A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water

Two media (mCP medium and Tryptose Sulphite Cycloserine (TSC) agar) were evaluated for recovery of Clostridium perfringens in environmental and part-treated drinking water. For laboratory strains of Clostridium , mCP was more selective and specific for Cl. perfringens than TSC, but was markedly less efficient for the enumeration of both vegetative cells and spores. For samples of river water and part-treated drinking water, TSC recovered significantly greater numbers of Cl. perfringens than mCP. In contrast to previous reports, there was a significant number of false presumptive positive and negative isolates on mCP. TSC is a more suitable medium for the routine monitoring of water supplies for the presence of Cl. perfringens .     

Enumeration of Bacillus cereus in Foods

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.     

New Quantitative, Qualitative, and Confirmatory Media for Rapid Analysis of Food for Clostridium perfringens

A selective and differential medium, Shahidi-Ferguson Perfringens agar (SFP agar), and a confirmatory medium, lactose-motility agar (LM agar), were developed for the enumeration and identification of Clostridium perfringens in foods. These media provide a rapid, specific, and direct diagnosis of C. perfringens. SFP agar contains sodium metabisulfite and ferric ammonium citrate to demonstrate H(2)S production and egg yolk to demonstrate lecithinase production by C. perfringens. On SFP agar, C. perfringens produces black colonies, 2 to 3 mm in diameter, surrounded by zones of opaque precipitate. The typical colonies are confirmed on LM agar. Enumeration and identification are completed within 48 hr. All of the ingredients of SFP agar are stable to heat and storage conditions. SFP agar also contains two antibiotics, kanamycin and polymyxin B, which are inhibitory to many bacteria commonly occurring in foods. A comparative study of SFP agar and noninhibitory media showed that SFP agar did not inhibit any of the 16 strains of C. perfringens tested. Recovery of C. perfringens added to foods averaged 90.6% for SFP agar as compared with 69.8% for sulfite polymyxin-sulfadiazine (SPS) agar (BBL) and 60.2% for SPS agar (Difco). The colonies on the SFP agar, were much larger and were consistently black. Of 464 food samples tested, C. perfringens was found in 27 samples with SFP agar and in 5 samples with SPS agar (Difco), with a recovery ratio considerably higher on SFP agar. SFP agar is a more specific presumptive medium for the enumeration of C. perfringens and in conjunction with LM agar should save considerable time, effort, and materials toward the final identification of the species.     

Salt extends the upper temperature limit for growth of food-poisoning bacteria

Inclusion of NaCl into the growth medium raised the upper temperature limit of growth of the following organisms: Staphylococcus aureus (two strains), Salmonella senftenberg, S. typhimurium, Escherichia coli, Streptococcus faecalis, Bacillus cereus, Clostridium sporogenes, C. perfringens (two strains). The magnitude of the response varied with the culture, the largest being 3.5 degrees with B. cereus cells. The spores of B. cereus were not protected by salt but clostridial spores behaved as the vegetative cells (response of 2.5 degrees). The optimal salt concentration for the protective effect varied with the organism ranging from 0.2 M for the Gram-negative organisms to 1.0 M for S. aureus.     

Immunity to Aerosol Challenge in Guinea Pigs Immunized with Gamma-Irradiated Venezuelan Equine Encephalitis Vaccines

An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 mug of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.     

The Influence of High Concentrations of Carbon Dioxide on the Germination of Bacterial Spores

The influence of carbon dioxide at 1–55 atm on the germination of Clostridium sporogenes, Clostridium perfringens and Bacillus cereus spores in a complex medium was studied. The germination studies at atmospheric pressure were done in the pH range 5.2–6.7. Controls at the same pH were done in 100% nitrogen. Carbon dioxide at atmospheric pressure (1 atm) inhibited the spore germination of B. cereus spores but strongly enhanced the germination rate of those of the clostridia. Spore germination of Cl. sporogenes and Cl. perfringens was inhibited completely at 10 atm and at 25 atm, respectively. The germination rate in carbon dioxide or nitrogen was generally higher at pH 6.7 than at 5.2–6.0.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

Enumeration and Identification of Bacillus cereus in Foods: I. 24-Hour Presumptive Test Medium

An egg yolk-polymyxin medium (KG) for rapid enumeration of Bacillus cereus is described. The test is presumptive in that differentiation of B. cereus (and closely related organisms) from other species is based on the formation of turbidity in the agar surrounding the colonies of the cereus group organisms. The medium is formulated to encourage sporulation and release of free spores for serological confirmatory tests within the 24-hr incubation period. The production of turbidity in egg yolk and free-spore production by 25 strains of B. cereus on KG agar were measured. The recovery of food poisoning strains of B. cereus inoculated into nonsterile food slurries was assessed. A comparison of KG agar and mannitol-egg yolk-polymyxin-agar indicated that the two media were comparable in their abilities to recover low levels of B. cereus from naturally contaminated foods. Since KG agar enhances spore formation by B. cereus, thus permitting early serological testing, its use in screening food products is advocated.     

Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media

In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method. Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work. The C. perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media. No statistically significant differences were found between C. perfringens spore counts on TSCF compared with those of other methods used. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C. perfringens spore recovery in groundwater samples. Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C. perfringens spores from groundwater samples.     

Diagnostic properties of three conventional selective plating media for selection of <Emphasis Type="Italic">Bacillus cereus</Emphasis>, <Emphasis Type="Italic">B. thuringiensis</Emphasis> and <Emphasis Type="Italic">B. weihenstephanensis</Emphasis>

The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol–egg yolk–polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin–egg yolk–mannitol–bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Clostridium perfringens type A: certain characters of epidemiologic significance.

Survival of spores of Cl. perfringens type A was significantly greater than that of vegetative cells in acid pH (pH 1.2). Survival of spores in soil varied from strain to strain. Time required for 5 million spores to be reduced to 500 per gram of soil varied from 2 to 8 months with an average of 4.5+/-2.3 months. Quantitative and qualitative heat resistance studies revealed that a majority of the Indian and all the American strains tested were heat-sensitive. These characters of Cl. perfringens have an important bearing on the epidemiology of food poisoning due to this agent.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation.

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages

AIMS: Clostridium perfringens is recommended as a suitable indicator bacterium for human enteric viruses, Giardia cysts and Cryptosporidium oocysts in finished water and in the assessment and evaluation of water treatment. Several agars and confirmation procedures were evaluated in parallel with the Australian/New Zealand Standard (AS/NZ) Method for the enumeration of Cl. perfringens from treated and untreated sewage samples. METHODS AND RESULTS: The current AS/NZ method utilizes tryptose sulfite cycloserine agar (TSC), lactose gelatin medium (LG) and nitrate motility medium (NM) at an incubation temperature of 37 degrees C. Sixty treated and untreated sewage samples were used to evaluate TSC agar, membrane Cl. perfringens agar (mCP), Perfringens agar (OPSP) and Perfringens agar with 4-methylumbelliferyl phosphate (OPSP-MUP) for enumeration of Clostridium. An incubation temperature of 44 degrees C for 24 h was used for comparison. Confirmation procedures were also evaluated using 103 isolates and included LG and NM, ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) with MUP (ONPG-MUP) and phosphatase reagent (PR). OPSP compared favourably with TSC agar. One false negative result was obtained from each of the LG/NM and ONPG-MUP procedures. No false results were obtained using the PR confirmation procedure. CONCLUSIONS: OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a simple and rapid method for isolating and enumerating Cl. perfringens from sewage samples and confirmed results can be reported more quickly due to shorter analytical turnaround times.     

ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in feces.

As the second phase of an international comparative study for the enumeration of Clostridium perfringens, four methods were compared for "total" and spore counts of C. perfringens in fecal specimens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserine) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. In both the total and spore count procedures, the confirmed C. perfringens counts in method D were lower than in methods A, B, and C. Little differences among methods were found in the percentages of presumptive colonies confirmed as C. perfringens. The nonspecific counts in methods A and D were generally greater than in B and C, but nonspecific microorganisms did not interfere in the enumeration of C. perfringens spores by any of the four methods. In overall performance, methods B and C were superior to A and D. The mean C. perfringens spore count was only 0.17 log lower than the mean total count. Spore counts alone are, therefore, adequate in investigations of C. perfringens outbreaks.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.