Tryptose Sulfite Cycloserine Agar Research Articles

Evaluation of Three Different Selective Media for Enumeration of Clostridium perfringens in Untreated and Treated Wastewater.

Current and emerging legislation in North Carolina and other regions calls for the enumeration of Clostridium perfringens as a surrogate indicator for protozoan parasites in various types of waters. Past studies that have evaluated selective media for the detection of this bacterium have provided limited, conflicting, and inconclusive results. In this study, membrane filtration was used to enumerate C. perfringens as culturable spores or total culturable cells in 19 samples of untreated and 25 samples of partially treated wastewaters on 3 candidate media, Tryptose Sulfite Cycloserine Agar (TSC), CP ChromoSelect Agar (CPCS), and membrane Clostridium perfringens Agar (m-CP) in parallel, and the results were compared. Presumptive isolates from each agar were further subjected to phenotypic confirmation tests for acid phosphatase production and stormy fermentation to further determine the performance of each agar. The CPCS agar was determined to have the highest enumerative capacity of total C. perfringens cells when compared to both TSC agar and m-CP agar (p-value < 0.05), but there was no significant difference in its ability to detect spores when compared to TSC agar (p-value > 0.05). The overall specificity of CPCS agar as determined by agreement of results from both confirmation tests was 0.81, while the specificity of TSC agar was only 0.28. Based on its performance, ease of preparation and use and consistency of colony characteristics, CPCS agar is recommended as the preferred medium for C. perfringens enumeration in wastewater.

Microbiological Studies on Clostridium Perfringens Type (A)

Background:Clostridium perfringens, a rapid-growing pathogen known to secrete an arsenal of >20 virulent toxins, has been associated with intestinal diseases in both animals and humans throughout the past century. Gas gangrene is synonymous with myonecrosis and is a highly lethal infection of deep soft tissue, caused by Clostridium species, with Clostridium perfringens being the most common. Clostridium perfringens is one of the most common cause’s gas gangrene that is associated with broadspectrum antibiotic treatment. The objectives of the present article were focusing on the diagnosis of Clostridium perfringens from wound swabs from patients suffering from gas gangrene associated with antibiotics treatment and determining the best antibiotics that can inhibit the growth of Clostridium perfringens.Materials and methods: Four hundred isolates were collected from Fever Abbassia hospital in Cairo, Egypt. Clinical samples were collected from swab wounds of limbs. Samples were taken during the period of the first of Oct 2017 till the end of Oct 2018. From four hundred cases collected, Two hundred twenty-eight identified as gas gangrene (57%), One hundred twenty-eightidentified as Clostridium perfringens type(A) (56.1%) confirmed by biochemical tests (Gram stain, Catalase test, Motility test, Skim milk test, Urease test, Indole test, Nagler reaction.) and Multiplex PCR. In this study, we are used different media such as (Thioglycalate broth, Reinforced Clostridial Agar, Blood agar, TryptoseSulfiteCycloserine Agar, Robertson's cooked meat media). The antibiotic sensitivity against CL.perfringens is assayed by two methods, disc diffusion Method and Determination of (MIC) using E-Test by following the reference standards established by the Clinical and Laboratory Standards Institute ( CLSI) and British Society for Antimicrobial Chemotherapy (BSAC). The antimicrobial susceptibility test was performed for all of the isolates (128) by disc diffusion method using antibiotics (Chloramphenicol (30 μg /disc), Cefoxitin (10 μg /disc), Clindamycin (2 μg /disc), Metrondiazole (5 μg /disc), Tetracyclin (30 μg /disc), Rifampin (5 μg /disc), Penicillin (10 μg /disc) and Piperacillin (30 μg /disc) and by E- test (MIC) (0.016- 256µg/1ml) method using antibiotics (Chloramphenicol, Cefoxitin, Clindamycin, Penicillin G, Tetracycline and Piperacillin) on blood agar and incubate anaerobically in Anaerobic jar for 24 hrs. at 37 ˚C. As Rifampin and Metrondiazole breakpoints (E-Test) for Clostridium perfringenhave not yet been established by either EUCAST or CLSI.Results and conclusion: In this study, isolates Clostridium perfringens from selective media TSC Agar or Blood agar and doing biochemical tests, the antibiotics susceptibility test showing that disc diffusion method and MIC method give the same result. Susceptibility tests were done on (128) isolates of CL.perfringens by E-Test to determine MIC.Our results revealed that the percentage of sensitivity of CL.perfringens to( Chloramphenicol , Cefoxitin, Clindamycin, Rifampin, Penicillin G, Metrondiazole and Tetracyclin) are (53.9% , 53.1% , 52.3 % , 41.4 % , 35.9 % , 21.1 % , 14.1 % ) respectively . On the other hand, the data revealed that CL.perfringens was resistant to PRL ( 100%). Future advanced research should participate in the prevention of C. perfringens gas gangrene outbreaks

Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens)

The main use of black soldier fly larvae (Hermetia illucens) is currently as an animal feed ingredient. While the bacterial community of the larvae has been characterised repeatedly via sequencing, microbiological safety assessment based on culture-dependent techniques is still scarce. This study focused on the occurrence of the spore-forming foodborne pathogen Clostridium perfringens during rearing and consecutive processing of the larvae, based on observations in a single rearing facility. C. perfringens vegetative cells and spores were determined, in addition to total viable counts, total aerobic spore counts and intrinsic parameters including pH, water activity and moisture content. All samples were obtained from an industrial production plant. In a preliminary experiment, substrate ingredients and dried larvae were analysed, but the larvae were produced with a previous batch of the substrate mixture. A second, more detailed, experiment was performed where all samples were collected sequentially from the same production run (substrate ingredients, substrate mixture, starting larvae, harvested larvae, residue, dried larvae and stored dried larvae). In the two experiments, (presumptive) C. perfringens, as determined on tryptose sulphite cycloserine agar, was found at low numbers in the ingredients and in the second experiment it was also found in the substrate mixture. Over the two experiments, total C. perfringens counts (i.e. vegetative cells plus spores) ranged between 3.0±0.1 and &lt;1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and &lt;1.0±0.0 log cfu/g. Interestingly, vegetative cells and spores of C. perfringens were below the detection limit in all larvae samples. Therefore, it appears that at this production site and based on the samples investigated, the pathogen did not colonise the larvae. However, these results indicate that insect producers should monitor this pathogen among others, and install good hygiene practices to avoid contamination.

Incidence and characterization of Clostridium perfringens isolates from milk and milk products

The present study was conducted to assess the incidence of Clostridium perfringens and to characterize the isolates from milk & milk products. The study included 250 samples of milk and milk products consisting 50 each of raw milk, pasteurized milk, shrikhand, khoa and lassi. The samples were overnight enriched in alternative thioglycollate broth followed by isolation on tryptose sulphite cycloserine agar. Out of 250 samples, 8.4% (21/250) had C. perfringens. The occurrence in raw milk was 16% (8/50), khoa- 12% (6/50), shrikhand-10% (5/50), lassi- 4% (2/50). Pasteurized milk samples were not positive for C. perfringens. All the isolates of C. perfringens were confirmed with 16S rRNA genes. The molecular study revealed that all the isolates had lecithinase (cpa) gene, while 5 isolates (raw milk-3, shrikhand2) showed the enterotoxin (cpe) gene. The presence of C. perfringens in milk and milk products revealed unhygienic and insanitary (merge in with sanitary) conditions which lead to contamination during production and processing of milk and milk products.

A comparative study on the use of selective media for the enumeration of Clostridium perfringens in poultry faeces

Isolation and fast detection of Clostridium perfringens is essential in veterinary medical diagnostics and veterinary research, as it allows to recommend suitable treatment options after antimicrobial resistance determination, and is essential to study pathogenesis. In this study four selective media were tested for the enumeration of, and selectivity towards C. perfringens in faecal samples from poultry. The routinely used Columbia agar with 5% sheep (CBA), Shahidi-Ferguson-perfringens agar (SFP), tryptose sulphite cycloserine agar (TSC), and a novel chromogenic medium, CHROMagar™ C. perfringens (CHCP), were tested. Overall, no difference in C. perfringens recovery could be observed between the selective media. The limit of quantification was 103 CFU/mL for all agars. CHCP showed the highest specificity, especially when low C. perfringens loads were present in the faeces, with TSC being the second most specific selective medium. Both CBA and SFP allowed considerable growth of other faecal microbiota and were not specific for C. perfringens. On CHCP, differentiation of C. perfringens from other faecal bacteria was straightforward due to the appearance of C. perfringens as orange colonies, with other bacteria being absent or appearing as blue/green colonies. On TSC, C. perfringens appeared as black colonies, but longer incubation periods were sometimes needed for the black colour to develop. Therefore, CHCP can be recommended when timely and easy identification and enumeration of C. perfringens from complex samples, such as faeces, is needed.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples.

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 °C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a "sandwiched", anaerobic environment. Agar plates are incubated overnight at 37 °C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 °C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15-20% of samples test positive for C. perfringens.

Effect of grape seed extract on heat resistance of Clostridium perfringens vegetative cells in sous vide processed ground beef

The heat resistance (57.5–65 °C) of a three-strain cocktail of Clostridium perfringens vegetative cells in sous vide processed ground beef supplemented with 0–3% grape seed extract (GSE) was quantified. The surviving cell population was enumerated on tryptose–sulfite–cycloserine agar. The decimal reduction (D)-values in beef that included no GSE were 67.11, 17.15, 4.02, and 1.62 min at 57.5, 60, 62.5, and 65 °C, respectively. Addition of 1.0% GSE resulted in concomitant decrease in heat resistance as evidenced by reduced bacterial D-values. The D-values in beef with added 1.0% GSE were 62.89, 13.70, 3.47 and 1.46 min at 57.5, 60, 62.5, and 65 °C, respectively. The heat resistance was further decreased when the GSE concentration in beef was increased to 2 or 3%. The z-values in beef with or without GSE were similar, ranging from 4.41 to 4.56 °C. The results of this study would be beneficial to the retail and institutional food service establishments in estimating re-heating time and temperature for sous vide processed ground beef to ensure safety against C. perfringens.

CLOSTRIDIUM PERFRINGENS IN DUCK FARMS, RETAIL OUTLETS AND IN CONTACT WORKERS IN EGYPT INCIDENCE AND TOXIN GENOTYPING

This study investigated incidence and toxin genotyping of Clostridium perfringens isolated from ducks and farm workers. Caecal contents of 125 ducks including apparently healthy (n=100) and diseased (n=25) ducks; and stool specimens of farm workers (n=25) were randomly collected from duck farms at Sharkia governorate, Egypt. Also, caecal contents (n=25) and meat (n=25) of ducks were collected from retail outlets at Sharkia governorate, Egypt. Samples were cultured on tryptose sulfite cycloserine agar supplied with Perfringens selective supplement then isolates were biochemically identified. Toxin genotyping of 17 C. perfringensisolates was performed by multiplex PCR. In duck farms, C. perfringens was isolated from 80% of healthy ducks, 84% of diseased one and 80% of in contact workers. In retail outlets, caecal contents and meat of ducks showed infection rates of 72% and 4%, respectively. The frequency distribution of cpa gene infarms was 80% in healthy ducks, 100% in diseased birds and 66.6% in apparently healthy workers. In retail outlets, cpa had frequency distribution of 66.6% in caecal contents and 100% in duck meat. Therefore, presence of C. perfringens type A and its associated cpa gene in caecal contents and meat of ducks confirmed its zoonotic hazard and public health implications.

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

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. 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. OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. 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.

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water

To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

The effect of grapefruit extract and temperature abuse on growth of Clostridium perfringens from spore inocula in marinated, sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag, marinated chicken breast that included additional 1.0% NaCl. The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 19 °C, C. perfringens levels were consistently about 2.5 log 10 CFU/g until 9.5 h regardless of the presence or absence of Citricidal®. However, storage of the unsupplemented marinated chicken samples and those with 50 or 100 ppm Citricidal® samples at 25 °C for more than 6 h resulted in rapid growth of C. perfringens, exhibiting 2–3 log 10 CFU/g increase at 7 h. Citricidal® at 200 ppm significantly ( p < 0.05) reduced the growth of C. perfringens at both 19 and 25 °C. The D-values obtained at 90 °C were significantly decreased ( p < 0.05) from 14.07 (no Citricidal®) to 9.20 min (200 ppm Citricidal®). Supplementing marinated chicken products with Citricidal® and the temperature abuses had no consistent effect on color, shear force or lipid oxidation. However, the organism may grow to unsafe levels if sous-vide products are poorly handled or temperature abused for a relatively long period. An extra degree of safety may be assured in such products by supplementation with 100 or 200 ppm Citricidal®. Industrial relevance: Since temperature abuse is a common occurrence during trasportation, distribution, storage or handling in grocery stores or by consumers, an extra degree of safety may be assured in marinated chicken products by supplementation 100–200 ppm Citricidal®. In such products, the temperature abuse should not have any consistent effect on color, shear force or lipid oxidation of products.

Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag marinated chicken breast that included 0%, 1.5%, 3%, or 4.8% sodium lactate (NaL; w/w). The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at 4, 19, and 25 °C. The total C. perfringens population was determined by plating diluted samples on Tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 25 °C, addition of 1.5% NaL was effective in delaying growth for 29 h. Increasing the NaL level to 4.8%, C. perfringens growth from a spore inoculum during storage at 25 °C for 480 h was not observed. At 19 °C, the growth was >6 log 10 cfu/g by 288 h in control samples. In samples with 3.0% or 4.8% NaL, the growth of C. perfringens from spores was dramatically restricted with little or no growth in 648 h at 19 °C. C. perfringens growth was not observed at 4 °C regardless of NaL concentration. The D-values at 55 °C ranged from 47.40 (no NaL) to 57.58 min (1.5% NaL). Cyclic and static temperature abuse of refrigerated products for ⩽20 h did not permit C. perfringens growth. However, temperature abuse of products for periods 24 h or longer in the absence of NaL led to growth of C. perfringens from a spore inoculum. An extra degree of safety may be assured in such products by supplementation with NaL at 1.5–4.8% NaL level.

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes ( cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2×10 4 colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens, but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.

Aerobic Incubation of Clostridium perfringens in the Presence of an Oxygen‐Reducing Membrane Fraction

ABSTRACTThe recovery and growth rates of C. perfringens were evaluated in media to which an oxygen‐reducing membrane fraction from E. coli was added. Each of three C. perfringens strains was incubated in Tryptose Sulfite Cycloserine agar, with and without the membrane fraction. Recoveries were greater on media incubated aerobically with the membrane fraction than on the other formulations incubated an‐aerobically or aerobically. C. perfringens ATCC 12917 reached stationary phase more rapidly in fluid thioglycollate medium (FTM) plus the membrane fraction than in FTM alone. Results showed that the membrane fraction allowed aerobic incubation for the recovery of C. perfringens.

Membrane filtration enumeration of faecal clostridia and clostridium perfringens in water

The use of egg yolk-free tryptose-sulphite-cycloserine (TSC) agar for the membrane filtration enumeration of Clostridium perfringens was evaluated using domestic sewage effluents and polluted surface waters. The medium recovered higher numbers of C. perfringens than a medium containing polymixin (mCP). Overlaying the membrane with TSC or cycloserine-free TSC did not improve recoveries. Supplementation of the medium with 50 mg l −1 neomycin sulphate resulted in significantly lower recoveries of C. perfringens. Comparison of two commercial formulations of TSC showed no difference in recoveries. A two tube confirmation procedure based on motility, nitrate reduction, lactose fermentation and gelatin liquefaction was found to be reliable to confirm C. perfringens from TSC plates. Although not as specific as mCP for the enumeration of C. perfringens TSC aappeared to be selective for sulphite reducing clostridia of predominantly faecal or urino-genital origin. A concept of faecal clostridia and C. perfringens (based on enumeration on TSC agar) analogous to that of faecal coliforms and E. coli is briefly discussed.

Enumeration and Characterization of Clostridium perfringens Spores in the Feces of Food Poisoning Patients and Normal Controls

The fecal spore enumeration method for confirming Clostridium perfringens as the cause of food poisoning was evaluated using strains implicated in nine outbreaks in the United States. Confirmed spore counts from 66 stool specimens were made on tryptose-sulfite-cycloserine (TSC) without egg yolk and trypticase soy-sheep blood (TSB) agars. Counts from outbreak stools on TSC agar ranged from 2.0 × 104 to 3.5 × 108 (mean ≥ 1.4 × 106/g) as compared with <103 to 5.0 × 105/g (overall mean 9.5 × 103/g) from normal stools. Similar results were obtained with TSB agar. Isolates from seven of the nine outbreaks were nonhemolytic and produced ≥100 ng enterotoxin/ml in spore broth, as measured by an enzyme-linked immunosorbent assay. Spores in stools from six of the outbreaks were heat-resistant and survived heating for 30 to 60 min at 100°C in cooked meat medium. Strains from the three remaining outbreaks were heat-sensitive and survived heating for only 15 min at 100°C. Enterotoxigenic isolates from all but one of the outbreaks were serotyped. In all instances, the predominant strain in specimens from an outbreak was of the same serotype, indicating that it was the causative strain. Reexamination of five specimens from each of three outbreaks after storage at −20°C for 6 months showed only a minimal reduction in the spore counts.

Rapid Detection of Clostridium perfringens: Comparison of Lactose Sulfite Broth with Tryptose- Sulfite-Cycloserine Agar

Abstract The lactose sulfite (LS) medium recommended for the detection and identification of Clostridium perfringens in foods was compared with a reference method using tryptose-sulfite-cycloserine (TSC) agar for the enumeration of this organism in a variety of foods and food ingredients. C. perfringens was detected and enumerated in 17 of the 54 samples examined with LS broth, but its presence could be confirmed in only 9 of the samples with TSC agar. In only 2 instances, C. perfringens was detected on TSC agar but not in LS broth. A positive response (FeS + and gas +) in LS broth incubated at 46°C always corresponded to the presence of C. perfringens; whereas the black colonies formed on TSC agar incubated at 37°C were frequently found to be Clostridium species other than C. perfringens. Thus, because of its highly selective nature, LS broth was superior to TSC agar for enumerating and confirming the small numbers of C. perfringens that were present in a majority of the samples. This was especially true when other Clostridia were also present. Besides its greater selectivity and sensitivity, LS broth had the additional advantages of requiring less work and giving confirmed results within 24-48 h compared with 3 days for the TSC agar method

Rapid Detection of Clostridium perfringens: Comparison of Lactose Sulfite Broth with Tryptose-Sulfite-Cycloserine Agar

The lactose sulfite (LS) medium recommended for the detection and identification of Clostridium perfringens in foods was compared with a reference method using tryptose-sulfite-cycloserine (TSC) agar for the enumeration of this organism in a variety of foods and food ingredients. C. perfringens was detected and enumerated in 17 of the 54 samples examined with LS broth, but its presence could be confirmed in only 9 of the samples with TSC agar. In only 2 instances, C. perfringens was detected on TSC agar but not in LS broth. A positive response (FeS + and gas +) in LS broth incubated at 46 degrees C always corresponded to the presence of C. perfringens; whereas the black colonies formed on TSC agar incubated at 37 degrees C were frequently found to be Clostridium species other than C. perfringens. Thus, because of its highly selective nature, LS broth was superior to TSC agar for enumerating and confirming the small numbers of C. perfringens that were present in a majority of the samples. This was especially true when other clostridia were also present. Besides its greater selectivity and sensitivity, LS broth had the additional advantages of requiring less work and giving confirmed results within 24-48 h compared with 3 days for the TSC agar method.

Comparison of Methods for Isolation and Confirmation of Clostridium perfringens from Spices and Herbs

Rapid Perfringens Medium (RPM), Perfringens Enrichment Medium (PEM) and Tryptose Sulfite Cycloserine agar medium (TSC) were compared for determination of Clostridium perfringens in spices and herbs. Of 147 samples, 62 (42%) contained C. perfringens when RPM was used; lower percentages of positive isolations were found with PEM (23%), TSC surface plate (19%) and TSC pour plates (26%). Heat-treatment of sample suspensions yielded additional isolates. C. perfringens was isolated by one or more of the techniques from 43 (80%) of 54 different kinds of spices and herbs and from 86 (59%) of 147 samples. Replacing glucose in RPM with raffinose and polymyxin and neomycin with cycloserine did not improve the efficacy of this medium. A good correlation was found between conventional confirmation tests for C. perfringens and tests for acid phosphatase, lecithinase, reverse CAMP test and an antiserum test.