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Vacuum-canned, commercial, mechanically deboned chicken meat was challenged with either Clostridium botulinum spores (20 strains of types A and B, proteolytic; final spore concentration of ca. 400/g of meat) or Salmonella enteritidis (ca. 104 CFU/g of meat) followed by irradiation to 0, 1.5, and 3.0 kGy and storage at 5°C for 0, 2, and 4 weeks. None of the samples stored at 5°C developed botulinal toxin; however, when these samples were temperature abused at 28°C they became toxic within 18 h and had obvious signs of spoilage, i.e., swelling of the can and a putrid odor. During 4 weeks of refrigerated storage the log10 of the population of S. enteritidis in nonirradiated samples decreased from 3.86 to 2.58. S. enteritidis CFU were detectable in samples irradiated to 1.5 kGy at 0 weeks but not in samples irradiated to 3.0 kGy. Log levels of aerobic and facultative mesophiles increased during 4 weeks of refrigerated storage from 6.54 to 8.25, 4.03 to 8.14, and 2.84 to 5.23 in samples irradiated to 0, 1.5, and 3.0 kGy, respectively. Based on taxonomic analyses of 245 isolates, the bacterial populations depended upon radiation dose and storage time. The change was predominantly from gram-negative rods in nonirradiated samples to gram-positive streptococci in samples irradiated to 3.0 kGy and stored for 4 weeks. Spoilage organisms survived even the 3.0 kGy treatment.

Radiation survival curves were determined for 7 strains of Enterococcus faecium , 10 strains of E. faecalis , and 8 strains of the proteolytic variety of E. faecalis . The D values (i.e. the doses giving 90% reduction of viable counts) ranged from 5.0-47 kGy for the E. faecium strains, 3.5-21 kGy for the E. faecalis strains, and 3.0-4.5 kGy for the proteolytic variants of E. faecalis . The survival curves were linear for most strains but some exhibited significant non-linear trends.

A new process involving acid-blanching and addition of EDTA to the canning brine (ABC) was compared with the standard commercial method (SCM) for the control of Clostridium botulinum spores in canned mushrooms. Fresh mushrooms were uniformly inoculated under vacuum with 105 spores of proteolytic strains of Clostridium botulinum Types A and B and processed either by the ABC or SCM method. Compared to the SCM, the ABC process significantly reduced the spoilage and toxigenesis of canned products. At Fo values of 0.5, 2, and 4, the ABC when compared to the SCM process reduced percent spoilage from 91.4 to 51.5, 17.6 to 6.3, and 12.1 to 2.9, respectively. No botulinal toxicity occurred except at Fo of 0.5 when the number of toxic cans was reduced from 54.3 to 30.3 by the ABC, compared to SCM.

Bacteria in non-fat dried milk (NDM) were enumerated by a method involving preliminary solubilization of the milk proteins in 0.015 N NaOH followed by centrifuging, washing in the NaOH, and microscopically examining stained smears. The method was used to enumerate bacteria in samples of NDM obtained from government surplus stocks or from local retail sources. Bacterial counts from surplus NDM ranged from 4.64 × 105 to 2.83 × 106/g (the mean and median were, respectively, 6.23 and 2.84 × 106/g). Counts from retail samples ranged from 4.48 × 105 to 2.42 × 107/g (mean and median were 5.57 and 2.85 × 106/g). The predominant bacteria in some samples were paired streptococci; other samples contained rod-shaped bacteria, some with identifiable spores. Comparison of this method with the Levowitz-Weber method indicated that it produced fewer artifacts, was applicable to NDM samples containing a wider range of bacteria, and did not require the use of the potentially carcinogenic tetrachloroethane.

Comminuted bacon, processed to contain target levels of 40 μg NaNO2/g and 0, 0.25 or 0.75% sucrose or 0.75% glucose, was inoculated with a mixture of spores of 20 strains of Clostridium botulinum (400 spores per g) and was canned under vacuum. Portions were irradiated using 137Cs at doses of 0, 0.19, 0.38, 0.75, 1.12 and 1.5 Mrad. Cans were incubated for 1, 2, 4 or 8 wk at 30°C. Some cans of nonirradiated bacon without or with 0.25% sucrose became toxic in 2 wk; with 0.75% sucrose, toxin production was delayed to 8 wk. Bacon irradiated at 0.75 Mrad, made with or without sucrose, became toxic in 2 to 4 wk, whereas most cans of bacon irradiated at 1.5 Mrad remained toxin-free for the 8-wk incubation period. A comparison of bacon made with 0.75% sucrose or glucose showed no difference between the sugars in the rates of toxin production by C. botulinum in irradiated cans of bacon. Irradiation at 0.19 Mrad increased the rate of toxin formation over nonirradiated bacon in sugar-containing (0.75%) bacon, but had...

Several short-chain alkynoic and aikenoic acids and esters were screened for inhibition of gas production by Clostridium botulinum in cans of comminuted, nitrite-free, temperature-abused bacon. The most active compounds were propiolic (2-propynoic) acid, methyl and ethyl propiolate, 2-propenoic acid, methyl and ethyl propenoate, and mono- and dimethyl and ethyl fumarate. Maleic acid, methylmaleic acid, ethyl maleate, 2-methyl propenoic acid, trans-2-methyl crotonic acid, 3-methyl-2-butenoic acid, 3-methylallyl alcohol, vinyl crotonate, methylmalonic acid, dimethyl glutarate, 2,4-hexadien-1-ol, cis-3-hexenoic acid, 4-pentenoic acid, trans-2-pentenoic acid and ethylidene acetic acid were less active. A more comprehensive study on the inhibition of toxin formation by C. botulinum in comminuted nitrite-free bacon was done by comparing equimolar quantities of some of these compounds with NaNO2 at 120 μg/g and sorbic acid at 9 and 18 mM (0.10 and 0.20%). These studies showed that propiolic acid was more effective than either nitrite or sorbic acid. Compounds in this study less active than nitrite or sorbic acid were 3-methylallyl alcohol, 2,4-hexadien-1-ol, dimethyl glutarate and methylmaleic acid.

Survival of Salmonella dublin , Salmonella senftenberg 775W, Staphylococcus aureus 196E, and S. aureus 184 was studied during processing of an inoculated beef, nonfermented snack sausage. No viable staphylococci or salmonellae were detected in sausages that had been heated at an internal temperature of 53.9-55.0 C or 57.8-58.9 C for 3.5 h followed by drying at 21 C and 50-55% relative humidity for 4 days. Heating at an internal temperature of 51.1-52.2 C for 3.5 h followed by drying did not produce a salmonellae- or staphylococci-free sausage.

Eleven analysts tested contaminated reconstituted (1:10) dry milk powders for penicillin residues using spores of Bacillus stearothermophilus var. calidolacris (Delvotest P method). Three types of responses were noted: positive, negative, and questionable. Prediction equations indicated that 95% of the time, analysts unfamiliar with the technique could detect positive results if penicillin concentrations in samples were 0.010 unit/ml or higher and positive and questionable results if the penicillin concentrations were 0.008 unit/ml. Increasing the reconstitution ratio from 1/11 to 1/4 increased the chances of detecting penicillin in milk powder. Penicillinase added to reconstituted penicillin-contaminated milks in all instances produced negative responses.

Strains of Clostridium botulinum types A, B and F were grown aerobically in metabiotic association with several aerobic and facultative microorganisms. In unbuffered media Enterococcus faecalis and Pediococcus cerevisiae produced enough acid to prevent toxin formation by C. botulinum but in buffered media toxin was formed. Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Yersinia enterocolitica, strains of Serratia, Pseudomonas, Alcaligenes and 35 unidentified strains from mechanically deboned chicken homogenate produced conditions favorable for toxin production under aerobic conditions. The metabiotes did not inhibit spore formation nor did toxin production correlate with the appearance of spores.

Salmonella senftenberg 775W added to frankfurter emulsion was killed during normal processing in the smoke house when internal product temperature was 71.1 C (160 F) or above. The thermal destruction point of S. senftenberg 775W in frankfurters (temperature at which no viable cells were detected) was a function of the length of time of the process rather than of the starting number of cells. Heating of frankfurters to 73.9 C (165 F) substantially reduced the total non-salmonella count. For total non-salmonella bacterial flora and salmonella, relatively little thermal destruction occurred below 43.3 C (110 F). The heating step can bring about a 7-log cycle decrease (10 8 to 10 1 /g) of bacteria present in the raw emulsion. The flora of this high-bacteriological-count raw emulsion was predominantly gram-negative rods. Variation in the number of bacteria (both total and salmonella) surviving at various temperatures during processing was attributed to slight variations in the temperature pattern of the smoke house during its operation. An integration process was devised which allowed calculation of exposure to temperatures above 110 F (43.3 C) on the basis of degree-minutes. Plots of degree-minutes versus log of surviving bacteria were linear. The salmonella plot had a greater slope than that of the total non-salmonella flora, indicating that salmonellae are more heat sensitive than the bacterial population as a whole. The predominant bacteria surviving the heating step were micrococci. These micrococci were able to increase in number in or on the frankfurters during storage at 5 C.

Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10 4 salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10 3 /g, S. dublin could not be recovered from started culture fermented pepperoni after 14 days of drying but persisted in the natural florafermented sausage. S. typhimurium (initial count, 10 4 /g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin , host adapted to cattle, or S. choleraesuis , host adapted to swine, had similar survival patterns in beef, pork, or beef-pork pepperoni. Heating salmonellaecontaminated beef-pork pepperoni (after fermentation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product.

Survival and destruction of Salmonella dublin and S. typhimurium added to Lebanon bologna was studied during manufacture of this sausage. During the aging period of salted cubed beef at 5 C, viable cell counts of S. dublin did not change over a 10-day period. Cell counts of S. Dublin were reduced 3 to 4 log cycles during the 4-day fermentation at 35 C; further reduction in the viable Salmonella count occurred during mellowing of the bolognas at 5 C. The number of S. typhimurium was usually reduced to an undetectible level before the end of the fermentation. Salmonella typhimurium was consistently more sensitive to the acid conditions of Lebanon bologna than was S. dublin. Introduction of an optional cooking step indicated that heating of bolognas to 51.7 C or above led to destruction of salmonellae. Unaged beef which was not inoculated with starter culture did not ferment and there was very little reduction in numbers of added salmonellae. Salmonellae were destroyed more rapidly in Lebanon bologna made from unaged beef with starter culture than in bologna made from aged beef (natural flora fermentation). Smoking also appeared to contribute to destruction of salmonellae. Four commercial Lebanon bolognas were tested for the presence of salmonellae but none were detected.

Plates incubated for 48 h showed significantly higher (0.01

Raw milk samples were diluted with distilled water or distilled water with added phosphate buffer as recommended by Standard Methods for the Examination of Dairy Products. The standard plate counts were higher in diluent without phosphate buffer with both high and low count milk. The higher counts were significant when analyzed by a nonparametric sign test or a t-test of differences but were not significant with an analysis of variance technique. Reproducibility was not statistically different in the two diluents. It is suggested that the use of phosphate buffer for raw milk bacteria counts be discontinued until information showing definite advantages is provided.

Treatment of milk with low-frequency ultrasound significantly increased the total bacterial counts and the counts of enterococci, coliforms, and staphylococci. Warming diluted milk for about 12 min at 30 or 40 C increased the counts of some organisms, but the heat produced by ultrasonic treatment did not account entirely for its effect. The ultrasonic effect was related to the energy output of the generator and to the energy absorbed by the treated materials.

Raw milk samples were plated and incubated at seven temperatures (2, 10, 20, 27, 30, 33 and 37 C). Lowest counts were obtained at 37 C. Plates at 33 C showed higher counts than at 37 C but were significantly lower than the other temperatures. The 10, 20, 27 and 30 C incubated plates were not statistically different in counts. When 32 C was substituted for 33 C, there was a significant difference (lower) only in the 37 C count. Colonies from 2, 20, 27, 33 and 37 C plates were picked and stabbed into plates subsequently incubated at these temperatures. All colonies developed best at 20 and 27 C. Evidence is presented that psychrophilic counts can be indirectly determined as the difference between counts at 27 C and 37 C.

Forty-six raw milk samples were analyzed for plate counts at 32 C by eight different laboratories; each using three mixing procedures for the initial dilution. These were: five inversions in a 5 sec period, 15 inversions in a 15 sec period, and the “standard” method of twenty-five, 1 ft long, vertical cycles in a 7 sec period. The standard method gave the highest bacterial counts (71.1 × 103/ml average) the 15-15 method was second highest (60.4 × 103/ml average) and the 5-5 method was lowest (57.8 × 103/ml average). The standard method gave significantly higher (P Tests of reproducibility (pooled average variances for each method) did not show any significant differences between mixing methods. There were significant differences in reproducibility between laboratories. There was evidence of interaction between mixing methods by samples and mixing methods by investigators.

Aspergillosis was induced experimentally in hatching chick embryos by dipping them in water containing spores of Aspergillus fumigatus or A. flavus . The addition to the dip water of antifungal compounds prevented the development of this syndrome. Of the compounds studied, amphotericin B was most effective, followed by pimaricin and nystatin. Sorbic acid was without effect.

Eighty-three samples of raw milk were assayed by the Standard Plate Count method with incubation periods of two and three days. The three-day incubation period gave higher counts (0.05 > p > 0.01). There were no appreciable problems (such as drying-out, spreaders, molds, etc.) encountered when plates were incubated the extra day. An optional three-day incubation period for the Standard Plate Count is recommended.

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.

A comparison was made of a horizontal versus vertical shaking procedure for raw-milk dilutions used in the Standard Plate Count for the enumeration of milk bacteria. No significant differences (P

Raw milk samples were diluted with buffered water and held at room temperature for periods up to 20 min before plating. There was an increase in counts at the 95% but not at the 99% level of significance. Most of this increase appeared at 10 min holding time. Interaction effects were highly significant (p < 0.01) between holding times and investigators and also between treatments and samples within investigators. It is suggested that the holding time of dilutions to be used for the standard plate count be no longer than 5 min.

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.

Twenty-five meat-and-bone meal samples were enriched with either selenite-cystine or tetrathionate and incubated for 1 and 2 days. Seven were previously found to be positive; of the other 18, 16 were positive for salmonella. The number of somatic serogroups per sample ranged from 1 to 11 with a mean of 3.8. Significantly more ( P < 0.01) group C 1 salmonellae were isolated using tetrathionate than selenite, whereas significantly more of groups G, 35, and Difco poly-valent D were isolated from selenite than tetrathionate. Seventy-six percent of the presumptive colonies from Brilliant Green agar showed a positive lysine decarboxylase reaction, and there were no differences between media or times of incubation. Ninety-four per cent of the lysine decarboxylase-positive cultures showed a positive somatic antiserum response; again there were no differences between times or enrichments although there were significantly more total positive serogroups at 2 days than at 1 day from tetrathionate but not from selenite. There were indications that certain serogroups preferred either one or the other enrichment. There were no differences in total positive samples with the two enrichments although neither alone was sufficient to identify all positives. Several lactose-positive salmonellae were recovered.

Inhibition of Clostridium botulinum by nitrite was potentiated by the addition of Fe(II) or Fe(III) to the culture medium. The effect of iron was more pronounced when nitrite was added after autoclaving.

Bacon was obtained from production lines of two local processing plants, H and M. The slices from 8 bellies from each plant were sequentially rearranged to form composite portions representative of each belly; these were analyzed for moisture-phase NaCl and for acceptability by a panel of judges using a 9-point hedonic scale. Samples were tasted shortly after processing and again after 6 weeks of storage at 4 or -23°C. The moisture-phase NaCl content ranged from 4.62 to 7.80% (c.v. = 18.2%) for bacon from plant H; the range for bacon from plant M was 3.25 to 10.05% (c.v. = 37.7%). The belly from plant M with the highest moisture-phase NaCl content also gave the lowest hedonic score of the bacon samples tasted before storage. The average hedonic scores for bacon from the two plants were different (p

Alcoholic extracts of spices were prepared and tested for inhibition of Clostridium botulinum in culture media. Mace (the outer covering of the seed of Myristica fragrans ) and achiote (annato, Bixa orellana ) were the most inhibitory of 33 spices studied. Also quite active were bay leaf ( Laurus nobilis ), white and black pepper ( Piper nigrum ) and nutmeg (the seed of M. fragrans ). Less active were rosemary ( Rosmarinus officinalis ), clove ( Eugenia caryophyllata ), oregano ( Oreganum vulgare ), turmeric ( Circuma longa ), thyme ( Thymus vulgaris ), and paprika ( Capsicum annuum ). Of the series C1 to C18, aliphatic straight chain alcohols of C14 or C16 chain-lengths were the most inhibitory against C. botulinum with a minimum inhibitory concentration (MIC) of 0.6 ppm. A plot of alcohol chain length versus MIC showed a highly significant (P < 0.01) cubic function.

The stimulation by yeast extract of acid production in milk by various lactobacilli was studied. It was found that supplementing milk with purine and pyrimidine bases and amino acids allowed nearly maximal acid production by Lactobacillus bulgaricus strain 7994, L. acidophilus 4796, 4356, and 4357, and L. leichmannii 326 and 327. Further supplementation with deoxyribotides allowed maximal acid production by L. acidophilus 204, but L. acidophilus 207 required adenosine or adenylic acid. L. casei strain 7469 showed no appreciable response to the amino acids or purine and pyrimidine bases, and is presumed to require an unidentified factor in corn steep liquor.

Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9- to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains.

Lactulose syrups were similar to sucrose syrups in water activity-lowering effects but were more inhibitory toward test microorganisms. Heat-treated commercial lactulose syrups were most inhibitory, whereas non-heat-treated pure lactulose was only slightly more inhibitory than sucrose.

Twelve straight-chain esters, C 5 to C 14 , C 16 , and C 18 , of p -hydroxybenzoic acid were prepared, and their melting points, solubilities in water at 25°C, infrared spectra, dissociation constants ( pK a ), and activities against Clostridium botulinum were determined. These studies also included four commercial straight-chain esters, C 1 to C 4 . The most potent activity was exhibited by undecyl and dodecyl esters, which are about 300 times as active as sodium nitrite. Quadratic and cubic equations were developed correlating the activity with pK a values and chain length of the esters, respectively.

A number of 5-nitrothiazoles with various substituents in the 2-position were tested for inhibition of Clostridium botulinum in a culture medium. Thiazole itself or 2-bromo- or 2-methylthiazole at 30 μg/ml did not inhibit the organism. An amino group in the 2-position of thiazole inhibited at 10 μg/ml. Substitution of a nitro group in the 5-position of 2-aminothiazole increased the inhibitory level to 0.12 μg/ml; acetyl-, propionyl-, or butyroyl-2-amino-5-nitrothiazole inhibited at 0.04 μg/ml. Benzoyl-2-amino-5-nitrothiazole inhibited at 0.16 μg/ml; this increased to 0.01 μg/ml when the benzoyl group carried a nitro group in the m - or p -position; a nitro group in the o -position, on the other hand, inhibited at 0.04 μg/ml. Unsaturated aliphatic acyls decreased inhibition. The greatest activity was exhibited by 2-nonanoyl- and 2-lauroylamides, with minimum inhibitory concentrations of 0.005 and 0.0025 μg/ml, respectively.

MANY workers have attempted to find a correlation between the antibiotic growth effect and the presence or absence of certain bacterial types in the intestinal tract. The microorganism most often implicated has been Clostridium welchii. Thus, Sieburth et al. (1951) found C. welchii in the cecal contents of turkey poults on normal rations. These were virtually eliminated by feeding 15 p.p.m. oxytetracycline. Lev et al. (1957) found C. welchii in the ceca of birds in infected, but not in uninfected quarters. Lev and Forbes (1959) reported that C. welchii depressed growth of germ-free birds. This depression was reversed when penicillin was fed. Other organisms have also been reported to decrease in numbers when antibiotics were fed. Dixon and Thayer (1951), Anderson et al. (1952), and Sieburth et al. (1954), reported decreases in numbers of enterococci in birds fed antibiotics. Slinger et al. (1954) associated the presence of enterococci in the…

SummaryPantethine and enzyme-hydrolyzed casein were found to supplant crude natural materials as sources of a presumed unidentified growth factor for several rumen bacteria and for avian Lactobacillus bifidus.

Microfermentation tests for members of the family Enterobacteriaceae were devised by using agar solutions in disposable, multi-welled, plastic trays. The tests could be made directly from isolated colonies picked from agar plates and represented a considerable saving in time, labor, and materials over the conventional methods. Tests were formulated for determining carbohydrate fermentations, citrate utilization, motility, amino acid decarboxylation, and production of H 2 S, indole, urease, and acetyl-methyl-carbinol.

THE bacterial flora of the chick’s intestinal tract has been studied by many workers. Most of these studies have been concentrated on the cecal flora of 8–12 week old birds. An entirely different aspect of the problem was presented by the investigations of Eyssen and DeSomer (1963). They showed that the most significant changes in young chicks occurred around the sixth day of life and were manifest by an inexplicable decrease in rate of gain accompanied by a decreased intestinal absorption of fats and carbohydrates. Addition to the diet of 20 p.p.m. virginiamycin increased the rate of gain and prevented the malabsorption of fats and carbohydrates. Pensack (1963) verified the results of Eyssen and DeSomer (1963) and showed that other antibiotics, such as chlortetracycline and penicillin, were effective in overcoming the malabsorption syndrome. This investigation was conducted to study the microflora of young chicks undergoing this malabsorption syndrome and…

The minimum pH for growth (MPG) in selenite-cystine enrichment medium was determined for a number of acids. The MPG for lactic, citric, hydrochloric, aspartic, malic, succinic, pyruvic, and tartaric acids was 5.80; acetic acid was considerably more inhibitory, giving a MPG of 6.30-6.40; the MPG's for fumaric and pyruvic acids were 6.00 and 5.70, respectively. The least inhibitory acid was α-ketoglutaric acid which gave a MPG lower than or equal to 5.1. Inoculum size had little or no effect on the MPG.

Mono- and dimethyl and ethyl esters of fumaric acid were evaluated for their antibotulinal efficacy in cans of comminuted nitrite-free bacon. At 0.125%, all were equal or superior to 120 ppm nitrite in preventing toxin formation in cans incubated at 30°C for 8 wk. No cans swelled or became toxic when mono- or dimethyl fumarate was added. With monoethyl fumarate, two cans out of twenty swelled but were nontoxic. The least effective ester was diethyl fumarate but its activity was equal to that of 120 ppm nitrite.

N-substituted aminodiamides of the general structure, RNHCO (CH2)x -CONH(CH2)yN(CH3)2, and the corresponding N-sulfopropyl derivatives were studied for their inhibiting effect on growth ofClostridium botulinum in culture media. With R = C16H33, more activity was observed with x = 2, 3, or 4 and y = 2, 3, or 4 than with R = C12H25 or C10H21 . There was little or no correlation of activity with the length of the x or y chains, but the addition of the sulfopropyl group resulted in decreased inhibitory activity. The C3 to C18 saturated aliphatic amines used for synthesizing the aminodiamides were effective inhibitors ofC. botulinum; tetradecyl-, pentadecyl-, and hexadecylamines were the most active with minimum inhibitory concentrations (MIC) of 0.8 μ/ml-The ratio of molecular weights of the diamides to the corresponding aliphatic amines indicated that most if not all of the inhibitory activity of the aminodiamides could be attributed to the amine moiety.

A rapid and sensitive assay for Perigo factor was developed using a medium of 0.5% yeast extract and tryptone, 0.2% glucose, 0.12% K2HPO4 and 0.1% cysteine HCI or sodium thioglycollate and vegetative cells of Clostridium botulinum type A. Yeast extract or tryptone, together with a reducing agent (cysteine, sodium thioglycollate, or glucose autoclaved with the medium), produced a Perigo inhibitor when autoclaved at 15 psi for 15 min with NaNO2. Tryptone was more active than yeast extract as a source of the Perigo inhibitor; of the reducing agents tested cysteine was more effective in producing Perigo-type inhibition than thioglycollate and either was better than glucose autoclaved with the medium.

The antifungal property of the gas phases of a number of alkenoic and alkynoic acids and esters was investigated by placing the compounds in desiccators containing suspended bread. The following compounds inhibited mold development at a concentration of 20 mg per 2.6-liter desiccator: dimethyl fumarate, diethyl fumarate, propenoic acid, methyl and ethyl propenoate, methyl and ethyl propiolate, methyl-2,4-hexadienoate, 2,4- hexadien-1-ol, diethylethylidene malonate, 4-penten-1-ol, and 2-cyclohexen-1-one. Other compounds were active at 200 mg per desiccator: 3-methallyl alcohol; allyl acetone; diallyl ether; propiolic acid; methyl, ethyl, and vinyl crotonate; ethyl-2,4-hexadienoate; methyl and ethyl-2-hexenoate; ethyl-3-hexenoate; 1,5-hexadien-3-01; methylmaleic acid; methylmalonic acid; and 1-penten-3-ol.