extensive hemorrhages on the serosal surface of all internal viscera typical of those previously observed in cases of moldy corn poisoning (9). No significant pathogenic bacteria were isolated in bacteriological examinations from such animals. During most of the winter, this herd had been "off-feed," with certain animals occasionally having elevated temperatures. The herd also had a high frequency of abortion, usually in the 5th or 6th month of pregnancy and, in general, failed to respond to the treatments of the local veterinarian. The moldy corn, originally planted but then not needed for silage preparation, was harvested in late October for storage as high-moisture ear corn. The major part of the crop was lower in moisture and filled a double, well-ventilated corn crib with an open center. Because of the large crop, the excess corn remaining after the cribs had been filled was placed in the center. The high-moisture corn picked later was then piled over the top of this drier corn. Feeding of this mixture of wet and dry corn began soon after harvest. Four thousand pounds of corn, 2,000 pounds of oats, and 600 pounds of soybean meal were ground and fed per week, along with additional nutrient supplements and salt. The farmer did not notice moldiness in the corn until late March, although some of the ground-up batches showed evidence of heating in February and March. By late April, the corn was visibly moldy. Large sample collections of this moldy corn were taken from the farm on April 28 and May 19 to be used for detailed biological and chemical analyses. This material was finely ground in a Wiley mill and stored frozen (-20 C) in large sealed polyethylene bags until processed. Microbiological analysis. The major fungi present in the moldy corn were isolated in pure culture by standard mycological techniques (11). Pure cultures maintained as single conidial isolates were stored on sterilized soil, cryogenically in liquid N2 or on potato dextrose agar slants at 5 C. For estimations of F. tricinctum populations, samples of ground moldy corn were blended aseptically for 2 min in 100 ml of sterile distilled water. A dilution series was prepared from this stock suspension, and the concentration of F. tricinctum propagules per g of moldy corn was estimated from numbers of typical colonies developing after 1 week on potato dextrose agar plates containing lactic acid and Tergitol NPX (Union Carbide Corp.) (10). Toxicological analysis. Random samples taken from the stored bags of ground moldy corn were mixed, oven dried (37 C), and extracted at room temperature four times with ethyl acetate (4 hr per extraction with 1 hr of constant shaking). Yield of the pooled oily extracts remaining after removal of the solvent amounted to 32.3 g per kg of dry corn. Oral toxicity of the extracts was determined by variously force feeding 0.5 or 1 ml of extract to 60-g albino rats (Holtzman Co., Sprague-Dawley strain). Because of the large amount of oil being fed to the 60-g rats, extracts from a second sample of the moldy corn were fed to 100-g rats (five rats per concentration) at a series of concentrations (0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml), and a 3-ml volume was kept constant by utilizing pure commercial corn oil (Mazola, C.P.C. International Inc., N.J.) as diluent and control (see Table 1). Dermal toxicity tests were carried out on 60-g rats by the procedures of Bamburg (Bamburg, Ph.D. thesis). Toxicity of major fungi. Pure cultures of isolates of each of the major fungal components of the moldy corn in 500-ml flasks on sterile moist corn were separately incubated at 25 and 8 C for 30 days. The colonized corn was then oven dried (37 C), ground, and stored frozen. Samples from each culture, at each temperature, were extracted with ethyl acetate as described above, and the extracts were force fed to 60-g rats (three rats per treatment; 2.5 ml per rat) (see Table 2). Solvent systems for chromatography. Three solvent systems for thin-layer and column chromatography (TLC) were used as follows: (i) tolueneethyl acetate, 3 Trimethylsilyl (TMS) derivatives. Small portions (1-5 mg) of various purified preparations were silylated by mixing with 0.08 ml of bis-trimethylsilyl acetamide and 0.02 ml of pyridine and allowing the mixture to stand at room temperature for several hours. Standard samples of TMS T-2 toxin and TMS T-2 tetraol (6) were prepared in the same manner. Gas chromatography. Gas-liquid chromatography (GLC) was carried out on an Aerograph HY-FR model 600D instrument equipped with a hydrogen flame ionization detector and an Aerograph linear temperature programmer model 326. The temperature program was started at the time of injection of the sample and continued throughout the run. The general chromatographic procedure and method of identification of peaks were carried out in a manner similar to that previously described (6). Preparation of columns. Gas chrom Q (80-100 mesh) was coated with either 2% QF-1 or 3% SE-30 by dissolving the desired amounts of the liquid phase in chloroform-methylene chloride (1:1, v/v) solution, adding the solid support and, after stirring the mixture thoroughly, evaporating to dryness at reduced pressure in a rotary evaporator. The coated support was further dried for 60 min in a 100 C oven and finally stored in a desiccator until needed. Coiled Pyrex columns (150 by 0.5 cm, inside diameter) were filled and preconditioned as previously described (6). Isolation of T-2 toxin. The ground, moldy corn (sample collected 19 May 1971) was extracted in 500-g portions in a Soxhlet apparatus with 1 to 1.5 liters of chloroform for a period of 15 to 20 hr. The chloroform solution was washed twice with about 800 ml of water each time, and the chloroform layer was then concentrated by evaporation to a thick brown oil. The oil was dissolved in 200 ml of methanolwater (4:1, v/v), and the lipids were separated by shaking out twice with 150-ml portions of Skellysolve B. The aqueous methanol layer was then diluted with 120 ml of water to make the methanolwater ratio 1:1, and this solution was twice extracted with 150-ml portions of chloroform-ethyl acetate, 1:1 (v/v). Evaporation of the chloroformethyl acetate solution left a light colored oil which was designated as preparation A. A 2.3-g quantity of preparation A obtained from 2 kg of the ground, moldy corn was further purified by passing through a silica gel column (2.2 by 64 cm) developed with solvent system I. The effluent was collected in 4-to 5-ml fractions. Every second fraction was monitored by TLC in solvent system I, with authentic T-2 toxin included on each plate for comparison. Fractions showing spots close to that of T-2 toxin were combined and, when evaporated, left 360 mg of oily product designated as preparation B, which was strongly positive in the rat skin test. A 60-mg portion of preparation B was further purified by preparative TLC on silica gel H plates, developed in solvent system II. A standard sample of T-2 toxin was included on each plate as a marker. Both T-2 toxin and the tetraol (below) were located on TLC plates by spraying with concentrated sulfuric acid followed by heating at 150 C. The area corresponding to the T-2 toxin was collected and exhaustively eluted with 3-ml portions of methanol. The eluted material was rechromatographed in the same manner on another similar preparative Silica gel H plate. Evaporation in a stream of nitrogen of the eluate from the second plate yielded 10 mg of residue, which was designated as preparation C. Another 20-mg portion of preparation B was saponified to convert any T-2 toxin present into the corresponding tetraol. For this purpose, the sample was dissolved in 4 ml of a 0.3 N solution of NaOH in ethanol-water (9:1). The mixture was held at room temperature for 12 to 15 hr, and then neutralized with 0.2 N alcoholic HCl. After evaporation of the solvent, the residue was extracted three times with a mixture of 0.6 ml of tetrahydrofuran and 1.2 ml of benzene each time. The extract was concentrated and applied to a silica gel H plate. After developing the plate three separate times in solvent system II, in which T-2 tetraol has an RF value of 0, a 1-to 2mm line 2 cm above the baseline (origin) was scraped away. The plate was then developed two times in solvent system HI in which T-2 tetraol has an RF value of 0.49. The silica gel between a point 0.5 cm above the base line and the line cut out 2 cm above the base line was collected and eluted with methanol-tetrahydrofuran (1: 1, v/v). Removal of the solvent in a stream of nitrogen left 2 mg of residue, designated as preparation D. RESULTS Fungal microflora. The major fungi present in these moldy corn samples were typical of those isolated in previous cases of moldy corn poisoning (Marasas, Ph.D. thesis; reference 9). The predominant fungi present were E. nigrum, Cladosporium herbarum, Acromoniella atea, and F. tricinctum. Certain ears contained almost pure cultures of F. tricinctum. Surfacedisinfected kernels, when plated on acidified potato dextrose agar, yielded mostly members of the mucorales (especially Mucor and Phycomyces). With the addition of Tergitol NPX to the medium, the mucorales were suppressed and F. tricinctum, F. roseum, F. moniliforme, E. nigrum, A. atra, and various penicillia (P. cyclopium, P. claviforme, and others) were the predominant fungi isolated. Where blended moldy-corn suspensions in dilution series were plated on the media, only Cephalosporium sp., C. herbarum, various yeasts, and F. tricinctum were the predominant fungi. Although F. tricinctum was not the predominant fungus, dilution plate colony counts indicated populations of the fungus in excess of 2 x 105 propagules per g of moldy corn in the April 28 collections. In the May 19 collection, however, the F. tricinctum populations, although detectable, were much lower. Toxicology of the moldy corn. Oily ethyl acetate extracts of the ground, dried moldy corn gave very toxic responses in the rat dermal assay (Bamburg, Ph.D. thesis). Sixty to 70 mg of preparation A (from 60 g of moldy corn) also gave a strong positive response. When force fed to 60-g rats, 1 ml of the ethyl acetate extract killed test animals in 3 hr (Table 1). Similar 1-ml doses in 2 ml of pure cSprague-Dawley strain (Holtzman Co.) unsexed rats; 60-g weight for sample 1 tests, 100-g weight for sample 2 tests. corn oil killed four of five 100-g rats in 24 hr. At higher doses (2.50 ml in 0.50 ml of corn oil), three of five rats died in 3 hr and the rest in 24 hr. The oral 24-hr mean lethal dose of the oily ethyl acetate extract was estimated by interpolation on a plot of the 24-hr mortality data in Table 1 to be 0.75 ml for the 100-g rats used, or 7.5 ml of body weight per kg. Toxicity of the major fungi. All the isolates of F. tricinctum from the moldy corn, when grown in pure culture, proved to be toxic, and 2.5 ml of the ethyl acetate extract, force fed, killed all test animals whether the cultures were grown at 8 C or at room temperature (24 C) ( Table 2). Pure cultures of Phycomyces sp. (24 C), P. claviforme (8, 24 C), P. cyclopium (24 C), A. flavus (24 C), and E. nigrum (8 C) also contained ethyl acetate-extractable toxins. Detection of T-2 toxin. When preparation B was converted to the TMS derivative and subjected to GLC on the SE-30 column, the chromatogram illustrated in Fig. 1 was obtained. The GLC conditions used are indicated in the figure legends. Although Fig. 1 indicates that the sample chromatographed was a complex mixture, a peak was apparent in the position expected for T-2 toxin. Preparation B was similarly chromatographed on the QF-1 column and again showed an apparent T-2 peak, although the portion of the mixture possibly 24,1972 consisting of T-2 toxin appeared much smaller in this case (Fig. 2). This peak was collected by preparative GLC on QF-1, and the material so obtained gave a strong positive skin test after 24 hr. In view of the difference in peak areas from the two columns, it appeared that the apparent T-2 toxin peak from the SE-30 column might have been contaminated with other materials. To further purify this material, and since the TMS derivative of T-2 toxin is sufficiently stable for preparative GLC purification, the peak corresponding to TMS T-2 toxin from the SE-30 column was collected, dissolved in a small amount of pyridine, and reinjected into the QF-1 column. The result ( Fig. 3) shows that by this procedure, the material was further separated into at least four components. The peak with longest retention Preparation D (saponified preparation B), when subjected to GLC on QF-1, produced a peak corresponding in retention time to T-2 tetraol (Fig. 4). This result provides strong confirmatory evidence that the material chromatographing as T-2 toxin in Fig. 1 to 3 actually was T-2 toxin, at least in part. This peak was collected and its mass spectrum obtained (Fig. 5). Preparation C, the most highly purified sample, was also silylated and chromato- Fig. 6. When the T-2 toxin peak of this chromatogram was collected and subjected to mass spectroscopy, the spectrum obtained was very similar to that of TMS T-2 toxin (Fig. 7). Since T-2 toxin (C2,H4O0, molecular weight 466) has one free hydroxyl group, it forms a mono-TMS derivative which has a molecular weight of 538. This is the molecular ion of both the standard and field samples (Fig. 7). The fragmentation patterns of the two samples are likewise very similar. Peaks appearing in the spectrum of the field sample which are absent from the standard are undoubtedly due to trace impurities. The mass spectra of the isolated and authentic T-2 tetraol samples (Fig. 5), however, are almost identical, no doubt because the unknown was more highly puri-fied. Therefore, on the basis of the chromatographic, spectral, and biological properties, the substance was definitely identified as T-2 toxin. The approximate concentration of T-2 toxin in the original feed was roughly estimated from peak areas in the GLC tracing of preparation C (Fig. 6) by comparison of peak areas produced by the sample and by standard T-2 toxin (6). The estimate of 2 mg/kg is undoubtedly low because of unavoidable losses during the purification procedure. DISCUSSION The moldy corn analyzed in this case of suspected moldy corn poisoning was one of the most toxic field samples ever examined in this laboratory. The presence of toxic strains of F. tricinctum in the samples at very high levels and the fact that one of the trichothecenes (T-2 toxin) could be positively identified at concentrations of at least 2 mg pier kg of dry corn strongly suggest a major causal relationship. Because of the complex microbiology of the samples, it can be assumed that other toxins were also present and contributed to the toxicity of the samples. The demonstration of the toxin-producing potential in pure cultures of isolates of other species from this corn indicates this also. Grove et al. reported that daily injections of 0.1 mg of T-2 toxin per kg of body weight were lethal to a 650-pound steer after 65 days (5). A dairy cow consuming 10 to 15 kg of this moldy corn per day would be obtaining at least 20 to 30 mg of T-2 toxin daily, although by the oral route. Whether this level would be sufficient to account for the observed toxicity in the herd concerned cannot be determined at this time, as oral mean lethal dose values for T-2 toxin in cattle have not yet been determined. By comparison with other animals, however, this appears to be a good possibility, particularly in view of the fact that the actual T-2 content of the mixed moldy grain sample examined might have been considerably higher than 2 Atg/g, and also since some of the feed was more highly contaminated with mold than other portions. The individual cattle affected may have consumed the more highly contaminated feed.

v3-fos

2018-04-03T00:25:45.305Z

1972-02-01T00:00:00.000Z

11720228

s2

Analysis of Free Amino Acid Pools in Fungal Mycelia Free amino acid pools derived from three different types of fungal mycelia have been analyzed by the method of Heathcote and Haworth by using thinlayer chromatography. The preliminary extraction was carried out with boiling water and interfering proteins; peptides and salts were first removed by means of an ion-retardation resin. As far as determined, the results obtained represent the first quantitative analysis of fungal amino acid pools. Free amino acid pools derived from three different types of fungal mycelia have been analyzed by the method of Heathcote and Haworth by using thinlayer chromatography. The preliminary extraction was carried out with boiling water and interfering proteins; peptides and salts were first removed by means of an ion-retardation resin. As far as determined, the results obtained represent the first quantitative analysis of fungal amino acid pools. Although an extensive range of microorganisms, algae, and higher plants have been examined for their free amino acid pools, little is known at the present time concerning the composition of the pools within fungal mycelia. Thornton and McEvoy (4) prepared free amino acid pools of several fungal mycelia by different methods, the most successful of which consisted of extraction into boiling water. They were unable, however, to obtain good chromatographic separation of the amino acids in these extracts because of the interference of proteins, peptides, and salts. Desalting with acid butanone improved the chromatographic picture, but the interference from peptides remained. Consequently, these workers were unable to determine the amino acid composition quantitatively. Recently, we developed a method for the preparation of peptideand salt-free extracts of amino acids from biological materials by using an ion-retardation resin (2). The extracts may then be examined and the amino acids quantitatively determined by the thin-layer chromatographic technique of Heathcote and Haworth (3). In the present paper, the method has been applied to the mycelia of Heliscus submersus, Tetracladium setigerum, and Aspergillus fkavus. MATERIALS AND METHODS Chromatographic equipment. Unoplan apparatus (Shandon Scientific Co., Ltd., 65, Pound Lane, London, N.W.10., Great Britain) was used in the preparation of chromatographic plates. Shandon chromotanks were used in the development of the chromatograms. Thin-layer plates (20 by 20 cm) were prepared from washed cellulose as described by Haworth Chromogenic reagents. Ninhydrin-cadmium acetate reagent was used to detect the a-amino acids and isatin-cadmium acetate reagent, the imino acids. The reagents were prepared as previously described by Heathcote and Haworth (3). Chromatographic columns. The columns for desalting were prepared from glass tubing (1.5 by 30 cm). Indentations were made about 1 cm from one end to support a glass wool plug which in turn supported the resin. The flow rate was controlled by means of a Hoffmann screw clip attached to a length of rubber tubing at the base of the column. The growth medium used for the cultivation of mycelia from A. flavus consisted of yeast extract (30 g), glucose (100 g), and distilled water (1 liter): Cellulose. MN300 cellulose was obtained from Macherey Nagel and Co. Ltd., Agents Camlab (Glass) Ltd., Cambridge. Ion-retardation resin. The ion-retardation resin used was Bio-Rad AgliA8, 50 to 100 mesh (batch no. 5145-16 B-2198 obtained from Calbiochem Ltd., 10 Wyndham Place, London W.1.). Densitometer. The instrument used was a "Chromoscan" double-beam densitometer (Joyce Loebl & Co. Ltd., Gateshead-on-Tyre, Great Britain) with thin-layer attachment, and the reflectance mode of operation was used throughout the work. 349 Inoculation and incubation of the organisms. The organisms H. submersus and T. setigerum were grown on slopes of malt-agar and A. flavus on slopes of Czapek-Dox at 23 C for 14 days. After this time, a quantity of mycelium was removed from each culture, homogenized in sterile water (5 ml) with a Waring Blendor (10-ml attachment bottle), and then used as the inoculum. Two drops of each individual inoculum were then introduced aseptically into 24 Roux bottles containing sterile medium (50 ml), and surface growth was carried out at 23 C for 13 days in a stationary horizontal position. It had been previously established that after 13 days all the organisms were in the logarithmic phase of growth. Nevertheless, tests were carried out to confirm the presence of excess glucose (Benedict's test) and unused peptone (biuret test) in the media to ensure that growth was active and not nitrogen-limited. After this time, the separate mycelia were harvested, combined, and then washed three times with 100 ml of sterile water. They were dried rapidly by suction and weighed. The total amounts of dried mycelia were 1.35 g/liter for H. submersus, 1.14 g/liter for T. setigerum, and 2.38 g/liter for A. flavus. Extraction of the free amino acid pools from mycelia. The dried mycelium was divided into nine portions. Each portion was weighed and made to 5 ml with distilled water, homogenized by means of a Waring Blendor for 5 min, and then made to 10 ml with distilled water. Three of these portions were extracted three times by boiling under reflux for 15 min, after which they were cooled and centrifuged and the mycelia residues were set aside. Each supernatant fluid was concentrated to a volume of 2 ml under reduced pressure at 40 C, and this procedure was repeated for the remaining six portions, three being refluxed for 30 min and three for 45 min. The water extracts were then passed individually down a column of Bio-Rad AG11A8 as described below to remove interfering protein, peptide, and salt before examination of their amino acid content on thin layers of cellulose. The mycelia residues remaining after water extraction were taken up in 5 ml of butan-2-one containing 6 N HCl [5% (v/v) ], homogenized for 15 min, and then centrifuged. In each case, the supernatant fluid was preserved, and the extraction was repeated twice. The three supernatant fluids were pooled and evaporated to dryness in a current of air. The solid remaining was dissolved in distilled water (2 ml), and a fractional amount (5 gliters) was put directly on thin layers of cellulose. Desalting procedure. A column (1.5 by 12.5 cm) of Bio-Rad AG11A8 (50 to 100 mesh) ion-retardation resin was prepared from an aqueous suspension and washed with water (100 ml) to remove impurities. The mycelial extract (2 ml) was applied to the column followed by a solution of sodium chloride [1 ml of 1% (w/v)], and the amino acids were eluted from the resin with distilled water (19 ml) at a flow rate of 2 ml/min. The first 13 ml of eluate was discarded, and the next 6 ml which contained the amino acids was collected and analyzed by thinlayer chromatography. The column was regenerated by washing with distilled water (100 ml) at a fast APPL. MICROBIOL. flow rate (10 ml/min). Thin-layer chromatographic procedure. The chromatograms were developed in the first dimension until the solvent front had reached 13 cm from the origin (2.5 hr), after which time they were dried in a current of cold air for 15 min and heated at 60 C for a further 15 min. The chromatograms were then cooled and developed in the second dimension until the solvent front had reached 13 cm from the origin (2.5 hr). The layers were next heated to dryness at 60 C for 15 min, allowed to cool, and sprayed with chromogenic reagent until they appeared translucent. The colored amino acid complexes were fully developed by heating at 60 C for 15 min and al- lowing the chromatograms to stand in the dark at room temperature for 4 hr. The imino acid complexes were developed by heating at 90 C for 10 min and allowing to stand for 1 hr. RESULTS AND DISCUSSION It has been known for some time that the extraction of fungal mycelia with boiling water is the most efficient of the available procedures. The chromatographic analysis of this type of extract has proved very difficult for the determination of free amino acid pools because of interference by salts and peptides. The thinlayer chromatographic patterns obtained with boiling water extracts of H. submersus, T. setigerum, and A. flavus are shown in Fig. 1-3, and it is clear that the picture is greatly distorted. Desalting of the extracts by no means clarifies the chromatographic pattern since peptide still interferes with the resolution of several amino acids and they cannot all be estimated quantitatively. However, complete separation of all of the naturally occurring amino acids and some other ninhydrin-positive compounds is possible when the boiling water extracts are treated with Bio-Rad AG11A8 before thin-layer chromatography. The improved resolution is shown clearly in Fig. 4-6, and no difficulty was found in determining the individual amino acids quantitatively after this treatment. The results of extraction with boiling water for various periods of time are given in Table 1. For most amino acids, the aqueous extraction appears to be complete after 15 min, but further extraction of the mycelial residues with acid butanone results in small additional amounts of tryptophan. The full results for the amino acid pools of the three organisms are presented in Table 2. The pool in H. submersus (656.9 ,ug/250 mg) was found to be about half that of T. setigerum (1,187 tg/250 mg) and considerably less than that of A. flavus (1,562.2 Mg/250 mg). An interesting feature of the metabolism of H. submersus is the unusually large percentage (53.4%) of basic amino acid which is present in the pool. Arginine alone accounts for 34%. In the case of T. setigerum, alanine, glycine, and histidine account for 46% of the total amino acids. A more balanced distribution of amino acids was found in the pool from A. flavus, and a larger content of tryptophan (4.5%) was present than occurred in the other two organisms. These two facts, together with the unexpected finding of arginino-succinic acid, may be related to a more advanced metabolism in this organism.

v3-fos

2018-04-03T04:52:26.660Z

1972-01-01T00:00:00.000Z

40656889

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 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from 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--* on March 23, 2020 by guest http://aem.asm.org/ Downloaded from 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 on March 23, 2020 by guest http://aem.asm.org/ Downloaded from 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

2020-12-10T09:04:11.642Z

1972-09-01T00:00:00.000Z

237234513

s2

Identification of a Germination System Involved in the Heat Injury of Bacillus Subtilis Spores Bacillus subtilis A spores were injured by exposure to heat treatments of 110 to 132 C. Injury was demonstrated by the inability to form colonies on fortified nutrient agar (FNA) unless the medium was supplemented with CaCl2 and Na2 dipicolinate (CNA). A preliminary heat treatment fully heat-activated the spores, was not lethal, and did not prevent injury by subsequent secondary heat treatment. Exposure of heat-activated spores to 122 C reduced germination in FNA. The primary germination agents in FNA were identified, and a defined germination medium of glucose, NaCl, l-alanine, and sodium phosphate (GNAP) was developed. Germination of heat-activated spores in GNAP was equivalent to germination in FNA. Injury measured by colony formation on FNA and CNA was correlated to injury measured by reduced germination in both FNA and GNAP. Inactivation of the FNA and GNAP germination systems by secondary treatment exhibited similar kinetics. Therefore, injury expressed as the inability to form colonies on FNA involved alteration of the GNAP germination system. The detection of surviving spores is required for thermal process evaluation, and the inability to accurately predict the survivors quantitatively has both economic and public health significance. Survivors of thermal processes may be injured and unable to grow under cultural conditions that are satisfactory for unheated spores. Little information is available as to whether germination, outgrowth, or vegetative cell growth is affected in heat-induced spore injury. Levinson and Hyatt (11) reported that for Bacillus megaterium at least 94% of the spores heated at 75 to 85 C retained germinability but were unable to form colonies due to damage to the cell-division process. Less than 6% were nonviable because of the lost ability to germinate. Campbell et al. (4) reported the isolation of germination mutants of B. stearothermophilus from spores heated at 121 C. Unlike unheated spores, the injured spores and their progeny were unable to form colonies on a minimal medium unless certain amino acids were added. Colony formation was I Paper no. 3633 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Ra-leigh, N the criterion of injury and survival; therefore, the authors could not distinguish between germination, outgrowth, or vegetative cell growth as the stage affected by the apparent mutations. Cassier and Sebald (5) observed improved recovery of heated Clostridium perfringens spores when egg yolk emulsion or lysozyme was added to the plating medium. Duncan et al. (6) recently reported that lysozyme germinated the injured C. perfringens spores whose normal germination system had been inactivated by heat treatment. Edwards et al. (7,8) reported that B. subtilis A spores were injured by exposure to ultrahigh temperatures (UHT). The injured spores did not form colonies on a fortified nutrient agar unless the medium was supplemented with CaCl2 and Na2 dipicolinate. This suggested that B. subtilis A spore injury involved the germination systems. The objectives of this investigation were to identify the germination system(s) active when B. subtilis A spores were enumerated in FNA and to determine whether this germination system(s) was involved in the UHT injury. Preliminary reports of these findings have been given previously ( (7). The inoculated slants were incubated at 44 C for 24 hr and stored at 2 C. Media. The plating media were fortified nutrient agar (FNA) and FNA supplemented with 44 mM CaCl2 and 40 mm sodium dipicolinate (K & K Laboratories, Inc., Plainview, N.Y.) (CNA). The composition and preparation of the FNA and CNA were described by Edwards et al. (7). Constituents of the germination media were autoclaved separately at 121 C for 15 min and stored at room temperature. Preparation of spore suspensions. Portions (0.5 ml) of a 16-hr nutrient broth (BBL) shake culture grown at 45 C were dispensed into 100 large petri dishes (150 by 22 mm) containing 100 ml of FNA, and were spread over the entire agar surface. The plates were incubated upright for 24 hr at 44 C in a water-jacketed, natural convection incubator, inverted and incubated for an additional 24 hr, and then stored at 4 C for 18 to 24 hr. The growth was harvested with cold, sterile deionized water, and the spores were washed and purified by centrifugation according to the procedure of Edwards et al. (7). To obtain spore crops of less than 1% vegetative cells required 10 to 18 centrifugations and washings. The spore crops were stored at 2 C in deionized water. Heat treatments. The heat activation or primary treatment was 90 C for 60 min. The spores were suspended at a concentration of 107/ml in 25 mm Na phosphate buffer, pH 7, and were heated in a covered water bath. The secondary or damaging heat treatments were at 110 to 132 C. The spores were suspended in 25 mm Na phosphate buffer, pH 7, at a concentration of ca. 106/ml. When germination was measured as a reduction in optical density (OD), the suspension for both the primary and secondary treatments contained ca. 109 spores/ml. This suspension was dispensed in 0.04or 0.05-ml quantities into glass capillary tubes (Kimble 34507; 0.9 to 1.1 by 90 mm) using a syringe equipped with a repeating dispenser (Hamilton Co., Whittier, Calif.), and the capillaries were sealed in a gas flame. The capillaries were placed in a wire mesh basket and immersed in a constanttemperature oil bath (Colora Ultra-Thermostat, Germany). The heat treatment was terminated by plunging the capillaries into an ice bath. Exposure time was measured with an electric timer (Precision Scientific, Chicago, Ill., model 69230) calibrated in 0.1-sec units. The temperature change during comeup was measured with a copper-constantan thermocouple implanted in a sealed, water-filled capillary tube and was recorded on a Speedomax G recorder (Leeds & Northrup Co., Philadelphia, Pa.). The heat contribution during come-up was calculated by the method of Halvorson (10) assuming a ZD of 10 C. Six seconds were required for temperature equilibration, and the contribution during come-up was equivalent to 3 sec at bath temperature. When germination was measured by change in OD, the capillaries containing the heated spores were washed, both ends were broken off, and the contents were blown into a small test tube. The spores were diluted 1 to 10 with deionized-distilled water and used in the germination experiment. When injury was measured by colony counts or germination was measured by loss of heat resistance, two capillaries that each contained 0.05 ml of heated spore suspension were washed, rinsed in sterile deionized water, and crushed in a 99-ml phosphate buffer dilution blank (3). The samples were then dispensed in germination media or were further diluted as described in Standard Methods for the Examination of Dairy Products (3), plated in triplicate on FNA or CNA, and incubated for 18 to 24 hr at 44 C. Germination. Germination was measured as a loss of heat resistance or as a reduction in OD at 625 nm. When the germination medium contained agar, the agar was melted, cooled to 43 C, and added to the germination mixture. Germination was initiated by the addition of spores to the germination mixture after the system had been allowed to equilibrate to germination temperature. When measured as loss of heat resistance, germination was terminated by exposing the entire mixture of substrate and spores to 90 C for 15 min and then diluting the mixture immediately before colony count with CNA. RESULTS AND DISCUSSION Effects of multiple heat treatments on the apparent viability of B. subtilis A spores measured on FNA or CNA are shown in Fig. 1. Treated spores received a primary or heat-activation treatment (90 C for 60 min) prior to exposure at 121 C. Germination in FNA required heat activation and the primary treatment fully activated the spores. The number of primary treated spores enumerated on FNA and CNA was approximately equal to the direct microscopic count (data not presented). The plate counts of untreated spores on CNA were similar to those of primary treated spores plated on FNA. This indicated that the Ca dipicolinate (CaDPA) in CNA medium germi- nated the spores directly and without previous heat activation. Riemann (12) also observed the germination of B. subtilis A spores by CaDPA. The primary treatment was not lethal as shown by equivalent plate counts on CNA before and after primary treatment. The injury induced by the secondary treatment was demonstrated by the difference between the apparent number of survivors enumerated on FNA and CNA. After a secondary treatment, a spore was considered dead if it was unable to form a colony on CNA, and a viable spore unable to form a colony on FNA was considered injured. The presence of CaDPA in CNA overcame the damage of injured spores but had no apparent effect on the additional cellular damage in dead spores. This was also observed by Edwards et al. (7,8) for spores heated in skim milk. The action of CaDPA as a germination agent for B. subtilis A spores and the requirement for outgrowth and vegetative cell growth for colony formation by injured spores on CNA strongly indicated that the site of injury was the spore germination system. Heat-activated spores that received a primary treatment appeared to be more resistant to the damaging effects of the secondary treatment. Similar observations were made by Carawan (M.S. thesis, North Carolina State University, Raleigh, 1970) who reported that a sublethal heat treatment protected B. subtilis A spores against thermal inactivation by subsequent UHT treatments. However, up to 90% of the primary treated spores surviving exposure at 121 C were injured. Therefore, the primary or heat activation treatment required for germination in FNA did not interfere with a study of the influence of secondary treatments on spore germination. Data on germination in FNA and CNA by primary treated and primary plus secondary (121 C for 6 sec) treated spores are shown in Fig. 2 and 3, respectively. Germination was measured as the loss of resistance to heat treatment at 90 C which also served to melt the agar when germination was carried out at 30 or 37 C. The controls indicate germination in 2% agar. With primary treatment only (Fig. 2), germination in FNA and CNA at 43 C were similar and greater than 90% germination occurred in 2 to 4 hr. Germination in FNA increased with increasing temperatures. The extent of germination in CNA, however, was much less influenced by temperature. This indicated that germination by CaDPA was either unaffected by temperature or increased with decreasing temperature, and thus com-pensated for the reduced germination in the FNA base of the CNA medium. Riemann (12) reported that the optimum temperature for CaDPA germination of B. subtilis A spores was between 10 and 30 C, and germination at 45 C was greatly reduced because of crystallization of the CaDPA. After secondary treatment (Fig. 3), germination in FNA at 43 C was reduced by ca. 50%. A direct relationship between germination temperature and the extent of germination in FNA also existed for secondary treated spores. In CNA, however, secondary treated spores exhibited an inverse relationship between germination temperature and the extent of germination. This reflects reduced germination of the spores by CaDPA above the 30 C. The injured spores were unable to germinate at 43 C in the FNA base of the CNA medium. About 80% of the spores capable of germination in CNA after primary treatment germinated in CNA at 30 C after primary plus secondary treatment. Complete germination of injured spores for plate counts at 43 C when CNA was used to enumerate survivors (Fig. 1) apparently occurred because of the lower temperatures during and after solidification of the CNA medium in the pour-plate technique. It was apparent, therefore, that the inability of injured B. subtilis A spores to form colonies on FNA was due to the inability of these spores to germinate unless CaDPA was added to the medium. The involvement of germination in heat injury necessitated identification of the germination system(s) that responded to FNA constituents and was suppressed by secondary heat treatment. This required evaluation of the FNA constituents, alone and in combination, for germination of uninjured and injured spores (Table 1). Germination in FNA after primary treatment or primary plus secondary treatment was similar to that shown for 90 min in Fig. 2 and 3. Deletion of minerals, Gelysate, or NaCl had no effect on the germination of spores that had received only a primary treatment. Removal of beef extract slightly reduced germination, and deletion of glucose severely retarded germination. Minerals, Gelysate, and especially glucose appeared to be important for the germination of spores that had also been subjected to a secondary treatment, although only a fraction of the survivors were able to germinate in the total or complex medium. Glucose was the only individual FNA constituent that by itself stimulated germination of heat-activated (primary treated) spores. glucose increased germination, and germination in glucose, NaCl, and beef extract approached the germination observed in FNA for primary treated and primary plus secondary treated spores. Other FNA constituents in combination with Amino acid analysis (Beckman amino acid analyzer) of FNA indicated the presence of 11 amino acids at the following concentrations: arginine, 1.65 mM; lysine, 0.76 mM; alanine, 0.62 mM; leucine, 0.49 mM; glycine, 0.44 mM, methionine, 0.33 mM; phenylalanine, 0.32 mM; tyrosine, 0.24 mM; isoleucine, 0.22 mM; serine, 0.19 mM; glutamic acid, 0.02 mm. For germination of primary treated and primary plus secondary treated spores in defined media, the media constituents were at the concentrations found in FNA; glucose, 0.55 mM; NaCl, 140 mM; Na phosphate, 2 mM; amino acids as given above. In glucose and the full complement of amino acids plus NaCl and phosphate, germination approached that observed in FNA. Maximal germination in L-alanine required the presence of other FNA constituents, especially glucose and NaCl. The defined germination medium (GNAP) used in future experiments therefore contained glucose, 0.55 mM; NaCl, 140 mM; L-alanine, 0.62 mM; and 37 Na Phosphate, 2 mm. After secondary treatment the extent of germination was reduced, as was the influence of the germination medium composition. Data describing the germination of B. sub-8 Fig. 4. The primary treatment increased germination in the defined medium by about threefold as had also been observed for colony formation in FNA (Fig. 1). After a primary and secondary treatment, germination in GNAP was reduced by 55 to 60% from that observed for primary treated spores. This agreed well with the 57% reduction in germination after 90 min in FNA (Fig. 2 and 3). A comparison of the effects of secondary treatment on the germination of heat-activated spores in FNA and GNAP is shown in Table 2. After primary treatment, the spores germinated to about the same extent in FNA and in GNAP. Injury induced by each secondary treatment and measured on FNA and CNA was accompanied by similar but reduced germination in both FNA and GNAP. After a primary treatment and a secondary treatment of 122 C for 6 sec, 82% of the spores retained viability as measured on CNA. Of these survivors, however, only 41% and 38% germinated in FNA and GNAP, respectively. These values correspond to reductions in germination activity (relative to the controls) of 52% and 59% for FNA and GNAP, respectively. These values are in agreement with others obtained when germination was measured as a loss of heat resistance (Fig. 2, 3) FIG. 4. The influence of primary (P) and secondary (S) heat treatments on the germination of Bacillus subtilis A spores in the defined medium. Open circles are control spores that received no heat treatment. P, 90 C for 60 min; S, 122 C for 6 sec. bPercentage of the survivors unable to form colonies on fortified nutrient agar (FNA). c Germination measured as the percentage of the survivors losing heat resistance during 90 min at 43 C in FNA or a defined germination medium of glucose, NaCl, L-alanine, and sodium phosphate (GNAP). OD (Fig. 4). Injury measured as reduced germination was greater than injury measured by colony formation on FNA and CNA. This may have been due to slow germination in FNA via a germination system not requiring germination stimulants and activated by the secondary treatment (2). This germination of injured spores would increase the plate counts on FNA thus reducing the apparent injury, especially since injury studied here does not appear to involve outgrowth or vegetative cell growth. The reductions in FNA-and GNAP-stimulated germination activity after exposure to secondary treatment had similar kinetics (Fig. 5). This confirmed that the GNAP medium included the germination agents active in FNA and verified the existence of the classical Lalanine-glucose germination system(s) operative in these spores (9). Also, a significant amount of the injury observed on FNA can be attributed to the effects of secondary heat treatments on the GNAP germination system(s). These findings on damage of a spore germination system are consistent with other findings reported by Adams and Busta (1). These alterations of this germination system(s) can be studied using the defined medium. Insight into the mechanism by which lethal heat treatments alter spore germination characteristics would improve understanding of spore germination and death. Such information should also permit establishment of recovery conditions more suitable for the detection of injured spores.

v3-fos

2020-12-10T09:04:12.894Z

1972-12-01T00:00:00.000Z

237230443

s2

Comparative Studies on the Detoxification of Aflatoxins by Sodium Hypochlorite and Commercial Bleaches Cultures of Aspergillus flavus and aflatoxins were destroyed by a commercial bleach (Clorox; active ingredient, NaOCl) or analytical reagent grade NaOCl at 7.0 × 10-3 M NaOCl in 5 days. Addition of Clorox or NaOCl at 2.8 × 10-3 M to the fungal growth medium prior to inoculation completely inhibited the fungal growth. Aflatoxin production was inversely proportional to the logarithm of NaOCl concentration and time of treatment. Clorox and NaOCl were equally effective on aflatoxins, but fungal cells were lysed more readily by Clorox than by NaOCl. Mycelia older than 8 days lysed more readily than younger ones. Most conidia survived concentrations below 1.4 × 10-3 M. The lowest effective concentration for a 2-hr treatment was 8.8 × 10-3 M which is well below the Clorox concentration recommended for routine laboratory decontamination of aflatoxins. Mice and rats injected with aflatoxins and aflatoxins incompletely destroyed by Clorox died within 72 hr and had typical liver and kidney damage caused by aflatoxins. However, animals injected with NaOCl or Clorox or Clorox-destroyed aflatoxin extracts survived and showed no obvious liver or kidney damage. It has been well established that aflatoxins are the most potent naturally occurring mycotoxins that possess carcinogenicity to many warm-blooded animals, and they are considered potential threats to human health (1,5,18,22,26,27). The toxigenic strains of Aspergillus flavus, A. parasiticus, and A. fumigatus, etc., have been frequently detected and characterized from many food commodities and agricultural crops (1,2,5,14,15,20,21,25,26,29). Although several chemical agents have been suggested to be effective in destroying aflatoxins (9), in-depth studies about their effectiveness and mechanisms of action against these carcinogens are not yet available. To date, the most common and convenient treatment practiced in laboratories heavily engaged in aflatoxin research is the one that was recommended in 1965 by Fischbach and Campbell (11) and by Stoloff and Trager (24). In this procedure, household bleach, which contains 5 to 6% NaOCl, is applied to decontaminate surfaces of lab equipment, work areas, and personnel. In general, this treatment has proved very effective. However, the high concentration of bleach employed in this procedure often causes difficulties in toxin assays and skin injuries to personnel. Although a one-tenth dilution of bleach was recommended in the original procedure for "general dishwashing" in order to minimize skin irritation, the effectiveness of the diluted bleach in the destruction of aflatoxins as well as its direct effect upon the toxigenic fungi were not investigated. The purposes of the present study were to determine: (i) whether an aflatoxigenic strain of A. flavus was capable of producing aflatoxins after various treatments with bleach; (ii) at what levels of concentration of bleach treatment was no longer effective; and (iii) whether the destructive effect of household bleach was in any way comparable to that of the pure chemical, sodium hypochlorite (NaOCl). MATERIALS AND METHODS Culture. Cultures of A. flakus Fc3, an isolate bearing close morphological resemblance to type culture A. flavus NRRL 2999, were used throughout this study. This particular isolate was chosen because it emits, under long ultraviolet (UV) exposure at 365 nm, strong, characteristic fluroescences, i.e., bluish when grown on Czapek agar and dull green when 885 grown on yeast extract agar (YES). All cultures were maintained at 4 C on either standard Czapek or YES agar slants. Preparation of inoculum. Cultures were grown on YES containing 2% yeast extract and 5% sucrose (28) for 5 days at 26 C. This is a modification of the YES medium originally developed by Davis, Diener, and Eldridge (7). Spores from mature colonies were aseptically collected into a sterile solution of 0.02% Tween-20 and 1% NaCl. After gentle shaking, samples of spore suspensions were counted with a Coulter type B electron particle counter (23) with upper and lower threshold settings of 50 and 20, respectively, in order to accommodate the small size of the conidia of A. flavus. The trace amounts of contamination from NaCl and Tween-20 inherited from the counting solution had no effect on either fungal growth or subsequent production of aflatoxins. Liquid cultures were started by inoculating 2 ml of spore suspension containing an average of 5.6 x 104 conidia/ml into 125-ml long-neck Erlenmeyer flasks which contained 20 ml of sterile YES medium per flask. Clorox and NaOCI treatments. Cultures were treated with either commercial Clorox or pure NaOCl. These experiments can be conveniently grouped into three categories. (i) Effect of the concentration of NaOCI. Five-day-old cultures were treated for a duration of 10 min with eight different concentrations of NaOCl ranging from 3.5 x 10-2 M to 3.5 x 10-I M. (ii) Effect of length of treatment. Five-day-old and 12-day-old cultures were treated with 2.1 x 10-3 M NaOCl and Clorox, respectively, for five different time exposures ranging from 120 to 1 min. (iii) Effect of treatment commenced at various stages of fungal growth. As many as seven concentrations of NaOCl or Clorox were used to treat the cultures as follows: (i) the additives were added to the growth media before inoculation, and fungal dry weight and aflatoxins were determined 5 days after inoculation; (ii) the additives were introduced to the cultures 4 days after initiation of growth, and assays of dry weights and toxins were made at the end of an additional 4-day period of growth; and (iii) commercial Clorox was introduced into cultures at the end of 8 days of growth, and assays were made at 24 and 48 hr thereafter. Assays of aflatoxins, fungal dry weights, and biological activity. (i) Aflatoxins. The procedure used for the extraction of aflatoxins was that of Lee (17) with a minor modification to suit this study. A chloroform to fungal filtrate, volume ratio of 4 to 1 was adopted for preparation of aflatoxin extracts from the fungal cultures. Both fungal mycelia and culture filtrates were extracted together. UV spectrophotometric and thin-layer chromatographic (TLC) analyses were used for characterization of aflatoxins. Spectrophotometric analysis of UV absorption patterns and approximation of amounts of aflatoxin produced in each of the fungal extracts were made with a Cary model 15 ratio-recording spectrophotometer. In general, the molar extinction coefficient (E) of aflatoxin B, in chloroform, which is 23,015 at a wavelength of 363 nm, and a 1-cm light path were used to approximate total aflatoxin production. This method of approximation for the crude aflatoxin extracts is similar to the quantitative estimation method by applying co-chromatography with aflatoxin standards on TLC gel plates. TLC analyses of the aflatoxin extracts were performed by using a standard Desaga one-dimentional ascending technique with a solvent system of chloroform and acetone at 4 to 1 ratio on Adsorbsil-1 gel plates. The gel plates were heated at 115 C for 30 min before spotting to render a good separation of the aflatoxin components. To obtain a 15-cm solvent front, a development time of 45 min was necessary. Detection of the aflatoxin components was made by viewing through a Desaga UV lamp at 366 nm, and the chromatograms were photographed for record keeping. (ii) Fungal dry weights. After each treatment, fungal masses were harvested from the chloroformextracted cultures and dried at 95 C until constant weights were obtained. All fungal weights reported are the average of three replicates. (iii) Biological toxicity. To determine the toxicity of extracts of the treated fungal cultures in comparison with that of aflatoxin controls, mice and rats were injected intraperitoneally with typical and atypical aflatoxin extracts. After the animals were killed by the toxins, their lungs, livers, kidneys, and brains were examined for injury. RESULTS Because of the close similarity in UV absorption spectra and chemical and biological properties of the individual aflatoxin components, no attempt was made to quantify the separate components of the aflatoxin mixtures obtained from the treatments (Fig. 1). However, the aflatoxin extracts were characterized with UV spectrophotometry and conventional TLC at each step in the experiments. Effect of pure NaOCI. The results presented in Fig. 2 show that fungal mass and aflatoxin production are inversely proportional to the logarithm of NaOCl concentrations. At a NaOCl concentration of 3.5 x 10-2 M, both aflatoxin and fungal mass were completely destroyed. At lower concentrations of NaOCl, aflatoxins were reduced more effectively than fungal mass. Effect of pretreatment of the growth medium. As shown in Table 1, five days after pretreatment of the medium with either commercial Clorox or pure NaOCl at 2.8 x 10-3 M, total inhibition of both fungal growth and aflatoxin production was achieved. However, at 2.1 x 10-3 M, only commercial Clorox completely inhibited both fungal growth and aflatoxin production. Although the inhibitory effects on aflatoxin production by pretreatment with both agents were similar, there was less 0006 YANG VOL. 24,1972 to destruction by Clorox than those in the younger cultures. However, the fungal cells of the older cultures were more readily lysed than those of the younger cultures. This is particularly evident with treatments at lower concentrations of Clorox. Cultures that were many months old were generally easily lysed by either commercial Clorox or NaOCl. Effect of time of treatment. Effect of time of treatment results are presented in Fig. 3. Although slightly greater reduction of aflatoxins resulted from treatment with Clorox than from treatment with NaOCl, the patterns of decline in the production of aflatoxins were very similar. A 1-min treatment with either agent resulted in almost a 30', reduction in aflatoxins. However, complete destruction of aflatoxins was not obtained with these treatments in less than 2 hr. Again, treatment with Clorox was much more effective in destroying fungal cells than was treatment with NaOCl. Nevertheless, for a 2-hr period, only 60% of the fungal cells were lysed by Clorox. At least 4 hr were required to complete the destruction of aflatoxins, and additional time was necessary to complete the lysis of fungal cells at this concentration. It was also noted that aflatoxins extracted after a 2-hr treatment with either agent did not change appreciably in their characteristics (Fig. 5). As depicted in Fig. 4, the UV absorption curves of aflatoxins extracted from cultures treated with NaOCl or Clorox were atypical. No absorption spectra were detected with extracts from cultures treated with these agents at concentrations greater than 8.8 x 10-3 M. Curve A represents extracts from cultures treated at 8.8 x 10-3 M. It shows a complete loss of the major peak at 363 nm. However, it still shows a peak near 240 nm. Curve B represents extracts from cultures treated at 3.5 X 10-3 M or 2.1 x 10-3 Mfor 2 hr or longer. The major peak at 363 nm was drastically reduced, but a pronounced peak near 240 nm occurred. Curve C, which represents extracts from cultures treated at 1.4 x 10-3 M, shows a close similarity in its UV absorption pattern to that of the control, curve D. When chloroform extracts of the cultures treated with NaOCl or Clorox were co- Fig. 5 to illustrate this comparison. All extracts chromatographed, except group B in which complete destruction occurred, developed the four major aflatoxin components, Bl, B2, Gl, and G2. Group C, extracts from cultures treated with 8.8 x 10-3 M Clorox, produced a blue fluorescent spot that was not present in the control, group A. It developed close to the solvent front with a RF X 100 value of 72. Group D, which includes extracts from cultures that received 3.5 x 10-3 M or 2.1 x 10-3 M NaOCl for at least 2 hr, produced a small, purplish fluorescent spot with a RF x 100 value close to 5 that was not present in group A. Group E, extracts treated with low concentrations of Clorox or NaOCl (1.4 x 10-3 M or below), developed two unusual fluorescent spots, one bluish with an RF x 100 value close to 22 and the other brownish with an RF x 100 value close to 8.0. that were not present in group A. The unusual fluorescent spots detected in these experiments could well be the degradation or reaction products of normal aflatoxins with sodium hypochlorite. However, they occurred in very minute amounts, and further chemical characterization of them was not possible. Biological toxicity. Young, male, white Swiss-Webster mice injected intraperitoneally with aflatoxins extracted from cultures treated with Clorox showed typical toxic responses similar to those of controls. Injection of mice with single-dose concentrations of aflatoxin extract equivalent to 2.1 and 1.8 mg/kg of B, killed the animals in 48 and 72 hr, respectively. Lower doses permitted some of the mice to survive for somewhat longer periods of time. As observed by others (4,8,16,18,19), liver damage and hemorrhage were the principal effects. Kidney damage was also observed in this study. Lesions induced by control aflatoxins, or by aflatoxins from cultures treated with either NaOCl or Clorox below concentrations of 8.8 x 10-3 M, showed a periportal zone of necrosis which developed during a 3-day period after dosing. This was accompanied by a marked biliary (bile duct) proliferation. Also, mice were killed by injections with extracts from cultures treated with NaOCl or Clorox at concentrations greater than 8.8 x 103 M. Neither liver nor kidney damage was observed in this case. Injection with pure NaOCl or Clorox alone did not kill mice even after prolonged periods of dosing. When male, adult Sprague-Dawley rats were tested in the same manner, the results were similar to those obtained with mice. A somewhat higher dosage was required for rats. The acute toxicity of the aflatoxins on the young mice in this study was higher than that reported by other workers from toxin diet feeding experiments (16,18). This discrepancy might well be due to the difference in the age of animals and route of toxin administration. Comparative studies on the chronic toxicity of sublethal doses of toxin extracts from cultures treated with NaOCl or commercial Clorox in various ways are now in progress. DISCUSSION The results confirm that high concentrations, 5 to 6% or 0.67 to 0.81 M, of NaOCl will completely destroy aflatoxins in a very short time (11,24). Most of the fungal cells so treated were lysed within half a day at these concentrations. The results from experiments with additions of either NaOCl or Clorox into cultures at different stages of growth have clearly demonstrated that intact aflatoxins can be produced by A. flavus Fc5, especially from those cultures treated at low concentrations, i.e., 3.5 x 10-4 M and below. Prolonged culturing of the fungus will eventually abolish whatever effect NaOCl or Clorox has on the fungus and aflatoxin production. There was not a great difference in the destruction of aflatoxins by treatment with VOL. 24,1972 889 APPL. MICROBIOL. either NaOCI or Clorox. However, Clorox proved to be much more effective than NaOCl in destroying the fungus. This is particularly true with older mycelia. Could this be merely due to an overall weakening in fungal mycelia during the process of aging or, rather, is it the result of some synergetic reaction(s) brought about by chemically complexing certain fungal autolytic products with NaOCl in aged culture? Any answer must be merely conjectural at the present. Certainly, the possibility that ingredients in Clorox other than NaOCl which might contribute to greater effect on lysis of fungal cells cannot be ruled out. It is not surprising to find the resistance of aflatoxins exhibited against the low level treatment of NaOCl, because those carcinogens are chemically very stable and well known in their resistance to a host of physical and chemical treatments such as heat, UV and gamma radiation, as well as many other chemicals (6,9,10,13,28). The changes in UV absorption spectra in relation to concentrations of NaOCl and Clorox indicate the possibility of some kind of stepwise alteration in the molecular structure of the aflatoxins. The lowest effective concentration of Clorox against both fungus and aflatoxins was 3.5 x 10-3 M, which is about one-half the concentration of the one-tenth dilution of Clorox that has been recommended for routine use in decontamination. Therefore, a one to nine dilution of the Clorox should provide safe cleanup, provided it is done thoroughly. The author wishes to express his appreciation to Franklin

v3-fos

2017-10-19T14:38:09.084Z

1972-04-01T00:00:00.000Z

26133953

s2

Salmonella Testing of Pooled Pre-Enrichment Broth Cultures for Screening Multiple Food Samples A method has been described for testing multiple food samples for Salmonella without loss in sensitivity. The method pools multiple pre-enrichment broth cultures into single enrichment broths. The subsequent stages of the Salmonella analysis are not altered. The method was found applicable to several dry food materials including nonfat dry milk, dried egg albumin, cocoa, cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-enrichment broth cultures were pooled without apparent loss in the sensitivity of Salmonella detection as compared to individual sample analysis. The procedure offers a simple, yet effective, way to increase sample capacity in the Salmonella testing of foods, particularly where a large proportion of samples ordinarily is negative. It also permits small portions of pre-enrichment broth cultures to be retained for subsequent individual analysis if positive tests are found. Salmonella testing of pooled pre-enrichment broths provides increased consu- Positive test results are relatively rare among the many samples of food ingredients and products which are analyzed for Salmonella. Therefore, a test procedure that gives a single negative answer for many negative samples offers an important saving in the analytical effort required for quality control. These studies support the conclusion that Salmonella pre-enrichment broths can be pooled for analysis without loss in sensitivity to Salmonella in the individual samples. MATERIALS AND METHODS Salmonella analyses were carried out as described in the Bacteriological Analytical Manual (BAM) (2) except for the pooling of pre-enrichment broths described in the following paragraph. The procedure for Salmonella testing of pooled pre-enrichment broths is illustrated in Fig. 1. Samples are individually pre-enriched followed by transfer from multiple pre-enrichments to single seleni'e and tetrathionate broths at the enrichment stage. During pooling, a portion of each pre-enrichment is transferred to a sterile culture tube and retained at 4 C for later reference to the individual samples. If the Salmonella test of the pooled preenrichment broths is positive, individual tests on the retained samples can be made to determine which sample or samples contributed Salmonella to the pool. Pre-enrichment broths consisted of 0.5% lactose broth for all foods except nonfat dry milk, for which sterile distilled water containing 0.002% Brilliant Green was employed. Salmonella-positive test samples were prepared by blending the dry test food with one of three dry inocula. Inoculum 1 was a dry enzyme drain cleaner which had been found to contain multiple Salmonella serotypes including E,,z1o; Gz19 (S. cubana); and C, g complex. Inoculum 2 was a freeze-dried, skim milk suspension of serotypes S. anatum, S. binza, S. tennessee, S. Worthington, S. cabana, S. braenderup, and a B1,g complex. Inoculum 3 consisted of four laboratory isolates of serotypes G,z,, (S. cabana); E1,b; E1,l complex; and C1,l complex grown in a sterile aqueous suspension of cottonseed flour and freeze dried. RESULTS AND DISCUSSION Dried egg albumen, cocoa, and nonfat dry milk were inoculated with inoculum 1 at levels ranging from 6 salmonellae per 100 g to 3,000 salmonellae per g. Pre-enrichment broths of the Salmonella-inoculated samples were analyzed both individually and after pooling with 5, 9, and 14 pre-enrichments of uninoculated The most-probable-number estimate (1) of the original Salmonella inoculum (inoculum 1 containing serotypes Ezlo; G,z29; and C,g complex) was 93,000/g with 95% confidence limits ranging from 15,000 to 380,000/g. The inoculation level represents a suitable dilution of inoculum 1, into an otherwise Salmonellanegative sample, to provide the levels as shown. samples. The positive inoculated samples were detected in pooled pre-enrichment broth cultures as effectively as when they were analyzed individually ( Table 1). The lvel of inoculation affected the recovery of Salmonella equally for both individual and pooled analyses. Detection of Salmonella in cocoa was less sensitive than in the other two test materials. A possibly sim-ilar antimicrobial effect of cocoa on strains of S. gallinarum and S. typhimurium was reported by Busta and Speck (3). Similarly, Salmonella was detected in wheat flour, coconut, and cottonseed flour samples without loss in sensitivity, with both inoculum 1 and inoculum 2, when single pre-enrichments were pooled with from 9 to 24 pre-enrichments of uninoculated samples. Pooled samples versus pooled pre-enrichment broths. Silliker (4) reported a loss in sensitivity when several samples were pooled in a large container of pre-enrichment broth as compared to individual sample analysis. This is not surprising since, to be detected, the desiccated and debilitated Salmonella cells in a dried food sample must grow out in pre-enrichment broth in competition with other bacteria in the sample. If many food samples are pooled at the pre-enrichment stage, the salmonellae face competition from the flora of all the samples in the pool. If, on the other hand, the samples are individually pre-enriched, the actively growing salmonellae might be expected to compete more favorably with organisms introduced from other samples when pooled at the enrichment stage. In addition, pooling pre-enrichment broths avoids the hazards and inconvenience of handling very large flasks of culture. A study was made of Salmonella detection in pooled samples and pooled pre-enrichment cultures in the presence of added interfering organisms, i.e., non-Salmonella bacteria having colonies that resemble Salmonella on the selective agar media. The study was carried out with nonfat dry milk and egg alblmin, using inoculum 3. About 500 cells each of four interfering organisms, Pseudomonas sp., Proteus sp., a lactose-positive Citrobacter freundii, and a lactose-negative Bethesda-Ballerup were added at the pre-enrichment stage. Pooled samples were prepared by placing nine 25-g Salmonella-negative samples and one 25-g inoculated sample together in a single jar containing 2,250 ml of pre-enrichment broth. A pool of 10 samples was considered to be a practical maximum for convenient handling. Pre-enrichment pooling was carried out as previously shown in Fig. 1 except that 1 positive and 14 negatives were included in the pool. Table 2 compares pooling of samples and of pre-enrichment broth cultures for nonfat dry milk and dried egg albumin at six levels of Salmonella inoculation. The results show that a higher number of positives was recovered by pooling pre-enrichments rather than by pool- ing samples. In addition, pre-enrichment pooling . lded positives at lower levels of inocula-'on. Similar results were found in tests with -almonella-inoculated cocoa, shredded coconut, and wheat flour. The pooling of broth cultures permits small portions of each individual culture to be retained for subsequent analysis if a positive test is detected following enrichment, thus maintaining the individuality of each test sample. On the other hand, the direct pooling of food samples into pre-enrichment results in the loss of individual sample identity. Number of isolates examined. Two or more colonies are picked from each positive selective plate for biochemical and serological testing by the BAM Salmonella method (2) to increase the chances of detecting Salmonella. By following this procedure, 180 isolates could be tested if 15 samples were tested individually, whereas only 12 isolates could be tested at the same rate of picking from a pool of 15 samples. To test the importance of the reduced number of isolates, 238 isolates were picked at the rate of one colony per positive selective plate from the pooled pre-enrichment culture tests of the three food materials: dry milk, egg albumin, and cocoa. Forty-four per cent of the isolates were Salmonella. In a similar evaluation of the tests on flour and coconut, 60% of 72 isolates picked at the rate of two colonies per plate were Salmonella. The high proportion of Salmonella indicated that pooled preenrichment provided an adequate number of isolates to detect the presence of Salmonella. Whether pooled pre-enrichments are less sensitive than individual tests in the presence of interfering organisms probably is not known. For the present, we would recommend that, if numerous non-Salmonella colonies that resemble Salmonella on selective agar media are encountered in the pooled test, the retained pre-enrichment cultures should be tested individually. Alternatively, problems with interfering organisms may warrant the use of direct selective enrichments, two selective enrichments, or possibly even a reduction in the incubation time of the pre-enrichment cultures. Effect of volume of pre-enrichment broth transferred. The BAM method (2) for Salmonella detection in egg products calls for the transfer of 1 ml of pre-enrichment broth culture to 10 ml of enrichment broth. In the multiple pooling of pre-enrichment broth cultures that has been described, 1-ml quantities of cultures were transferred, generally, to a volume of enrichment broth determined by the number of samples to be pooled to maintain a fixed pre-enrichment-to-enrichment ratio. Thus, 150 ml of enrichment broth was used to receive 15 1-ml poolings, 100 ml for 10 1-ml poolings, etc. However, for routine laboratory operations, it is more desirable to dispense enrichment broth at a constant volume. Since the number of samples to be pooled may vary considerably from time to time, it is important to know whether variations in enrichment ratio would significantly affect the detection of Salmonella. Accordingly, a study was undertaken to test the effect of 1/10, 1/50, and 1/100 enrichment ratios on the recovery of Salmonella in inoculated food systems. The study included evaluation for both pooled samples and pooled pre- a Chi-square tests and tests for linear trend between the number of positive tests and the enrichment ratio indicated no significant differences at the P = 0.05 level. enrichment broth cultures. Each pooled preenrichment test was comprised of 15 25-g samples, whereas each pooled sample test consisted of 10 25-g samples. Six different tests were included for each ratio. Table 3 summarizes the results of this study. No significant differences in detection were noted among the three ratios tested as measured by the chi-square test and the test for linear trend. The tentative conclusion drawn is that the enrichment ratio over the range investigated and for the food samples tested (nonfat dry milk, dry egg albumin, and cocoa) was not critical to Salmonella detection. We have also examined the effect of 1-ml and 2-ml transfer volumes of the pre-enrichment cultures on Salmonella detection and found no significant differences in recoveries. This finding has prompted the standard use of a 1-ml transfer for routine Salmonella analyses.

v3-fos

2019-04-02T13:02:27.216Z

1972-05-01T00:00:00.000Z

90637049

s2

CARBOHYDRATE AND ACID COMPOSITION OF FINNISH BERRIES The study comprised six wild and seven cultivated berries grown in Finland, one imported berry and the rhubarb. The samples were analysed for sugars, starch, hemicel ulose polysaccharides, cellulose, crude lignin, titratable and total acidity and organic salts, crude protein, crude fat and ash. Five berry species were furthermore analysed for the amount and composition of seeds. The sugar content of the berries is some 35—55 per cent of the dry matter, hemicellulose plus cellulose is 10—20 per cent, and crude lignin 3—lo per cent. A considerable proportion of the last two groups occurs in the seeds. The total amount of plant acids varies within a range of 10—20 per cent, and 10—30 per cent of the acids is in the form of salts. Titratable acid amounts to some 70—90 per cent of the total acid content. Seed content and the composition ofseeds varies greatly. In some berries seeds account for one-quarter of the dry matter, and seeds may have a fat content that is nearly 30 per cent of the dry matter. The main components in the dry matter of berries are carbohydrates, primarily sugars, and non-volatile organic acid, i.e. plant acids. There is, however, little published information on the composition of berries. Money and Christian (1950) quated analytical data of fruits, including many berry fruits. These data include total sugars and titratable acidity. Similar information is provided in Osborn’s (1964) table. Kuusi (1969) has published certain parameters of Finnish berries though no carbohydrate values. Like the above authors, she expresses the acidity as a simple titration result, the so-called titratable acidity. Certain textbooks and tables of foodstuffs (e.g. Souci et al. 1962, Drews 1968) contain similar data on some berries. Whiting (1958) has studied the non-volatile acids of some berries, determining both the titratable acid and total acid content. The objective of the present study was to investigate the main composition of Finnish berries. The samples were analysed for sugars, starch, hemicellulose, cellulose, crude lignin, titratable and total acidity, organic salts, crude protein, crude fat and ash. Five berry species were furthermore analysed for the amount and composition of seeds. Materials and methods Materials. The samples were obtained from Helsinki vegetable market and food stores. The berries accepted for analysis were ripe, fresh and of good quality. The The main components in the dry matter of berries are carbohydrates, primarily sugars, and non-volatile organic acid, i.e. plant acids. There is, however, little published information on the composition of berries. Money and Christian (1950) quated analytical data of fruits, including many berry fruits. These data include total sugars and titratable acidity. Similar information is provided in Osborn's (1964) table. Kuusi (1969) has published certain parameters of Finnish berries though no carbohydrate values. Like the above authors, she expresses the acidity as a simple titration result, the so-called titratable acidity. Certain textbooks and tables of foodstuffs (e.g. Souci et al. 1962, Drews 1968) contain similar data on some berries. Whiting (1958) has studied the non-volatile acids of some berries, determining both the titratable acid and total acid content. The objective of the present study was to investigate the main composition of Finnish berries. The samples were analysed for sugars, starch, hemicellulose, cellulose, crude lignin, titratable and total acidity, organic salts, crude protein, crude fat and ash. Five berry species were furthermore analysed for the amount and composition of seeds. Materials and methods Materials. The samples were obtained from Helsinki vegetable market and food stores. The berries accepted for analysis were ripe, fresh and of good quality. The only exception was the cloudberry which appeared to have been picked half-ripe and had ripened in the dish. Berries of poor appearance were discarded from the samples. From three berry species, two different samples were analysed. The sample blueberry I represents the first ripe berries on the market, while blueberry II was picked three weeks later. Samples I and II of strawberry and raspberry were obtained at a few days interval. In addition to Finnish berries, also an imported berry, the Hungarian raspberry, was investigated. Besides berries, also rhubarb was analysed. Preparing of samples. All berries were »tailed» before weighing, and the gooseberries also »topped». The hard berries were rinsed in water and dried with paper. The rhubarb was peeled and chopped. The berries were crushed in a dish and the majority of the samples was freeze-dried. Other samples were dried in a vacuum oven at 40°C. No difference was noted between the two drying methods in the analysis, and the method is therefore not indicated in the tables. The dried samples were ground with an IKA analysis mill. A sieve mill is not suitable for this type of material. A seed sample was prepared from five berry species, and the proportion of seed in the dry matter was determined. Seeds were separated using a Top Drive macerater, sieves and water. Methods. The carbohydrates were determined by Salo's (1965a) method. The hemicellulose determination was, however, carried out directly on ethanol-extracted residue. No starch was traced in the berries, and the occurence of tructosan was considered so unlikely that it was not tested. Titratable acidity, total acidity and organic salts were determined by the methods of Salo and Kotilainen (1969). The only exception was that the cation determination was carried out with flame spectrophotometry on a number of the samples, and with atomic absorption spectrophotometry on the rest. The latter determination is faster since cations can be determined directly from aqueous extraction. Crude protein was determined by the Kjeldahl method. Crude fat was determined by the conventional ether extraction method. The dry matter was determined by drying at 100°C and the ash by incineration at 700°C. Results and discussion Carbohydrates. Table 1 presents the carbohydrate contents of the berries. Like all other analysis results of the present paper they are expressed as a percentage of dry matter. The results reveal, firstly, that sugars form the biggest dry matter group in berries. The amount of sugars, monosaccharides and sucrose together, usually varies within the range of 35-55 per cent. The bulk of the sugars is in the form ofmonosaccharides (glucose and fructose). The fact that cloudberry has a lower sugar content than the other berries was probably due to the sample having ripened in the dish. No starch was found in the berries, not even in their seeds. Berries have a relatively high content of hemicellulose, cellulose and lignin. A comparison of the figures of Tables 4 and 5 shows that a considerable proportion of these cellwall substances derives from the seeds. Especially xylans and mannans belong to the seeds. Seed plants usually have mannans only in traces (Salo 1965 b), whereas mannose was found to form about two-thirds of the hemicellulose sugar units in the seeds of currants and gooseberries. In the seeds of other berries mannose is replaced by xylose. The high content of uronic acid, galactose and arabinose indicates that the hemicellulose of the seedless part ofberries principally belongs to the pectin group. Crude lignin is surprisingly abundant in many berries. Rhubarb differs from berries on many points; as regards carbohydrates the most essential difference is the low sugar content. The papers mentioned in the introduction have assembled figures on total sugars, indicating the mean, maximum and minimum values calculated on fresh weight. Comparison is difficult because also the dry matter contents are mean values of several samples. The only conclusion that can be drawn is that the sugar contents of the present study are well within the variation ranges of the data quoted in the literature. No comparable figures were found in the literature for carbohydrate groups other than the sugars. Plant acids and organic salts. Table 2 titration result of water extract. Column 2 shows the total acidity, i.e. the acid result after salts had been converted into acid form by cation exchanger. The cation exchanger also changes the possible inorganic salts into corresponding acids, and column 3 gives the sum of the phosphate, chloride and nitrate corrections, mainly composed of phosphates. The corrected total acidity is shown in the table both as equivalents and calculated as citric acid. Berries, like plant materials in general, contain many organic acids. In berries citric acid is found to predominate (Whiting 1958, Kushman & Ballinger 1968, whereas malic acid predominates in rhubarb. Also oxalic acids occurs in rhubarb, but forms only 10-30 per cent of the total acid quantity. Furthermore, almost half of it is not soluble in water (Blundstone & Dickinson 1964, Allison 1966). Table 2 also shows the amounts of four water soluble cations, and the organic salt quantities calculated on the basis of the sum of cations. The percentages reveal that the majority of the acids in berries (some 70-90 per cent) is in free form. Furthermore it can be seen that this figure is in agreement with the titratable acidity obtained by simple alkaline titration. The titratable acidity suffices, therefore, for a relatively precise determination of the free acid quantity in berries. In vegetables and feeds the share of free acid in total acid is much lower and less uniform (about 10-50 per cent) (Salo & Kotilainen 1969). For this reason also their titratable acidity/total acidity ratio varies within wide limits. The acid content of most berries is of the order of 10 -20 per cent of the dry matter. Cranberry, currants, gooseberry and raspberry represent the highest acid content, cloudberry and blueberry the lowest. The only sample offoreign berries in the table, the Hungarian raspberry, differs essentially from the Finnish: its acid content is about half that of the Finnish, while it has more sugar than the Finnish raspberry. The same finding was recorded in a comparison of Finnish and imported apples (Salo & Korhonen 1972). It is difficult to compare the reports in the literature with the present results for the reason already indicated for sugars. If a comparison of titratable acidity of fresh weight is carried out, the mean values for the same berry species in Kuusi's (1969) tables are of the same magnitude as those now reported, excepting the lingonberry and cranberry for which Kuusi quotes a higher acid content. In the tables of Money and Christian (1950) and of Osborn (1964) the titratable acidity values are somewhat lower than the present values. The same is true of rhubarb (Blundstone & Dickinson 1964, Allison 1966). Whiting (1958) determined both total acids and titratable acids for some of the berry species included in the present study. His figures, on the whole, were also slightly lower than the present values; the acid content of raspberry, however, was closer to the Finnish than the Hungarian. The share of titratable acidity in total acidity was close to that recorded in the present study. General information on the composition of berries. Table 3 presents general information on the composition of berries. The differences in cell-wall carbohydrates, lignin and protein contents are partly due to differences in the seed content, as can be seen from Tables 4 and 5, and partly to the structural differences of the berry species. The analysis results suggest that the seed content is highest in cloudberry which, however, was not examined for seeds. The differences in crude fat content are due to differences both in the seed content of various berries and in the fat contents of the seeds (cf. Table 4). The fat values are inaccurate, because e.g. citric and malic acid are dissolved in ether. The last column shows the sugar/acid ratio. This figure reveals clearly the difference between the Finnish and Hungarian raspberry. It also reveals the difference between the berries and rhubarb. It can furthermore be seen that the difference in composition between different samples (I and II) of the same species are not great. The riper berries seem to contain some more sugar and less protein than the less ripe ones, whereas no difference can be seen in the acid contents. The seeds. When berries are eaten a large quantity of seeds, up to a quarter of the dry matter, is also eaten. In the human body the berry seeds escape digestion. Owing to the high seed content the physiological calorie value of berries is lower than might be expected from the chemical composition of the berries. The seed content and composition of seeds was determined for five berry samples. The dry matter content of seeds was about 60-65 per cent. Table 4 shows the seed content and their composition calculated on dry matter. A low content of sugar and acid is the characteristic common to the seeds of all five species. Otherwise the composition of seeds varies a great deal from one species to another. The currants and gooseberries are alike: the main component of the hemicellulose is mannan, in the other species it is xylan. No starch occurs in seeds; the energy reserve consists of fat which in currant and gooseberry seeds amounts to 25-30 per cent of dry matter. The seed residue obtained from currants in the berry processing industry would make valuable animal feed owing to its high fat and protein content, if drying and grinding the residue were an economic proposition. Table 5 shows the composition of the seed!eis part ofberries, calculated by subtraction. The sugar and acid contents are naturally higher compared with the whole-berry sample, while the proportion of cell-wall components and mostly also that of protein has fallen. In currants and gooseberries, the »tops» of the berries apparently raise the cell-wall contstituent percentage of the seedless part. The crude fat content is not calculated because the ether extract partly consists of substances other than lipids.

v3-fos

2018-04-03T00:50:38.892Z

1972-07-01T00:00:00.000Z

26913463

s2

Gas-volume measurement system for evaluating effectiveness of antimicrobial compounds. Yeast spoilage was followed by measuring gas volume produced inside a sealed bag of inoculated fruit. Volume of gas produced correlates with plate counts. Osmophilic fermentative yeasts, the major cause of spoilage in fruit juices and dried fruits, produce gas during the logarithmic growth phase. This phenomenon has been used in the rapid, simple, nondestructive method described below in evaluating the effectiveness of chemical antimicrobial agents for fruit products. Plate-counting the number of organisms present was too cumbersome and was not convenient for analyzing large numbers of samples. Gross examination of the samples for visible signs of spoilage was not satisfactory because it was too insensitive. Our method, though indirect, was found to correlate reasonably well with actual plate counts and to be reliable in determining onset of spoilage. The procedure used is to put the treated, inoculated fruit product (juice or solid) into a polyester bag and heat-seal it after collapsing the bag to remove as much air as possible. In some applications, the inoculated fruit is sealed in the bag and the treatment is added later. For example, a gaseous preservative is added after the bag is sealed by puncturing the bag near the corner with a syringe needle and then resealing it below the puncture. Obviously, more elaborate gas-volume measuring techniques (such as the Warburg respirometer) are available. The advantage of this method is its simplicity. An initial volume measurement is made by submerging the bag in water and measuring the displaced volume (Fig. 1). Further volume measurements are made at appropriate intervals during the period of incubation. The volume data are then plotted (Fig. 2). The logarithmic portion of the curve is determined by extrapolation to the base volume to establish 14( the onset of logarithmic growth. The length of time elapsed until the inflection point was used to gauge the amount of preserving effect of the treatment. This system greatly reduces the necessity of frequent measurements. The important element used in this technique is the bag. We employ Scotchpak heatsealable polyester film bags. These bags have a low permeability to gases and water vapor so that any volume measured is equivalent to the gas produced within the bag. Appropriate controls indicate that the gas is a result of microbial action. Commonly we used 6-by 11inch (ca. 15.2 by 27.9 cm) bags which have a maximum volume of about 1 liter and contain 100 g or 100 ml of product. For smaller samples we used smaller bags. Figure 2 shows the influence of 200 Mg and 500 Mg of potassium sorbate per ml, each added to 100 ml of reconstituted white grape juice inoculated with Saccharomyces cerevisiae, as compared with an untreated control. The potassium sorbate delays the onset of logarithmic growth and thus increases the storage life. Increasing levels of potassium sorbate provide longer preservation (Fig. 2). The viable yeast count of the grape juice in the bags is also shown (Fig. 2). The viable count correlates best with bag volume at the longer incubation times. For the 500 Mg of sorbate/ml treatment the correlation coefficient is 0.99 for seven observations, and for 200 Ag it is 0.92 for six observations. The correlation between bag volume and viable count for all the data in Figure 2 is 0.87. All these correlations are significant statistically (P = <0.01). The correlation for the four observations in the control is 0.75, which is not significant. The lag between viable count and bag volume is due, in part, to the amount of carbon dioxide required to saturate the product. With 100 ml of juice, this calculated volume is about 70 ml of carbon dioxide (1). Another factor is the lag between growth and gas production in microbial growth. The reproducibility of the measurements we have observed is also shown in Figure 2. The first volume measurements of nine bags for the sorbate-treated juice (500 gg/ml), before the volume inflection, ranged from 123 to 130 ml over a period of 73 hr. This is less than 6% variation in measurement when no control was made over pressure or temperature. At higher bag volumes, the repeatability of bag measurements is the same, so the percentage of variation is less. This technique has also been used to examine the spoilage of inoculated and rehydrated dried fruit. In some cases where spoilage is very slow and gas volume is not noticeable for a month or longer, it is possible to observe yeast growth before a definite increase in bag volume is detected. With prolonged incubation, the slow transfer of gases through the plastic bag wall may influence the volume of gas and thus would make this test inappropriate.

v3-fos

2020-12-10T09:04:12.443Z

1972-03-01T00:00:00.000Z

237235268

s2

Tenuazonic Acid, a Toxin Produced by Alternaria alternata' Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxi-genic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria. Mycelia of Alternaria are common and sometimes abundant under the pericarps of wheat (1,5), and the outer portions of weathered kernels of wheat, barley, oats, and sorghum contain relatively massive amounts of it. The fungus is also prevalent in peanut fruits during their development (4). Palyusik (8) found an isolate of Alternaria tenuis that when grown in pure culture and fed to geese was lethal to them, and Doupuik and Sobers (2) reported that 31 of 96 isolates of Alterraria longipes from tobacco when grown in pure culture and fed to chicks were lethal to them. According to Lucas (7) both of these species should now be designated Alternaria alternata (Fries) Keissler. Forgacs and Carll (3) fed Alternaria-infested grain to chicks and produced diarrhea and various other symptoms in the young chicks and hemorrhaging and gizzard lesions in older ones. The work here reported was undertaken to develop further information concerning the possible toxicity of this almost ubiquitous fungus. MATERIALS AND METHODS Isolates of Alternaria from grains, seeds, and other plant materials in which it is common were grown on a 1:1 mixture of autoclaved moist corn-rice substrate for 14 to 20 days at 23 to 25 C, after which the fungus-infested grain was dried, ground, and fed either as the sole ration or as 50% of a balanced toxicological diet (12) to 50-g weanling female white rats (Rattus norvegicus). The toxin was originally extracted from Alternaria-infested corn-rice and purified partially by thin-layer chromatography. The material to be used ing, with 5 liters of water, 2 kg of corn-rice infested by an isolate of A. alternata from peanuts. The aqueous extracts were reduced to 100 ml in a flash evaporator and then extracted three times with 100 ml of butanol saturated with water. The butanol extracts were dried under vacuum, dissolved in 10 ml of water, and loaded onto a 5 by 60 cm column of Sephadex G-10. The material was eluted with distilled water at a rate of 5 ml/min. The eluant was monitored with an LKB Uvicord column monitor, and 10-ml fractions were collected. The first ultraviolet absorbing peak was found in fractions collected between 450 and 600 ml and had the ultraviolet spectrum characteristic of the toxin. These fractions were pooled, dried under vacuum, dissolved in 5 ml of ethanol, and streaked silica gel PF 254 preparative plates. The plates were developed in chloroformmethanol (90: 10), the top 2 cm of the ultraviolet absorbing band was scraped off, and the substances present were eluted with ethanol. After being chromatographed three times in this manner, the ethanol solution of the ultraviolet absorbing material was concentrated under a stream of nitrogen and dried under vacuum to give 75 mg of a white powder. A standard solution was made by dissolving 5.0 mg of the isolated material in 1.0 ml of absolute ethanol. Ultraviolet spectra were taken in water, absolute ethanol, 0.09 N NaOH, and 0.09 N HCl at a concentration of 10 jsg/ml. For infrared spectroscopy, 0.6 mg of the substance was thoroughly ground with 50 mg of spectral-grade KBr and converted to a pellet in a micropress. The infrared spectrum was obtained on a Perkin Elmer model 257 grating infrared spectrometer. In addition, a portion of the purified material was chromatographed on silica gel PF 254 plates using the acid system toluene-ethyl acetate-formic acid (5:4: 1), to yield the free acid. The free acid was eluted with ethanol, dried under vacuum, and dissolved in chloroform to give a 10% solution. An infrared spectrum of this solution as well as a chloroform blank was taken on the same spectrometer using a 0.1-mm salt cell. MERONUCK ET AL. A 10-mg sample was dried at 40 C under vacuum for 48 hr and submitted to Clark microanalytical laboratory (Urbana, Ill.) for combustion analysis. A 10-mg sample was also submitted to emission spectroscopy. For chemical degradation studies, 20 mg of the substance was placed in a tube containing 0.5 ml of 2 N HCl. The tube was sealed and heated for 24 hr at 100 C. The tube was cooled and opened, and the hydrolysate was diluted to 2 ml with water. One milliliter of this was made to 10 N with NaOH and cooled to 0 C, and 20% I2 in KI was added in 10% excess. After 1 hr the solution was filtered to remove the iodoform and brought to pH 7 with HCl, and the excess iodine was removed with 10 N sodium thiosulfate. This solution was then extracted three times with one volume of butanol, and the butanol extracts were concentrated to 1 ml under nitrogen. One microgram of each of 22 amino acids was co-spotted with 5 pliters of the butanol solution on silica gel G (0.5 mm layer) and developed to 15 cm with butanolacetic acid-water (75:15:10). Ninhydrin in butanol was used to make the amino acids visible. One microgram of each of the amino acids which co-chromatographed with the unknown in the above system, 5 uliters of the unknown, and 5 pliters of the unknown plus 1 gg of known amino acid were spotted onto Silica Gel G plates and developed for 6 hr with methyl ethyl ketone-pyridine-water-acetic acid (70: 15:15:2) in an S chamber with the top closure removed. Thirty-four Alternaria isolates from various sources were tested for their ability to produce the toxin isolated from the peanut isolate. The isolates were grown on corn-rice substrate (1: 1) for 2 weeks at room temperature, and then dried, ground and extracted with water (5:1) overnight. The water extracts were analyzed by ultraviolet spectroscopy for the presence of the toxin. If absorption maxima at 239 and 278 nm were obtained, the toxin was reported as present. In addition, samples were extracted with diethyl ether (5:1). The ether extracts were concentrated and streaked on Silica Gel G plates (0.5 mm thick). After development in chloroform-methanol (90: 10), the plates were examined under long-wave ultraviolet light (340 nm). The bright blue fluorescent band near the solvent front was marked; the silica was scraped from the plate, and the substance was eluted with ethanol. The eluant was examined by ultraviolet spectroscopy for the presence of alternariol monomethyl ether maxima at 335 to 342 (wide band), 301, 290, 257, and 220 nm; these absorption maxima match those reported earlier by Rosett et al. (9). A sample of sorghum and a sample of soybeans, both of which were naturally colonized by Alternaria, were assayed for the toxin. A 100-g sample of each was ground in a Stein mill, 50 ml of water was added, and concentrated hydrochloric acid was used to adjust the pH to 2.0 or less. The sample was then extracted twice with 500 ml of ethyl acetate. The extracts were collected by filtration and concentrated first in a flash evaporator then under a stream APPL. MICROBIOL. of nitrogen to a volume of about 2 ml. This was then streaked onto preparative silica plates and developed with toluene-ethyl acetate-formic acid (5:4: 1). The plate was then examined under short-wave ultraviolet light (254 nm), and the materials running at the same RF as the toxin were obtained by scraping the silica from the plate and eluting the substances with ethanol. The ethanol solution was examined by ultraviolet spectroscopy for the toxin. The eluted materials were then chromatographed again in the same manner, and the spectroscopic procedures were repeated. RESULTS Fifty-seven of the 85 isolates of Alternaria grown on a corn-rice substrate and fed to rats were lethal to one or both rats within 10 days, and 54 or 64% were lethal to both rats to which they were fed ( Table 1). All of the rats that succumbed to the Alternaria-infested rations lost weight, and those that survived gained weight and consumed approximately as much feed as did the controls ( Table 2). Postmortem lesions in the rats included hemorrhaging in the gastrointestinal lumen and anorexia. The partially purified material originally found in ethanol extracts of Alternaria-infested corn-rice proved fatal to 50-g rats when administered at a dosage of 100 to 200 mg/kg. This material showed ultraviolet absorbance maxima at 238 and 279 nm in ethanol, and reacted with ethanolic ferric chloride to give a deep red color. The material isolated for use in the characterization study had the same above characteristics and consisted of 75 mg of a white powder having an extinction coefficient of 598 for a 1% ethanolic solution (at 278 nm). Ultraviolet absorption maxima of the isolated substance and tenuazonic acid described by Stickings (11) are presented in Table 3. The most pertinent features here are the shift from 238 to 220 nm in acid, indicating an enolate to enol conversion and the nearly identical absorptions of the isolated compound and tenuazonic acid. The lack of an absorption maximum at 217 nm in the spectrum of the isolated toxin when measured in ethanol can be accounted for if the isolated material was in the form of a salt. The ratios between the extinctions at 278, 238, and 220 nm are the same for both tenuazonic acid and the isolated toxin. The infrared spectrum of the isolated substance is presented in Fig. 1 KBr pellet and as the free acid in chloroform is presented in Table 4. The infrared spectrum of the toxin in either form is similar to that of tenuazonic acid. Particularly the amide absorption at 1,617 cm-', the NH absorption at about 3,045, and the absorptions in the region below 1,600 cm-I are similar in both materials. No published spectra of tenuazonic acid were available for direct comparison. The combined results of emission spectroscopy and combustion analysis are presented in Table 5. The combustion analysis data and emission spectra data are consistent with the material being a mixed K, Ca, and Mg salt of tenuazonic acid. In the chemical degradation study, only iso-leucine co-chromatographed with the unknown amino acid obtained. This result is again consistent with results with tenuazonic acid by Stickings (11). Water and ether extracts of corn-rice infested with selected isolates from the sources listed in Table 6 were analyzed for the presence of salts of tenuazonic acid and for alternariol monomethyl ether. Of 34 isolates of Alternaria tested, 23 were toxic ( Table 6). Twenty of these 23 isolates produced tenuazonic acid, and 14 produced alternariol monomethyl ether. Of the 11 nontoxic isolates, 2 produced alternariol monomethyl ether, and none produced tenuazonic acid. Three toxic isolates did not produce tenuazonic acid, and one of these produced alternariol monomethyl ether. Presumably, toxins other than tenuazonic acid were involved in the toxicity of these isolates. Tenuazonic acid was not found in the two naturally infested samples examined. DISCUSSION Alternaria sp. have been widely reported as toxigenic, but this toxicity has not previously been attributed to any particular metabolite. In these studies a toxic substance was isolated and data were obtained to indicate that it was a mixed salt of tenuazonic acid. The lethal dose of 100 to 200 mg/kg was close to the 137 to 205 mg/kg lethal dose in female rats re- ported by Smith et al. (10). In addition, a survey of isolates from various sources showed that the tenuazonic acid was produced by 20 of 23 toxic Alternaria isolates. It thus appears that tenuazonic acid and its salts are major toxic metabolites produced by isolates of Alternaria from agricultural products. The evidence for the natural occurrence of this metabolite is negative but inconclusive. In view of b Calculated from C,1H,,03N. by feeding 20-day-old, the relatively large doses required to produce ill effects, it seems unlikely that tenuazonic acid represents a serious mycotoxin problem, but this possibility should not be discounted

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2020-12-10T09:04:11.401Z

1972-09-01T00:00:00.000Z

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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 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. 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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2020-12-10T09:04:17.449Z

1972-04-01T00:00:00.000Z

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Efficiency of Salmonella Isolation from Meat-and-Bone Meal of One 300-g Sample Versus Ten 30-g Samples Twenty-five meat-and-bone meal samples were analyzed for salmonellae, comparing a single 300-g to ten 30-g samples. Seventeen were positive using the larger sample; eighteen were positive with the smaller. The 300-g sample showed a significantly higher (P < 0.01) percentage of confirmed salmonellae at 2 days of incubation than at 1 day. The ten 30-g samples did not show changes at 2 days. At 2 days, the 30-g samples showed significantly fewer confirmed salmonellae than the 300-g sample; however, there was no difference at 1 day. Of 1,417 presumptive colonies picked, 1,215 (85.7%) were lysine decarboxylase-positive and 1,152 (81.3%) were agglutinated by one of the somatic antisera. There were no significant differences in diversity or total numbers of different somatic groups between the large and small samples. The choice of sample size for microbial analyses depends on a number of factors such as size of available analytical equipment, size of original lot of material, particle size, and personal preference. It is axiomatic that no sampling scheme is perfect unless the whole lot is assayed destructively. Even then, success in finding the contaminating microorganisms depends on the efficiency of the assay procedures. Most sampling schemes are based on statistical techniques, taking into account the basic assumptions of homogeneity of contamination, random sampling methods, and a high degree of isolation efficiency. A sample size of 25 g was recommended by the Food Protection Committee of the National Academy of Sciences (4) for routine work, with multiples of 25 g being used for foods with a high degree of consumer sensitivity. The Food and Drug Administration (7) also recommended 25-g samples. Thirty grams in 100 ml of enrichment medium was recommended as a sample size for isolating salmonellae from foods and feeds by Galton et al. (3). The United States Department of Agriculture (6) also recommended the use of a 30-g sample and 100 ml of enrichment. The American Public Health Association (1) suggested a 20-g sample and 80 ml of enrichment. IEastern Marketing and Nutrition Research Division, Agricultural Research Service, U.S. Department of Agriculture. 688 The effect of compositing on the isolation efficiency for salmonellae from egg products was reported by Silliker (appendix C of reference 4). Some efficiency was lost when a composited sample was used; the suggestion was made that more work was needed on the effect of compositing on Salmonella recovery. The State/Federal Salmonella Program of the United States Department of Agriculture adopted the Salmonella Uniform Methods and Rules of the United States Animal Health Association (2). In this program, ten 30-g samples of meat-and-bone meal are collected from each rendering plant and returned to cooperating laboratories for Salmonella analyses. The samples are assayed individually. From the standpoint of labor, equipment, and time, it would be advantageous to pool the ten samples into one composite. Determining the effects of such compositing on Salmonella isolation efficiency was the purpose of the present study. MATERIALS AND METHODS Meat-and-bone meal samples. Twenty-five meat-and-bone meal samples were obtained from rendering plants in Pennsylvania, Ohio, Indiana, Wisconsin, and Iowa. One plant was sampled five times over a 2-year period and was known to harbor a considerable Salmonella population (represented by samples 13, 15, 20, 21, and 22). Sample 12 was also a known positive. The other 19 were selected without knowledge of previous history of Salmonella contamination. The samples were collected in ap-proximately 2,000-g lots in plastic bags. Most of the samples were obtained from piles of meat-and-bone meal in bins or on the floor, but sometimes samples were taken directly from the conveyor lines. Each sample, prior to subdividing, was screened through a no. 12 standard sieve to remove the larger particles of teeth, bones, etc. Subdividing technique. Approximately 650 g of the screened meat-and-bone meal was thoroughly mixed, and a 300-g sample was removed and placed into a 4,000-ml beaker. The remainder was used for ten 30-g samples in 8-oz (240 ml), wide-mouth, screw-cap jars and for ten 3-g samples which were placed in test tubes (approximately 1.9 by 15.2 cm). Cultural procedures. A 3,000-ml amount of prewarmed (37 to 40 C) selenite-cystine broth was added to the 300-g sample. A 100-ml amount of the selenite medium was used for the 30-g and 10 ml for the 3-g sample. Samples were mixed thoroughly before incubation. The covers of the screw-cap bottles were placed on loosely. The tubes with the 3-g samples were covered with a 10-ml beaker. Incubation was at 37 1 C for 24 i 1 hr. The incubated samples were mixed and streaked onto Brilliant Green agar plates. The 300-g sample was streaked onto four different plates, and each of the 30and 3-g samples was streaked onto a single plate. Preliminary confirmation. To perform a statistical evaluation, 40 presumptive colonies were picked from each sample. Five colonies were picked from each of the four Brilliant Green agar plates streaked from the enrichment medium of the 300-g sample and two colonies from each of 10 plates streaked from the 30-g enrichments. When 20 presumptive colonies were not available, the difference was made up by picking lactose-positive colonies. The lysine-iron slants were incubated for 24 i 1 hr at 37 C. Production of a straw-colored butt was considered a negative Salmonella reaction, and these tubes were discarded. A neutral or alkaline butt, with or without H2S production, was indicative of a presumptive positive Salmonella culture. Final confirmation. The lysine-iron slants showing the presumptive Salmonella reactions were used for slide agglutination studies using grouped and individual somatic antisera. The antisera used were purchased from Difco and included the somatic groups A, B, CD, C2, D, El, E2, Es, E4, F, G, H, I, poly A, poly A-1, poly B, poly C, poly D, and Arizona polydiphasic. The single factors 18, 21, 30, and 35 were also used. The reactions first were checked with the polyvalent antisera and whenever possible were further assigned to a somatic group. RESULTS Positive samples. The number of positive meat-and-bone meal samples is shown in Table 1. The 300-g sample gave 17 positives out of the total of 25, and the 30-g samples yielded 18 positives. The difference was not significant. Several samples were positive when one sample was used but not the other. Sample 4 was positive with the 300-g sample but negative with the ten 30-g samples, whereas samples 11 and 17 were positive with the 30-and negative with the 300-g sample. The remainder were either positive or negative with both sample sizes. If only the first two colonies picked from the first plates of the large samples were considered, 15 of the 17 samples were positive. An estimate of relative contamination is also shown in Table 1. Based on the numbers of positive samples obtained with the 3-and 30-g portions, sample 8 showed the highest level of contamination. Other samples with high levels of salmonellae were numbers 1, 2, 5, 6, 9, 13, and 16. Positive colonies and somatic groups. Table 2 shows the numbers of lysine-positive cultures obtained from the different samples of meat-and-bone meal and the numbers of cultures which were confirmed as salmonellae by agglutination in somatic antisera. The results for the 300-g and ten 30-g samples can be compared directly because both had the same total weight of sample. The 3-g samples were not directly compared to the other samples. The table shows that there were more lysinepositive cultures isolated from the larger sample at both days. Of the total of 19 positive Ten Ten Ten Ten Ten Ten Ten Ten 300-g 30-g 3-g 300-g 30-g 3-g 300-g 30-g 3-g 300-g 30-g 3-g samples at 1 day, the 300-g sample showed more lysine decarboxylase-positive cultures in 14, whereas only two of the ten 30-g samples had more positives. At 2 days, the larger sample had more lysine-po6itives in 12 samples compared to 4 for the smaller. The larger sample also showed more somatic group-positive cultures than the smaller. Twelve samples at 1 day gave more positive cultures with the larger sample and four with the smaller. At 2 days of incubation, the larger sample was superior 11 times compared to 3 times for the smaller. Chi square analyses of confirmation ratios. Table 3 shows the statistical analyses of the preliminary confirmation ratios (proportion of lysine decarboxylase-positive cultures to total presumptive Brilliant Green agar colonies). The 3-g samples in this case were compared to the 30-g samples, since proportions were used rather than absolute numbers per se. There was no significant difference between the single 300-and the ten 30-g samples at 1 day, but there was a highly significant difference (P < 0.01) at 2 days in favor of the larger sample. The 30-g sample also showed a significantly higher preliminary con- firmation ratio than the 3-g sample at both days. The 300-g sample showed a significantly higher confirmation ratio at 2 days than at 1. There were no differences in days of incubation for the 30or 3-g samples. The fully confirmed lysine ratios (proportion of somatic-positive cultures to lysine decarboxylase-positives) are also shown in Table 3. There was a highly significant difference (P < 0.01) at 2 days of incubation in favor of the larger sample when the 30-and 3-g samples were compared. There was a less significant (P < 0.05) difference when 300-g sample was compared to the 30-g sample at 2 days (in favor of the 30-g) but not at 1 day. None of the other comparisons differed significantly. Table 3 also shows the differences obtained in the overall confirmation ratios (proportion of somatic-positives to presumptive colonies). There were highly significant differences comparing the 300-g to the 30-g samples at 2 days (in favor of the 300-g sample), the 30versus the 3-g sample at 1 and 2 days (in favor of the 30), and 1 versus 2 days with the 300-g sample (in favor of 2 days). The other differences were not significant. The fully confirmed overall ratios were nearly identical to the preliminary confirmed ratios. Numbers of different somatic groups. The numbers of different somatic groups of the positive samples of the 300-g samples varied from none (samples 11 and 17) to seven for sample 9. The number of somatic groups in ten 30-g samples varied from none (sample 4) to six in samples 8 and 9. The total somatic groups were 39, 43, 48, and 45, respectively, for 1 day, 300-g sample; 2 days, 300-g sample; 1 day, 30-g samples; and 2 days, 30-g samples. None of these differences was statistically significant. Somatic grouping of positive cultures. There were no significant differences in the isolation rates of any of the serogroups when 1 day of incubation was compared to 2 days, although a fairly high Chi square (4.21) was obtained with the H2S-positive E, group, in favor of 1 day over 2 days with the 300-g sample. Comparison of the 300versus the 30-g samples at 1 day of incubation showed a significant (P < 0.05) increase in the number of times that Arizona was recovered from the 30g sample over the 300-g sample. Arizona was, however, recovered from only 2 of the 19 positive samples. At 2 days, there were significant increases in group B and E2 isolations from the 30-g samples and in poly A-1 from the 300-g sample. Comparison of large and small samples for isolation efficiency. A summary was made of the number of times that the large sample was equal to, worse than, or better than the smaller samples, using either one, two, three, or four of the Brilliant Green agar plates of the 300-g series for the comparison. When the criterion was the number of positive samples out of the 19 samples tested, even with only one plate, picking five colonies, the 300-g sample was worse than the 30-g sample only three times and was the same 16 times. When all four plates were compared, the larger sample was better once, worse twice, and the same 16 times. When the criterion of isolation efficiency was the number of diverse somatic groups obtained, the 300-g sample, with only one plate, was worse 10 times, the same 7 times, and better twice. This gradually improved until, with all four plates, the large sample was worse 7 times, better 5 times and equivalent 7 times. There did not appear to be any significant changes at the second day of incubation. Lactose-positive salmonellae. No systematic studies were made on the number of times lactose-positive salmonellae could be isolated from the meat-and-bone meal samples. Some indication of their importance was obtained from the green colonies of the Brilliant Green agar plates which were picked to make up the total of 20 colonies. One group C1 lactose-positive culture was obtained from the 30-g sample; three serogroups (Arizona, C1, and G) were obtained from sample 2; five C1 cultures were obtained from sample 14. No further studies were made of these organisms. DISCUSSION It is conceivable that the size of sample could influence the recovery of one of a mixed group of microflora because of differences in growth rates due to inoculum size. These differences would be magnified when selective media were employed. Silliker's study (5) indicated that 16 of 389 lots of egg products were positive for salmonellae in both the 25-g individual sample and the 400-g composited samples. Twelve lots were positive in one or more of the 25-g samples and negative in the larger sample, whereas only one was positive in the large sample and negative in the small samples. The study reported here showed one lot positive with the 300-g sample and negative with the smaller samples; two of the 30-g samples were positive when the corresponding 300g sample was negative. When the number of different somatic groups was compared at one day of incubation, the large sample was superior five times and the smaller samples were superior seven times. There was an indication that more somatic groups might be isolated when smaller samples were tested, but this could have been because a larger number of plates was examined in testing the small samples. Statistical analysis of Silliker's data indicated that the agreement of the 25-and 40-g sample sizes would depend on the level of Salmonella contamination. With a low level of salmonellae, the two methods might disagree quite often, whereas with high levels they would generally agree. Similar results were found in this study. The superiority of the larger sample over the smaller samples in the percentages of colonies which were lysine decarboxylase-positive (presumptive salmonellae) and somatic group-positive (confirmed salmonellae), as shown in Table 2, was perhaps an indication of the efficiency of a larger size sample in eliminating false-positive salmonellae. This might be of importance in some cases in which the number of samples assayed may be cumbersome; the weeding out of false-positives would save some time. However, there were indications that, at 1 day of incubation, the smaller samples recovered more types of salmonellae than the large samples. In all the comparisons of the large and small samples, the same number of colonies was picked from each. To determine whether a sample was Salmonella-positive, only one plate (five colonies) from the large sample was needed to attain about the same efficiency as the smaller samples (total of 20 colonies). For determining the greater variety of serotypes, however, all four plates of the large sample were needed to approximate the efficiency of the ten smaller samples. The pooling of samples, as represented here by the large sample, would represent a considerable saving of time and labor by the analyst, even if all 4 plates and 20 colonies were analyzed. The results indicated that somatic groups B, E,, and Arizona occurred more frequently in the smaller samples and that group poly A-1 occurred more frequently in the larger samples. It should be noted that Arizona and the poly A-1 were found in only 2 of the 19 positive samples; E2 was in 4, and B was in 7. Furthermore, when multiple Chi squares were done, there was a probability (when P < 0.05) that an average 1 out of 20 of the comparisons was significant; in reality, the high Chi square value may have been due to chance. Further studies of this nature would be needed to es-tablish the validity of these values for Chi square. One way to obviate some of these difficulties is to set the a value lower, at 0.01. Under these conditions the chance of a random significant Chi square is only 1 in 100. Under these conditions, the only comparison among the above results that had a significant Chi square was group poly A-1; since this somatic group occurred in only 2 of the 19 positive samples, the validity even of this comparison may be questioned unless further studies on more samples are done. The presence of apparently lactose-positive salmonellae in meat-and-bone meal deserves further study. The green or greenish-yellow colonies on the Brilliant Green plates are routinely disregarded; however, these studies indicate that they may need to be more carefully considered. These data indicate that it is feasible to pool samples into one large sample. This will result in a considerable saving of labor and time with little loss of efficiency for determining the presence of salmonellae in a sample if two suspect colonies from the Brilliant Green agar plates are picked. To isolate the predominant serotypes from the meat-and-bone meal, more than 2 colonies should be picked, preferably 20 from 4 different plates.

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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. Images 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. 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.

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2020-12-10T09:04:12.766Z

1972-04-01T00:00:00.000Z

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Salmonella Testing of Pooled Pre-Enrichment Broth Cultures for Screening Multiple Food Samples A method has been described for testing multiple food samples for Salmonella without loss in sensitivity. The method pools multiple pre-enrichment broth cultures into single enrichment broths. The subsequent stages of the Salmonella analysis are not altered. The method was found applicable to several dry food materials including nonfat dry milk, dried egg albumin, cocoa, cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-enrichment broth cultures were pooled without apparent loss in the sensitivity of Salmonella detection as compared to individual sample analysis. The procedure offers a simple, yet effective, way to increase sample capacity in the Salmonella testing of foods, particularly where a large proportion of samples ordinarily is negative. It also permits small portions of pre-enrichment broth cultures to be retained for subsequent individual analysis if positive tests are found. Salmonella testing of pooled pre-enrichment broths provides increased consumer protection for a given amount of analytical effort as compared to individual sample analysis. Positive test results are relatively rare among the many samples of food ingredients and products which are analyzed for Salmonella. Therefore, a test procedure that gives a single negative answer for many negative samples offers an important saving in the analytical effort required for quality control. These studies support the conclusion that Salmonella pre-enrichment broths can be pooled for analysis without loss in sensitivity to Salmonella in the individual samples. MATERIALS AND METHODS Salmonella analyses were carried out as described in the Bacteriological Analytical Manual (BAM) (2) except for the pooling of pre-enrichment broths described in the following paragraph. The procedure for Salmonella testing of pooled pre-enrichment broths is illustrated in Fig. 1. Samples are individually pre-enriched followed by transfer from multiple pre-enrichments to single seleni'e and tetrathionate broths at the enrichment stage. During pooling, a portion of each pre-enrichment is transferred to a sterile culture tube and retained at 4 C for later reference to the individual samples. If the Salmonella test of the pooled preenrichment broths is positive, individual tests on the retained samples can be made to determine which sample or samples contributed Salmonella to the pool. Pre-enrichment broths consisted of 0.5% lactose broth for all foods except nonfat dry milk, for which sterile distilled water containing 0.002% Brilliant Green was employed. Salmonella-positive test samples were prepared by blending the dry test food with one of three dry inocula. Inoculum 1 was a dry enzyme drain cleaner which had been found to contain multiple Salmonella serotypes including E,,z1o; Gz19 (S. cubana); and C, g complex. Inoculum 2 was a freeze-dried, skim milk suspension of serotypes S. anatum, S. binza, S. tennessee, S. Worthington, S. cabana, S. braenderup, and a B1,g complex. Inoculum 3 consisted of four laboratory isolates of serotypes G,z,, (S. cabana); E1,b; E1,l complex; and C1,l complex grown in a sterile aqueous suspension of cottonseed flour and freeze dried. RESULTS AND DISCUSSION Dried egg albumen, cocoa, and nonfat dry milk were inoculated with inoculum 1 at levels ranging from 6 salmonellae per 100 g to 3,000 salmonellae per g. Pre-enrichment broths of the Salmonella-inoculated samples were analyzed both individually and after pooling with 5, 9, and 14 pre-enrichments of uninoculated The most-probable-number estimate (1) of the original Salmonella inoculum (inoculum 1 containing serotypes Ezlo; G,z29; and C,g complex) was 93,000/g with 95% confidence limits ranging from 15,000 to 380,000/g. The inoculation level represents a suitable dilution of inoculum 1, into an otherwise Salmonellanegative sample, to provide the levels as shown. samples. The positive inoculated samples were detected in pooled pre-enrichment broth cultures as effectively as when they were analyzed individually ( Table 1). The lvel of inoculation affected the recovery of Salmonella equally for both individual and pooled analyses. Detection of Salmonella in cocoa was less sensitive than in the other two test materials. A possibly sim-ilar antimicrobial effect of cocoa on strains of S. gallinarum and S. typhimurium was reported by Busta and Speck (3). Similarly, Salmonella was detected in wheat flour, coconut, and cottonseed flour samples without loss in sensitivity, with both inoculum 1 and inoculum 2, when single pre-enrichments were pooled with from 9 to 24 pre-enrichments of uninoculated samples. Pooled samples versus pooled pre-enrichment broths. Silliker (4) reported a loss in sensitivity when several samples were pooled in a large container of pre-enrichment broth as compared to individual sample analysis. This is not surprising since, to be detected, the desiccated and debilitated Salmonella cells in a dried food sample must grow out in pre-enrichment broth in competition with other bacteria in the sample. If many food samples are pooled at the pre-enrichment stage, the salmonellae face competition from the flora of all the samples in the pool. If, on the other hand, the samples are individually pre-enriched, the actively growing salmonellae might be expected to compete more favorably with organisms introduced from other samples when pooled at the enrichment stage. In addition, pooling pre-enrichment broths avoids the hazards and inconvenience of handling very large flasks of culture. A study was made of Salmonella detection in pooled samples and pooled pre-enrichment cultures in the presence of added interfering organisms, i.e., non-Salmonella bacteria having colonies that resemble Salmonella on the selective agar media. The study was carried out with nonfat dry milk and egg alblmin, using inoculum 3. About 500 cells each of four interfering organisms, Pseudomonas sp., Proteus sp., a lactose-positive Citrobacter freundii, and a lactose-negative Bethesda-Ballerup were added at the pre-enrichment stage. Pooled samples were prepared by placing nine 25-g Salmonella-negative samples and one 25-g inoculated sample together in a single jar containing 2,250 ml of pre-enrichment broth. A pool of 10 samples was considered to be a practical maximum for convenient handling. Pre-enrichment pooling was carried out as previously shown in Fig. 1 except that 1 positive and 14 negatives were included in the pool. Table 2 compares pooling of samples and of pre-enrichment broth cultures for nonfat dry milk and dried egg albumin at six levels of Salmonella inoculation. The results show that a higher number of positives was recovered by pooling pre-enrichments rather than by pool- ing samples. In addition, pre-enrichment pooling . lded positives at lower levels of inocula-'on. Similar results were found in tests with -almonella-inoculated cocoa, shredded coconut, and wheat flour. The pooling of broth cultures permits small portions of each individual culture to be retained for subsequent analysis if a positive test is detected following enrichment, thus maintaining the individuality of each test sample. On the other hand, the direct pooling of food samples into pre-enrichment results in the loss of individual sample identity. Number of isolates examined. Two or more colonies are picked from each positive selective plate for biochemical and serological testing by the BAM Salmonella method (2) to increase the chances of detecting Salmonella. By following this procedure, 180 isolates could be tested if 15 samples were tested individually, whereas only 12 isolates could be tested at the same rate of picking from a pool of 15 samples. To test the importance of the reduced number of isolates, 238 isolates were picked at the rate of one colony per positive selective plate from the pooled pre-enrichment culture tests of the three food materials: dry milk, egg albumin, and cocoa. Forty-four per cent of the isolates were Salmonella. In a similar evaluation of the tests on flour and coconut, 60% of 72 isolates picked at the rate of two colonies per plate were Salmonella. The high proportion of Salmonella indicated that pooled preenrichment provided an adequate number of isolates to detect the presence of Salmonella. Whether pooled pre-enrichments are less sensitive than individual tests in the presence of interfering organisms probably is not known. For the present, we would recommend that, if numerous non-Salmonella colonies that resemble Salmonella on selective agar media are encountered in the pooled test, the retained pre-enrichment cultures should be tested individually. Alternatively, problems with interfering organisms may warrant the use of direct selective enrichments, two selective enrichments, or possibly even a reduction in the incubation time of the pre-enrichment cultures. Effect of volume of pre-enrichment broth transferred. The BAM method (2) for Salmonella detection in egg products calls for the transfer of 1 ml of pre-enrichment broth culture to 10 ml of enrichment broth. In the multiple pooling of pre-enrichment broth cultures that has been described, 1-ml quantities of cultures were transferred, generally, to a volume of enrichment broth determined by the number of samples to be pooled to maintain a fixed pre-enrichment-to-enrichment ratio. Thus, 150 ml of enrichment broth was used to receive 15 1-ml poolings, 100 ml for 10 1-ml poolings, etc. However, for routine laboratory operations, it is more desirable to dispense enrichment broth at a constant volume. Since the number of samples to be pooled may vary considerably from time to time, it is important to know whether variations in enrichment ratio would significantly affect the detection of Salmonella. Accordingly, a study was undertaken to test the effect of 1/10, 1/50, and 1/100 enrichment ratios on the recovery of Salmonella in inoculated food systems. The study included evaluation for both pooled samples and pooled pre- a Chi-square tests and tests for linear trend between the number of positive tests and the enrichment ratio indicated no significant differences at the P = 0.05 level. enrichment broth cultures. Each pooled preenrichment test was comprised of 15 25-g samples, whereas each pooled sample test consisted of 10 25-g samples. Six different tests were included for each ratio. Table 3 summarizes the results of this study. No significant differences in detection were noted among the three ratios tested as measured by the chi-square test and the test for linear trend. The tentative conclusion drawn is that the enrichment ratio over the range investigated and for the food samples tested (nonfat dry milk, dry egg albumin, and cocoa) was not critical to Salmonella detection. We have also examined the effect of 1-ml and 2-ml transfer volumes of the pre-enrichment cultures on Salmonella detection and found no significant differences in recoveries. This finding has prompted the standard use of a 1-ml transfer for routine Salmonella analyses.

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2020-06-04T09:07:45.671Z

1972-09-01T00:00:00.000Z

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Variation in Density of Breeding Ducks Across the Aspen Parklands and Grasslands of Canada Godfrey, W. E. 1966. The birds of Canada. Nat. Mus. Bull. No. 203 Biol. Ser. No. 73, 428 pp. March, G. L. 1971. The biology of the Band¬ tailed Pigeon (Calumba fasciata) in British Columbia. Ph.D. Thesis, Simon Fraser Uni¬ versity. Peterson, R. T. 1961. A field guide to western birds. Houghton Mifflin. Co., Boston. Spalding, D. A. E., and M. J. Hampson, 1969. First Alberta record of the Band-tailed Pigeon. Can. Field-Nat. 83:282-284. Wilson, M. 1968. New sighting of Band-tailed Pigeon in Alberta. Blue Jay 26:181. VARIATION IN DENSITY OF BREEDING DUCKS ACROSS THE ASPEN PARKLANDS AND GRASSLANDS OF CANADA by Kees Vermeer, Canadian Wildlife Service, Edmonton, Alberta The aspen parklands and grasslands of the Prairie Provinces of Canada contain potholes, marshes and lakes and constitute the major breeding ground for most game duck species in North America. Excluding scoters, eiders, mergansers and oldsquaws, Crissey (1969) estimated that during 1955-64 an average of 47 percent of the North American duck population and 61 percent of the Canadian duck population utilized this region for breeding. This region has been divided into 10 survey strata (Fig. 1). Table 1 shows the biotic zone and area of each stra¬ tum. Stratum 25 is an exception to the other nine strata in that it is in the boreal forest. However, much of the boreal forest has been cleared in this stratum, making it similar for waterfowl observing to those encountered in adjacent parkland. For this reason it has been included here. The purpose of this study was to determine for each species the breed¬ ing pair density in each stratum and the preference for a biotic zone. Methods The annual breeding pair surveys have been conducted and published by the U.S. Bureau of Sport Fisheries and Wildlife. Detailed description of the methods used in the waterfowl breed¬ ing pair survey is given in the United States Fish and Wildlife Service manual: Standard Procedures for Population index = Aerial count of breeding ducks X Stratum area Surveyed area Visibility rate -Aerial count ground count of air: ground comparison transect Visibility rates are used to adjust breeding population indices as there are discrepancies between aerial and ground counts. Air:ground comparison transect survey techniques were not perfected until 1961. As no visibility rates were available for 1960, aver¬ ages of 1961-1969 rates were used for that year. To obtain figures on average breed¬ ing pairs per square mile for the period 1960-69: 1) breeding popula¬ tion indices were divided by visibility September, 1972 rates to obtain adjusted breeding popu¬ lations for each species by stratum and year; 2) the adjusted breeding populations were divided by the area 156 of each stratum to obtain numbers of breeders per square mile; 3) the re¬ sulting figures were divided by two to obtain breeding pairs per square mile; 4) the breeding pairs per square mile were averaged for the 10-year period. Breeding pair densities of ducks un¬ adjusted for visibility rates in the aspen parklands and grasslands were compared with those of the boreal forest north of that region. Results and Discussion Average breeding pair densities dur¬ ing 1960-69 for 10 species of ducks are shown ( Table 2) for each of the 10 strata. The densities for seven dab¬ bling duck species are illustrated in Fig. 2 and for three diving duck species in Fig. 3. Mallards, with the highest density over the entire region, averaged eight breeding pairs per square mile (Table 2). Pintails and Blue-winged Teal had the second highest density. For the 10 species the average density was about 30 breeding pairs per square mile. Stratum 27, in the parklands of Al¬ berta, had the highest density for the Mallard, Gadwall, American Widgeon, Shoveler, Green-winged Teal, and Lesser Scaup. Pintail densities were highest in Strata 26 and 28 in the Alberta grasslands. Blue-winged Teal densities were highest fn Strata 20 and 24, which may be related to the fact that those strata had the highest aver, age number of ponds in May of 1960-1969 of all strata. There was a highly significant correlation between Blue¬ winged Teal and May pond densities (r = 0.818). No positive significant cor¬ relation was found for the other species. The obtained breeding pair density for a species within a stratum probably depends on the interaction of many factors within that stratum, one of which is May pond density. May pond density may be the most im¬ portant factor influencing Blue-winged Teal densities but not those of other ducks. Densities of Mallard, Redhead, Canvasback and Lesser Scaup in dif¬ ferent strata in relation to the vegeta¬ tion of strata suggests some prefer¬ ence for aspen parkland. Unadjusted breeding pair densities for the 10 survey strata in the aspen parklands and grasslands are com¬ pared in Table 3 with those of survey strata in the boreal forest of northern Alberta, northern Saskatchewan, northern Manitoba, northeastern British Columbia and the Northwest Territories. As visibility correction factors for areas north of the aspen parklands and grasslands are based upon small samples and are of ques^ tionable accuracy, the densities can only be used on a comparative basis for the parkland-grassland region and the boreal forest to its north,. It can be seen that only the Lesser Scaup has a higher density in the boreal forest. That Lesser Scaup are the most numer¬ ous breeding duck in the boreal forest is also supported by observations in Alberta by Vermeer (1970), in Saskat¬ chewan by Townsend (1966) and in the Northwest Territories by Murdy et al. (1970). A small fraction of the Lesser Scaup density in the boreal forest is probably constituted by Greater Scaup (Aythya marila). Murdy et al. (1970) found that Greater Scaup in the Yellowknife area ap¬ peared to be chiefly confined to islands in Great Slave Lake. Vermeer et al. (in press) also found that Greater Scaup was the most common breeder on an island in northern Lake Winni¬ peg, which is 300 miles south of the breeding boundary for this species as indicated by Godfrey (1966).

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1972-04-01T00:00:00.000Z

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Steam Versus Hot-Water Scalding in Reducing Bacterial Loads on the Skin of Commercially Processed Poultry A comparison of two types of scalders was conducted to determine their effectiveness in reducing bacterial contamination of poultry carcasses. A conventional hot-water scalder and a prototype model of a steam scalder were tested under commercial conditions. Total plate counts from steam-scalded birds were significantly lower than the counts of water-scalded birds immediately after scalding and again after picking. No differences in the two methods could be found after chilling. Coliform counts from steam-scalded birds were significantly lower than the counts from water-scalded birds immediately after scalding. No significant differences in coliform counts were detected when the two scald methods were compared after defeathering and chilling. A comparison of two types of scalders was conducted to determine their effectiveness in reducing bacterial contamination of poultry carcasses. A conventional hot-water scalder and a prototype model of a steam scalder were tested under commercial conditions. Total plate counts from steam-scalded birds were significantly lower than the counts of water-scalded birds immediately after scalding and again after picking. No differences in the two methods could be found after chilling. Coliform counts from steam-scalded birds were significantly lower than the counts from water-scalded birds immediately after scalding. No significant differences in coliform counts were detected when the two scald methods were compared after defeathering and chilling. Scald water can be a source of bacterial contamination of broiler carcasses. Scald water, relatively free of microorganisms at the beginning of operation, increased to 1 million/ml at the conclusion of the day's operation (8). In other studies, the total bacterial counts of the scald water have been found to range from as low as 5,900 to 17,000 organisms/ml (9) to as high as 292 million/ml (4). The low numbers of bacteria obtained from scald waters in some studies could be attributed to a relatively high scald-water temperature (58 to 60 C), the low counts on some live birds, and the dilution effect of adding fresh scald water (2). Increasing the scald-water temperature resulted in an increased shelf-life of refrigerated poultry (10). MATERIALS AND METHODS The broilers used in this study were taken from the processing lines at a commercial broiler plant. One-half of the birds were scalded by using a conventional hot-water scalder at 55 C for 120 sec, and the other half were scalded with a steam-hot-water spray using a prototype of a steam scalder at a temperature of 53 to 55 C for 131 sec. Each of these scalding units was operated on separate defeathering and eviscerating lines. Swab samples to determine bacterial contamination were taken from the thigh area of the birds at three different positions along the processing line for each type of scalder. These were: position 1, immediately after scalding; position 2, after picking; and position 3, after passing through the chilling system. Templates for taking samples were prepared from glass tubing with an inside diameter of 49 mm and ground to a sharp edge to give a clearly defined area of 1.88 cm2 for each sample. Samples were taken from the thigh by swabbing with a sterile, commercially prepared, cotton-tipped swab which was rubbed over the area covered by the template, using parallel strokes and repeating at right angles (5). To reduce contamination, the templates were immersed in 70% ethanol for a minimum of 2 min between samples and air-dried before reuse. The swab samples were placed in test tubes containing 4 ml of 0.5% peptone broth. The tubes were stored immediately in an ice bath until plated (within 5 hr). Replicate samples from five birds each were taken at 7: 00, 9:00, and 11:00 AM and at 2:00 and 4:00 PM to test for the possibility of buildup of bacteria during the day. The samples were plated in duplicate on standard plate count (SPC) agar (Difco) for total bacteria count and on violet red bile (VRB) agar (Fisher) for detection of coliforms. SPC agar plates were incubated for 48 hr at 32 C, and VRB agar plates were incubated at 37 C for 20 to 24 hr according to Standard Methods for the Examination of Dairy Products (1). Counts were expressed as number of bacteria per square centimeter of surface area swabbed. Samples were taken on 4 different days during 2 weeks of sampling. Data were analyzed by analysis of variance for each method (7). Differences between treatment means were separated by Duncan's multiple range tests (3). RESULTS AND DISCUSSION The log counts obtained during the 4 days of sampling were averaged at each of the five time intervals throughout each day. The av-796 erage log counts of the water-scalded birds were higher than those of the steam-scalded birds at each sampling time at position 1 (Fig. 1). Bacterial counts on the carcasses scalded by each method increased at 4:00 PM over previous levels. The coliform counts remained relatively constant throughout the day at the three sampling positions, although some fluctuations in counts were noted. Both the scalding method and the time of sampling influenced the average counts for total bacteria present (P < 0.01). At position 2, after picking, the bacterial loads on birds scalded in hot water were larger than the loads from steam-scalded birds (Fig. 2). This trend continued throughout the day at each sampling time. Method of scalding influenced the total bacterial load on the broilers after picking (P < 0.01). Sampling time did not have an effect on bacterial load. The average log counts of total bacteria were similar for the two methods of scalding during the sampling intervals at position 3 (Fig. 3). Differences in bacterial counts between the two scald methods would not be expected to be apparent at this position. The bacterial loads on the birds had been washed off, diluted, and redistributed during the defeathering operation as well as being subjected to chlorination during the chilling operation. The total bacterial counts at position 3 may not be an indication of the shelf-life of the birds. It has been suggested that seeding of . Log averages of total and coliform bacterial counts per square centimeter from swab surface samples immediately after chilling using a continuous chilling system. carcasses with psychrophiles occurs in slush ice chillers although total bacterial count decreases (6). Moisture pick-up by the carcasses may provide a more favorable environment for psychrophilic growth. Under the present processing operations of wet-chilling, steamscalding would not be advantageous in decreasing bacterial loads. However, dry-chilling techniques are being introduced in some processing operations. In these operations, the

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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. 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- STORAGE OF P. CHRI dium loss of streptomycin yield was avoided during serial subculturing (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.

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Cultivation of Leptospires: Fatty Acid Requirements Both the parasitic and the saprophytic leptospires grow well on a pair of fatty acids (one saturated, the other unsaturated) if they contain at least 15 carbon atoms. The fatty acid requirements of serotypes of Leptospira interrogans were previously investigated utilizing a medium which contained "fatty acid-poor" fraction V bovine albumin (Pentex, Inc., Kankakee, Ill.) (3). We recently became aware that, although this albumin contained only trace amounts of free fatty acids, it was contaminated with 2.25 mg of lipid per g of albumin. These lipids markedly affected the lipid composition of leptospires (4). Accordingly, the fatty acid requirements were reinvestigated using a "lipid-poor" albumin which contained <50 jig of lipid per g of albumin (4). The medium used in this study is the same as that previously described except for the albumin component (3). Leptospires used in this investigation were from cultures in the logarithmic or early stationary phase of growth. Unless stated otherwise, a 1% (v/v) inoculum which yielded approximately 3 x 106 cells per ml was used. The results presented in this report were obtained from the third transfer in the test medium. In the absence of added fatty acids, none of the leptospires could be subcultured in the medium. Growth was measured daily with a Coleman (model 7) photonephelometer calibrated with an arbitrary standard. The relationship between nephelometer reading and number of organisms was verified by periodic counts with a Petroff-Hausser counting chamber. All incubations were conducted at 30 C for 5 to 9 days. In the designation of fatty acids, when two numbers are used the first indicates fatty acid chain length, and the second indicates the number of double bonds. When three numbers are used, the first indicates position of double bond, the second, the fatty acid chain length, and the third, the number of double bonds. Both parasitic (eight serotypes) and saprophytic (eight serotypes) leptospires required fatty acids containing at least 15 carbon atoms. These results are in contrast to our earlier finding which indicated that the saprophytes could grow on fatty acids containing less than 15 carbon atoms (3), whereas the parasites required the longer chain fatty acids. The basis for this discrepancy was found to be the ability of the saprophytes to utilize shortchain fatty acids when very low levels of longchain fatty acids were present. The saprophyte patoc grew on 4 x 10-4 M 12:0 (lauric acid) if as little as 0.5 x 10-5 to 10-5 M 16:0 (palmitic acid) was provided. The concentration of longchain fatty acids associated with contaminating lipid of the fatty acid-poor albumin was calculated to be 10- Other studies carried out in the lipid-poor albumin medium further elucidated fatty acid requirements of the leptospires. The unsaturated fatty acid cis-9-18: 1 (oleic acid) was generally a poor substrate, especially for the parasites (Table 1), whereas the combination of cis-9-18: 1 and 16: 0 or 16: 0 alone were good substrates. The saturated fatty acid 18:0 (stearic acid) supported good growth of the saprophytes, but only a few of the parasites grew on this acid ( Table 1). The two parasites ballum and hardjo required a combination of a saturated and a cis-unsaturated fatty acid (16: 0 + cis-9-18: 1) for growth ( Table 1). The trans form of 9-18:1 (4 x 10-4 M) was found to substitute for the above pair of acids with an equivalent level of growth resulting (40 x 107 leptospires/ml). Similar results with trans-9-18:1 have been reported for the Kazan and Reiter strains of Treponema pallidum (1) and the goat mycoplasma strain -y (5), microorganisms which also require a pair of fatty acids for growth. Fatty acids (2) and fatty alcohols (T. Auran and R. C. Johnson, Bacteriol. Proc., p. 27, 1968) are the only two readily utilizable major carbon and energy sources known for the leptospires. Since the fatty alcohols were previously tested in a medium which was not lipid-poor, they were reinvestigated using the lipid-poor albumin medium. The results obtained with palmityl alcohol and oleyl alcohol were similar to those observed with the corresponding fatty acids (Table 1). In addition, a lipid analysis was conducted on patoc cells cultivated on palmityl alcohol. The fatty acid composition of the phosphatidyl ethanolamine of these cells was found to be the same as that of cells cultivated on palmitic acid (4), indicating that the fatty alcohols and fatty acids are metabolized in a similar manner. This work was supported by Public Health Service grant AI-06589 from the National Institute of Allergy and Infectious Diseases. We thank Brian Livermore for conducting the lipid analyses.

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Effect of Low-Roughage Diets on the Microflora and Lipid Metabolism in the Rumen Changing the diet of five lactating cows and one nonlactating cow from high to low roughage induced milk fat depression in the lactating cows and altered the composition of the rumen microflora. While the numbers of lactic and propionic acid-producing bacteria increased, the numbers of Butyrivibrio spp. decreased. The numbers of lipolytic bacteria and the in vitro lipolytic activity of the rumen fluid were also decreased, as was the extent of hydrogenation of linoleic and linolenic acids combined in soybean oil incubated in vitro with rumen fluid. It is suggested that among the bacterial population in the rumen the vibrios, which were adversely affected by the low-roughage diets, may contribute significantly to both lipolysis and hydrogenation in the rumen. Although most diets of ruminants normally contain high proportions of polyunsaturated acids as phospholipids and galactoglycerides, only small amounts of these acids are directly transferred to adipose tissue or to milk since most of them are metabolized to more saturated derivatives by the rumen microflora (31) before being absorbed from the small intestine. Hydrolysis of the complex dietary lipids is necessary before isomerization and hydrogenation of the fatty acids can take place (15). When cows are fed low-roughage diets, particularly those which depress milk fat secretion, the changes in the fatty acid composition of lipids of milk (25), blood plasma (9,21,27), digesta (29,33), and adipose tissue (30) suggest that hydrogenation in the rumen of dietary unsaturated acids may be reduced. Reductions in the numbers of ciliate protozoa have been associated with reduced hydrogenation (6), and lower numbers of these organisms and of the bacterium Butyrivibrio fibrisolvens, which also actively hydrogenates (23), are known to occur with lowroughage rations (4,18,24). In this investigation the effect of low-roughage diets, including milk fat-depressing diets, on the composition of the microflora and the metabolism of feed lipids in the rumen have been examined more fully. The effect of these diets on the rumen fermentation are reported elsewhere (Latham, Sutton, and Sharpe, in press). MATERIALS AND METHODS Animals and their management. Rumen contents from four lactating Friesian cows with perma-nent rumen fistulas were used for in vitro studies on hydrogenation. Their diet was progressively changed from a control of high roughage (8 kg of hay and 10 kg of concentrate cubes, 20% dry weight as crude protein, daily) to a milk fat-depressing low-roughage diet in which flaked maize was substituted for part of the hay and concentrates until the milk fat content was depressed to less than 2%. The final proportions (in kilograms) of hay, concentrates, and flaked maize varied for each cow and were respectively 1, 1, 9 (cow G10); 1, 6, 4 (cow G9); 1, 6, 4 (cow B20) and 1, 1, 5 (cow G27). Rumen contents from two more fistulated Friesian cows were used for in vitro studies on lipolysis. One (M21) in mid-lactation was treated similarly to the first four cows and received a final milk fat-depressing diet of 1, 2, 8 kg of hay, concentrates, and flaked maize, respectively, per day. The other cow (J), which was nonlactating, received an all-hay diet for 6 weeks and then a low-roughage diet of 20% hay and 80% flaked maize. Sampling. Milk yields were recorded, and daily composite samples of the milk were analyzed for total fat content by the Gerber method (2) and for fatty acid composition by gas-liquid chromatography (26) with either a Perkin Elmer 880 or 900 model chromatograph. For characterizing the microflora, single samples of well mixed rumen digesta were taken from each of the lactating cows when given the high-and the low-roughage milk fat-depressing diets. Characterization of the microflora. The Hungate (16) method of strict anaerobic culture as modified by Latham and Sharpe (17) was used throughout. All media for the enumeration of viable and lipolytic (tributyrin-hydrolyzing) bacteria and for the identification of isolates were based on medium 10 (4) as described by Latham, Sharpe and Sutton (18). Counts were made after 3 days of incubation, and the composition of the flora was determined by picking and subsequently identifying all the colonies (100-150) from suitable roll tubes used for the enumeration of the total viable population. Direct microscopic counts of formalized suspensions of protozoa in glycerol-water (1:1, v/v) were made in a Neubauer counting chamber (Hawksley & Sons Ltd., Lancing, England), and differential counts of bacteria were made by the Breed technique (1) by using Gram-stained preparations. Lipolysis studies. Two methods were used for determining lipolytic activity. (i) An agar diffusion method (19) was used to test the lipolytic activity of the bacteria from cow M21. The rate of hydrolysis of various triglycerides emulsified in buffered agar by test suspensions of bacterial cells was determined by measuring the diameter of the clear zones produced in the opaque gel at intervals during incubation at 30 C. Suitable suspensions of bacterial cells were obtained from 100 ml of rumen fluid which had been filtered through one layer of muslin. The filtered rumen fluid was centrifuged several times at approximately 300 x g for 5 to 10 min to remove the protozoa, and the supernatant fluid was centrifuged at 15,000 x g for 1 hr to sediment the bacteria. The bacteria were resuspended in 2.5 ml of 0.05 M sodium phosphate buffer, pH 7.0, and the amount of protein per milliliter of suspension was determined (20) so that in comparing the lipolytic activity between rations all values could be corrected to a unit value of 25 mg of protein per ml of suspension. (ii) An incubation method was used to determine the lipolytic activity of whole rumen contents from cow M21 and of the protozoal, bacterial, and cell-free rumen fluid fractions of rumen contents from cow J when each cow was given highand low-roughage diets. Sediments obtained by centrifuging at 300 x g (rich in protozoa but contaminated with feed particles and large numbers of bacteria) and 15,000 x g (rich in bacteria) were resuspended in their original volume of cell-free (15,000 x g supernatant fluid) rumen fluid. Duplicate flasks containing each of the rumen fluid fractions were incubated at 39 C in a water bath. Each flask was closed with a rubber bung adapted to gllow continuous stirring and gassing with 02-free nitrogen throughout the incubation. Emulsions of linseed oil or triolein were prepared by sonic oscillation (Dawe Soniprobe type 1130) in 0.05 M phosphate buffer, pH 7.0, containing 0.1% (w/v) gum acacia. The emulsion was purged with O2-free nitrogen, and 10% (v/v, final incubation mixture) added to one (experimental) of each pair of flasks to give 10 mM final concentration of triglyceride. The same volume of phosphate buffer containing only gum acacia was added to the remaining flasks (controls). After thorough mixing, 20-ml subsamples were removed from both the experimental and control flasks (0 hr), the reaction was stopped by acidifying to pH 2.0 with 50% HZSO4, and the sample was shell frozen with CO2-acetone. Additional subsamples were taken at 8 hr and treated similarly. All of the subsamples were then freeze-dried. Total lipid in the freeze-dried material was extracted in 2: 1 (v/v) chloroform-methanol, and the nonesterified fatty acids in the extract were separated by thin-layer chromatography (28) and estimated quantitatively by the colorimetric method of Duncombe (10). Hydrogenation studies. Samples of rumen digesta were taken 4 to 5 hr after feeding from animals on the control diet and again from the same animals after they were established on the low-roughage diet and showing depressed milk fat. These samples were taken directly into each of two sterile graduated flasks and immediately gassed with 02-free nitrogen. Sufficient anaerobic dilution fluid (3) was added to dilute the contents by one-third to give a final volume of 1 liter. At the same time additional rumen samples were taken for microbiological analysis. The flasks were incubated in a similar manner to that used in the lipolysis studies. A 5-ml amount of 10% soybean oil emulsion ("Intralipid": Paines and Byrne, Greenford, England) was injected into the contents of one (experimental) of the flasks. After allowing 2 min for thorough mixing of the emulsion with the digesta, 30-g subsamples were taken from both the experimental and control flasks (0 hr), and again after 1, 2, 4, and 6 hr of incubation. The weight of each subsample was recorded, and the reaction was stopped by immediately transferring the sample into 2:1 (v/v) chloroform-methanol for extraction of total lipids. At each time of sampling, the pH and total numbers of bacteria were also recorded. Extraction of total lipids and total fatty acids and determination of fatty acid composition. Total lipids from whole rumen contents in the hydrogenation studies and from freeze-dried rumen fluid fractions in the lipolysis studies were extracted in 2:1 (v/v) chloroform-methanol and washed with 0.88% (w/v) KCl solution as described by Folch, Lees, and Sloane Stanley (12). Total fatty acids were extracted from the total lipid samples after saponification, and their composition was determined by gas-liquid chromatography (26). RESULTS Milk fat. Although the composition of the low-roughage diets fed to individual animals differed, they -ll gave a marked depression in milk fat secretion. At the same time, the proportions of saturated fatty acids in the milk fat were decreased and the proportions of unsaturated fatty acids were increased (Table 1). Rumen microflora. With the exception of cow G9, the low-roughage diets increased the number of viable bacteria in all cows by 0.6 to 1.6 log units but decreased the number of lipolytic (tributyrin-hydrolyzing) bacteria by up to 3 log units (Table 2). At the same time, these diets reduced by various amounts the numbers of ciliate protozoa. Of the two cows used in the lipolysis studies, M21 lost its ciliates, and there was a reduction in the number of ciliates in cow J which was not quantified. Only one (G10) of the four cows used in the hydrogenation studies lost its ciliates, while in cow G27 they were reduced by 90%. However, the two remaining cows (G9 and B20) lost only 32 and 45% of their ciliates, respectively. The variation in the composition of the bacterial flora between cows given the lowroughage diets was considerable ( Table 3). The proportions of Selenomonas, Peptostreptococcus, Bifidobacterium, and Lactobacillus were particularly variable. Nevertheless, increases in lactogenic and propionogenic bacteria typical of low-roughage diets (4,18) were observed. The most consistent effect of the low-roughage diets on the bacterial flora was the severe reduction in the proportion of Butyrivibrio spp. Borrelia, which like Butyrivibrio is capable of hydrogenation (23,35), was also reduced. Rumen lipolysis. Preparations of mixed rumen bacteria derived from cow M21 when fed the high-roughage diet hydrolyzed buffered emulsions of both tributyrin and tripalmitin, but tributyrin was hydrolyzed almost 2.5-fold as fast as tripalmitin (Fig. 1). On the low-roughage diet the rate of hydrolysis of both triglycerides by similar preparations of mixed rumen bacteria fell to one-fifth of the previously observed rates. Incubations of whole rumen contents with emulsified linseed oil confirmed this apparent reduction in lipolytic activity (Table 4). Progressively less triglyceride was hydrolyzed as the roughage was reduced and the cereal content of the ration increased. In a separate experiment the lipolytic activity of the protozoal, bacterial, and cell-free rumen fluid fractions obtained from cow J given similar high- and low-roughage rations were compared. The bacterial fraction associated with the hay diet had the greatest lipolytic activity, hydrolyzing 87.6% of the added triglyceride (Table 5), whereas the protozoal and cell-free rumen fluid fractions hydrolyzed only 28.6 and 13%, respectively. The same fractions from animals on the low-roughage ration hydrolyzed 5.8, 34.9, and 5.7%, respectively, indicating a considerable decrease in lipolysis by the bacterial fraction offset to some extent by a slight rise in that of the protozc level of unto increased N concentrate Rumen were fed th tions of tot digesta we when they These diff higher diet; age rations of rumen i were appre acids of di incubation any differe C18 fatty controlanm metabolisn The patt similar for mean resu diets are s Over the 6tion of lino form of so, diet, 59 and 63%, respectively, of that obtained on the high-roughage ration. The overall reduced rates of hydrogenation on the low-roughage diet were significant at the 5% and 1% level for linolenic and linoleic acids, respectively. The pattern of hydrogenation also differed between the two diets; with the high-roughage diet the hydrogenation appeared to commence soon after addition of the soybean oil and to progress rapidly up to the end of the fourth hour of incubation, whereas with the low-roughage ration the most rapid hydrogenation occurred between the second and sixth hours. )al fraction. In both experiments the study (18), the composition esterified fatty acid of dietary origin terial flora of nonlactating with increases in the proportion of and low-roughage diets es in the diet. between cows fed the hydrogenation. When the cows present experiment the between-cow Le low-roughage diets, the concentraon the low-roughage ;al lipid and fatty acids in the rumen extended to the protozoa. ore two or three times greater than variability was not clear were fed the high-roughage diets. related to the high food erences can be accounted for by cows. ary intakes of lipid on the low-rough-It has been shown , and by increases in the contribution fats or oils are added microbial lipid (29). Although there rapidly adsorbed on food ,ciable amounts of oleic and linoleic ganisms (13,14); Czerkawski ietary origin present in the in vitro that levels of linseed oil systems, it has been assumed that ml of rumen fluid inhibited nces in the composition of the total Greater concentrations acids between samples from the this, while saturating i experimental incubations reflected rumen fluid (Demeyer, n of the added soybean oil. tion), have not been shown ;emns of hydrogenation obtained were In the present experiment, all four cows examined, and the total lipid and fatty lts for the high-and low-roughage used for the in vitro s hown in Fig. 2 and 3, respectively. lipolytic and hydrogenating *hr incubation period, the hydrogenagreater with the low-roughage lenic and linoleic acids added in the high-roughage diets, ybean oil was, on the low-roughage genation studies the acids in "Intralipid" during incubation with-rumen contents from cows fed a low-roughage diet. was added at the rate of 50 mg/100 ml of digesta, more than 800 mg/100 ml of digesta was added in the lipolysis studies. However, serial samples taken during the various incubation experiments revealed no major change in either the direct count or motility of bacteria and protozoa, indicating that even at the high substrate levels used in the lipolysis studies the added triglyceride had no apparent deleterious effect on the microflora. It is probable that the reduced lipolytic activity of the rumen digesta found with the low-roughage diets was related to the reduced numbers of lipolytic vibrios. These diets greatly reduced the viable counts of tributyrin-hydrolyzing bacteria, and approximately 70% of the tributyrinolytic bacteria isolated on a previous occasion in this laboratory from the same medium were found to resemble butyrivibrios. Five out of 25 of these strains were able to hydrolyze long-chain triglycerides up to triolein. In the present work, the strongly lipolytic vibrio Anaerovibrio lipolytica was not isolated, and subsequent work in this laboratory suggests that this and similar lipolytic organisms constitute no more than 1% of the viable population. This effect of diet on lipolysis is in agreement with recent observations of Demeyer (personal communication) who found that the inclusion of sucrose in the diet ot'a sheep or of glucose in an in vitro incubation of rumen fluid reduced lipolysis. It is not clear why the lipolytic activity of the protozoal fraction should have increased with the low-roughage diet. Clarke and Hawke (7) found that much of the lipolytic activity of this fraction could be transferred to the bacterial fraction after homogenization, and they therefore concluded that many lipolytic bacteria were attached to the particulate matter. Since on the low-roughage diet many starch granules will also sediment at the low "g" values used for separating the protozoal fraction of rumen fluid, they will, in so doing, bring with them even larger numbers of adhering bacteria among which may be actively lipolytic strains. As indicated by the changes in milk fat composition and from the results of the in vitro incubations, the hydrogenating activity of the rumen contents was also reduced by the lowroughage rations. Strains of Butyrivibrio fibrisolvens were the first rumen bacteria to be implicated in the biohydrogenation of polyunsaturated fatty acids and were found to hydrogenate to the monoene (23). Since then laboratory strains of Borrelia (35), a gram-negative micrococcus (22), a strain each of Eubacterium and Ruminococcus (34), and two strains of cellulolytic Clostridium spp. (32) have all been found to biohydrogenate in a similar manner. However, only two strains of bacteria have so far been reported to be capable in pure culture of the complete reductive process to the saturated acid (34). Under normal conditions of high-roughage feeding, Butyrivibrio fibrisolvens is by far the most numerous of all of these biohydrogenating organisms found in the rumen and in the present experiment comprised 25.0% of the viable population in cows fed the high-roughage diets. On the assumption that the isolates of Butyrivibrio and Borrelia included hydrogenating strains, the reduction in their numbers which occurred with the lowroughage diets is consistent with the decrease in hydrogenation observed in vitro. Tove and Matrone (30) reported that, compared with conventional diets, the reduction of the monoenoic to the saturated acid proceeded more slowly with high-concentrate diets. As most workers have not been able to isolate strains which can carry out this final reductive step, such organisms are unlikely to occur in the majority flora, yet, like the butyrivibrios, they would appear to be greatly affected by dietary changes. Although the protozoa-rich fraction of rumen contents has been shown to have biohydrogenating activity (5). the nule (l individual species of ciliate protozoa in this activity remains equivocal (31). However, studies on the plasma lipids of lambs with or without rumen ciliates indicate that dietary C,. unsaturated fatty acids are more effectively hydrogenated in the presence of ciliates (A. K. Lough, Proc. Nutr. Soc., 27:30A, 1968). It is therefore of interest that in the present work the reduction in hydrogenation induced by the low-roughage diets was no greater in the two cows which either lost or suffered a 90% reduction in the numbers of ciliates than in the remaining cows which retained between 55 and 70% of their ciliates. Because Hawke and Silcock (15) have recently shown that only nonesterified fatty acids can be hydrogenated, the present results suggest that, in addition to the adverse effects of low-roughage rations on the numbers of potential biohydrogenating organisms, a reduction in lipolysis may also contribute to the observed reduction in hydrogenation. ACKNOWLEDGMENTS The analysis of total fatty acid composition by A. J. Hall. the care of the animals by V. W. Johnson, and the capable technical assistance of Ann Siddell are gratefully acknowledged.

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2020-12-10T09:04:12.270Z

1972-10-01T00:00:00.000Z

237232498

s2

Common Antigenic Determinant in a Rumen Organism and in Salmonellae Containing the Antigen O4 Nineteen of 28 strains of rumen organisms isolated from a cow on a high roughage diet and identified morphologically as butyrivibrios, reacted to a low agglutinin titer with salmonella antisera, forming five groups. However only one strain reacted with polyvalent O salmonella antiserum. This strain reacted with O4 factor serum and with antisera to Salmonella strains containing the antigen O4, and agglutinin absorption tests showed the presence of an antigen identical to O4. When 16 further strains of butyrivibrio-like rumen organisms isolated from three cows and one steer were examined, one possessed an antigen similar to but not identical with the antigen O9, and two strains reacted with specific O6,7 factor serum but were not examined further. These four strains were presumptively identified by physiological tests as butyrivibrios. The possible site of antigenic stimulation by such organisms is discussed. Natural antibodies against Enterobacteriaceae, including a number of Salmonella strains, occur widely in the sera of animal species including ruminants (3,7). Sharpe, Latham, and Reiter (11) have shown that in the sera of ruminants relatively high titers of agglutinating antibodies occurred against strains of anaerobic bacteria isolated from the bovine rumen. This suggested that the reactions against Enterobacteriaceae might be due to antibodies against cross-reacting antigens in these organisms and in rumen strains. Antibodies against motile, curved, gram-negative chain-forming rods (butyrivibrio-like organisms) were particularly prevalent in the sera of the ruminants. This work describes the reaction of some of these organisms to antisera against some Salmonella serotypes. MATERIALS AND METHODS Organisms from the bovine rumen. Samples of well mixed rumen contents were removed from fistulated bovines, and organisms were isolated by the anaerobic techniques of Hungate (4), with the media and methods of cultivation described by Sharpe et al. (11). Strains of anaerobic, gram-negative, curved, motile rods in chains were identified microscopically. Presumptive identification of rumen strains. Four serologically reacting isolates were further examined, using medium M10 as basal medium for tests, for growth in relation to oxygen tension, sulfide production, minimum pH in glucose broth, gas production in glucose agar, starch hydrolysis, cellulose digestion (filter paper strip), and production of lactic acid (1,5,8). Salmonellae. Strains were obtained from W. Sojka, Central Veterinary Laboratory, Weybridge, and from the National Collection of Type Cultures. They were grown aerobically in nutrient broth at 37 C. Strains used for preparing antisera were checked at the Salmonella Reference Laboratory, Colindale, to confirm their antigenic patterns. Preparation of antisera: rabbits. Vaccines consisted of 18-hr cultures of washed cells, either suspended in 0.15 M NaCl + 0.2% (v/v) formaldehyde, or in 0.15 M NaCl and heated at 100 C for 60 min, optical density being adjusted to 0.55 absorbancy at 580 nm (1-cm light path). Rabbits were inoculated intravenously with 1 ml of vaccine every 3 to 4 days for a course of five to eight inoculations. Goats. Two milliliters of the heat-killed vaccines as above was given intravenously for a course of four to six inoculations. Commercial antisera. Polyvalent and factorspecific antisera were obtained from Wellcome Reagents Ltd., Beckenham, Kent. Agglutination tests. Suspensions for agglutination were either (i) suspended in 0.15 M NaCl + 0.2% (v/v) formaldehyde; (ii) suspended in 0.15 M NaCl and heated at 100 C for 60 min; or (iii) (later in the work) heated to 50 C for 30 min while suspended in ethanol, centrifuged, and resuspended in 0.15 M NaCl. Agglutination tests were read after overnight incubation at 50 C. 613 Agglutination absorption tests. Antisera were absorbed at a dilution of 1:5 or 1:10 with ethanoltreated packed cells, two parts cells to 10 parts diluted antiserum, for 2 hr at 37 C. Two absorptions were often necessary. RESULTS Isolates. Isolations were made from a sample of the rumen contents of cow N13 on a high roughage diet, which promotes the predominance of butyrivibrios (8). Twenty-eight isolates were microscopically identified as motile, gram-negative, curved rods in chains. Reaction of isolates to salmonella antisera. Formol-killed and heat-killed suspensions of these organisms were tested by tube agglutination with antisera (diluted 1: 20) prepared against Formol-killed vaccines of S. typhimurium, S. dublin, S. bovis morbificans, S. farmsen, and S. give. Formol-treated suspensions were also tested against 1:20 dilutions of polyvalent H antisera and heated suspensions against 1: 20 polyvalent 0 antiserum. Formol-and heat-treated suspensions both reacted similarly to the antisera against single salmonella strains. As the formolized suspensions did not react with the polyvalent H antisera, no further work was done with these suspensions. As these butyrivibrios possess only a single polar flagellum (1), the use of formolized or heat-treated suspensions, respectively, to determine the types of H or 0 antigens present, as with the salmonellae, is unlikely to be useful. Table 1 shows the reactions of suspensions of heat-treated cells of isolates at 1: 20 dilution of antiserum. With these antisera it was possible to group 19 of 28 of these isolates into five groups. However, the reactions occurred at the low titre of 1:20 and could not be considered specific. Only one strain, Nor 37, reacted strongly with the polyvalent 0 antiserum. On testing representative strains of the rumen isolates against eight different specific 0 factor sera, only Nor 37 reacted, giving a specific reaction with 04 antiserum. This strain was chosen for further work. Reaction of two rumen strains and salmonella serotypes to antisera to Nor 37 and salmonellae. Using heat-treated cells for vaccines, antisera were prepared against Nor 37 and also against S. abortus equi and S. altendorf (each of which possesses the 04 antigen). These antisera were tested against alcohol-treated suspensions of eleven Salmonella serotypes, Nor 37, and a rumen strain, 1L6-31, isolated from another cow. Table 2 shows the titers obtained and also titers against the serum from cow N13. Serum from this cow contained a low titer of antibodies to many of the salmonella strains. The preinoculation goat serum also showed the presence of antibodies to many of the salmonellae. However in the two rabbits' sera, antibodies to salmonellae were absent before inoculation. After inoculation with strain Nor 37 the antibodies raised in rabbit 417 reacted not only with the homologous strain but also specifically with strains of salmonella containing 0 antigen 4. In the goat a significant rise in titer occurred against these same organisms. Inoculation of rabbit 427 with S. altendorf resulted in the presence of antibodies reacting with Nor 37, salmonella serotypes containing the 0 antigen 4, and also serotypes containing 0 antigen 12. An antiserum against S. typhimurium also reacted in a similar pattern. The Wellcome 04 factor-specific antiserum agglutinated only Nor 37 and salmonellae containing the 0 antigen 4. Figure 1 shows the rise and fall of titers of agglutinating antibodies to Nor 37 and to three salmonella strains in a rabbit given' five inoculations of alcohol-treated cells of Nor 37. The higher titers observed with the homologous organism may be due to the presence of another specific antigen in this organism but not in the other strains, or to the other 0 antigens present in the salmonella strains partially obscuring the 04 antigen. Absorption of antisera. The effect of absorption of antisera by whole cells on agglutinating titers is shown in Table 3. When antiserum to Nor 37 was absorbed with S. altendorf all reactions to salmonella strains were removed, but some of the antibodies to Nor 37 remained, indicating that, in addition to antigen 04, another more specific antigen was indeed present in Nor 37 but not in the other strains. Absorption with nonreacting strains of S. dublin or 1L6-31 did not remove the reactions. When antiserum to S. altendorf was absorbed with Nor 37, titers were considerably lowered, but reactions, presumably to their common antigen 12, remained for the salmonella strains. Both Nor 37 and S. altendorf removed all the reacting antibodies from factor 04 antiserum. There is therefore a common antigenic factor in the rumen organism Nor 37 and in salmonella of group B containing the 04 factor. Reaction of further rumen strains to salmonella antisera. Sixteen more strains of anaerobic, motile, curved, gram-negative rods in chains isolated from three other cows and from a steer on different experiments were tested against the Wellcome polyvalent 0 antiserum and against antisera to S. typhimurium, S. dublin, and S. bovis morbificans, all diluted 1: 20. One strain, 1L6-31, reacted with the polyvalent 0 antiserum and also with antiserum to S. dublin. On further testing with factor-specific sera, 1L6-31 reacted with factor 09 to a titer of 160. Antisera against this strain raised in a goat and in a rabbit reacted to titers of only 80 to 160 with strains of S. strasbourg (09, 46), S. zega (09, 12), S. enteritidis, and S. dublin, whereas titers against the homologous organism were 5,120 and 2,560. However, absorption of factor 09 antiserum with cells of strain 1L6-31 failed to remove the reactions of the four salmonella strains, although some drop in titer occurred, and the reaction against 1L6-31 was completely removed. This suggests that the antigenic determinant present in 1L6-31 which reacted with the 09 factor antiserum was similar but not identical to the 09 antigenic determinant present in salmonella of group D. Two other strains of butyrivibrios, both from a steer, reacted with factor-specific serum 06, 7 to a titer of 1: 80. No further tests were done on these strains. Further identification of rumen strains. Strains Nor 37 and 1L6-31 and the two strains reacting with the 06, 7 serum produced a final pH in glucose broth of 5.0 to 5.4, none were cellulolytic, none produced lactic acid, only one produced sulfide, one produced gas from glucose, and two hydrolyzed starch. These characteristics were compatible with the strains being non-cellulolytic butyrivibrios. Production of antibodies to strain Nor 37 in animals by other routes of administration. Attempts were made to raise serum antibodies against strain Nor 37 in the rabbit and in the goat by oral infusion of large numbers of viable cells of the culture, given at twice-weekly intervals for 5 weeks. In the rabbit no antibodies were detected in the serum after this treatment. In the goat, which had an initial titer of 40, the titer rose to 160, but on further infusion did not rise any higher. Attempts were also made to raise antibodies to this organism in the ruminating calf by infusing twice weekly very large numbers of viable cells directly into the rumen of fistulated animals or into the duodenum of duodenumcannulated animals. No increases in the initial low titers (40-80) occurred with any of these calves. Bacteriological and serological examination of isolates from rumen contents indicated, however, that strain Nor 37 did not become established in the rumen flora of the animals inoculated. DISCUSSION Common antigenic determinants between unrelated organisms are not uncommon particularly with the 0 antigens of the Enterobacteriaceae, serological cross-reactivity being due to identical chemical structures in the polysaccharide of cross-reacting species (reviewed Kwapinski [61, Wilson and Miles [12]). If the rumen strain Nor 37 has the same 04 antigenic determinant as group B salmonellae it should contain abequosyl-mannosyl-rhamnosyl-galactose repeat units. It is of interest that the blood serum of an uninoculated cow and of two goats contained 0 antibodies to a variety of salmonella agglutinating antigens whereas that of rabbits did not. This may be correlated with the presence or absence of antibodies to rumen organisms in the serum of animals observed by Sharpe et al. (11), who showed that the two former species of animals possess such antibodies but the rabbit does not. Our results indicate that previous reports of the presence of antibodies to a wide range of Enterobacteriaceae, including salmonellae, in bovine serum, may be due to cross-reactions with identical or similar polysaccharides present in rumen organisms. Although only a small number of the butyri-vibrios tested (4 of 44) reacted initially with the polyvalent 0 serum (containing factors of groups A through G) and then with factorspecific sera, these strains were isolated from three out of five of the animals examined. Further work is necessary to determine the incidence of such cross-reacting strains. Failure to raise antibodies against strain Nor 37 with massive doses of live organisms by oral, direct ruminal or duodenal routes, may have been due to two factors: either a constant persistent stimulation is required over a long period of time and our treatment did not continue for long enough, or the organism must become established in the rumen for stimulation to occur. To elicit an immune response, organisms should penetrate or at least survive on the mucosal epithelia (10). For this, viable bacteria are necessary, as shown by the production of antibodies on ingestion of live but not heatkilled cells of S. enteritidis (2). As the caecum may be colonized by species of bacteria similar to those in the rumen and producing similar volatile fatty acid patterns (9), this could be the site of antigenic stimulation. Histological examination of the cecum wall showed clearly that it contained large numbers of plasma cells and mucosal scrapings were found to contain immunoglobulins (unpublished data). These observations may point to the origin of the antibody production in the cecum, as the rumen wall is devoid of plasma cells.

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