Inhibition of Clostridium botulinum Toxin Formation by C. sporogenes in Culture Media and in a Meat System.

Vegetative cells and spores of types A and B Clostridium botulinum and C. sporogenes were coinoculated into culture media and into mechanically deboned chicken meat. Botulinal toxin formation was inhibited; the degree of inhibition depended on the relative concentrations of the two microorganisms. Inhibition of toxin formation by C. sporogenes also occurred in the meat system, but not all strains of C. botulinum were affected.

Gamma Radiation Resistance of Clostridium botulinum 62A and Bacillus subtilis Spores in Honey.

Irradiation D values for the natural bacterial flora of two samples of raw (bulk) honey were 7.50 and 1.91 kGy; for two samples of retail honey the D values were 5.66 and 3.49 kGy. Irradiation D values of Clostridium botulinum 62A spores inoculated into three honey samples and into water were respectively, 8.11, 9.38, 12.77, and 2.07 kGy. Similar D values for Bacillus subtilis spores were 3.42, 3.35, 4.00, and 1.43 kGy. The radiation resistance of C. botulinum and B. subtilis spores in honey and in sugar syrups was a function of water content.

Inhibition of Clostridium botulinum by Spice Extracts and Aliphatic Alcohols.

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.

Effect of Acids on Selenite Inhibition of Salmonella typhimurium and Salmonella dublin.

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.

Use of pH gradient plates for increasing the acid tolerance of salmonellae.

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.

Effect of low-frequency ultrasound and elevated temperatures on isolation of bacteria from raw milk.

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.

Gamma Radiation Inactivation of Enterococci.

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.

Effect of added iron on the formation of clostridial inhibitors.

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.

Effect of antifungal compounds on aspergillosis in hatching chick embryos.

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.

Effect of type of enrichment and duration of incubation on salmonella recovery from meat-and-bone meal.

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 p-hydroxybenzoic acid n-alkyl esters.

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 degrees 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 Procedure for the Direct Microscopic Count of Bacteria in Non-fat Dry Milk.

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.

Acceptability of Bacon Stored Six Weeks at 4 or -23°C.

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<0.05). There were no significant differences due to storage condition for bacon from plant M, but bacon stored at 4°C from plant H had lower (p<0.05) average scores than the bacon sampled shortly after processing or that kept 6 weeks at -23°. Storage at 4°C caused a significant (p<0.01) reduction in hedonic score in one belly from each plant.

Inhibition of bacteria by lactulose preparations.

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.

Inhibition of Clostridium botulinum in Comminuted Bacon by Short-Chain Alkynoic and Alkenoic Acids and Esters.

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.

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.

Microfermentation series for identification of single colonies of Enterobacteriaceae.

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.

Inhibition of Clostridium botulinum by 5-nitrothiazoles.

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 mug/ml did not inhibit the organism. An amino group in the 2-position of thiazole inhibited at 10 mug/ml. Substitution of a nitro group in the 5-position of 2-aminothiazole increased the inhibitory level to 0.12 mug/ml; acetyl-, propionyl-, or butyroyl-2-amino-5-nitrothiazole inhibited at 0.04 mug/ml. Benzoyl-2-amino-5-nitrothiazole inhibited at 0.16 mug/ml; this increased to 0.01 mug/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 mug/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 mug/ml, respectively.