Growth and toxin production by Clostridium botulinum on sliced raw potatoes in a modified atmosphere with and without sulfite.

The ability of Clostridium botulinum type A or B spores to grow and produce toxin on fresh raw potatoes in a modified atmosphere with or without sulfite was investigated at 22 degrees C. Fresh, peeled, sliced potatoes, untreated or dipped for 2 min into 0.7% sulfite solution and drained, were surface-inoculated at several concentration levels with a mixture of C. botulinum spores, either type A or B. They were placed in a modified atmosphere (30% N/70% CO2) within oxygen-impermeable bags (200 g/bag) and incubated at room temperature (22 degrees C). Toxicity was tested on days 0, 3, 4, 5, 6, and 7. After incubation, the potatoes were blended and centrifuged, and the Millipore-filtered supernatant fluid was injected intraperitoneally into mice. Sensory evaluation, except taste, was also performed. Potatoes inoculated with C. botulinum type A spores but untreated with NaHSO3 became toxic in 4 to 5 days, which coincided with the sensory evaluation "unfit for human consumption". Potatoes treated with NaHSO3 regardless of inoculum size or residual SO2 levels appeared acceptable for human consumption through day 7, even though they were toxic after 4 days of incubation. Although toxicity from type B spores occurred later and in fewer test samples than toxicity from type A, some potatoes again appeared acceptable but were toxic. Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C. botulinum under these conditions.

Shelf Life of Modified-Atmosphere-Packaged Fresh Tilapia Fillets Stored under Refrigeration and Temperature-Abuse Conditions.

We investigated the shelf life of fresh Tilapia spp. fillets packaged in high-barrier film under both 100% air and a modified atmosphere (MA) of 75% CO2:25% N2, and stored under refrigeration (4°C) and abuse temperatures (8 and 16°C). The chemical spoilage indicators trimethylamine, K-value, and surface pH, as well as microbial counts, were compared with the sensory characteristics of spoilage. For fillets packaged under 100% air, the shelf life was 9 to 13 days at a storage temperature of 4°C, but decreased to 3 to 6 days at 16°C. However, the shelf life of MA-packaged fillets stored at 4°C increased to >25 days when the lag phase and generation time of the bacteria were extended. MA packaged fillets stored under temperature-abuse conditions (8 and 16°C) had a shorter shelf life. The trimethylamine content associated with onset of sensory spoilage for MA packaged fillets increased as storage temperature increased and differed for each temperature. The surface pH and K-values of MA-packaged fillets were not good indicators of spoilage onset.

Lipid globule staining to aid in differentiating Bacillus species.

The use of the lipid globule stain to aid in differentiating the Bacillus cereus group (i.e., B. cereus, B. cereus var. mycoides, and B. thuringiensis) from other Bacillus species was investigated. Smears from colonies grown on suitable agar were made on precleaned slides, stained, and examined microscopically for characteristic deep blue lipid globules. The study included a total of 649 cultures of Bacillus species plus 143 incompletely characterized Bacillus isolates from food. Only B. cereus, B. cereus var. mycoides, B. thuringiensis, B. megaterium, and B. sphaericus were consistently positive for lipid globules, although at times, a few cells of B. aneurinolyticus and B. thiaminolyticus were also positive. The lipid globule stain procedure is of value in differentiating Bacillus species, especially when performed by an experienced analyst and used in conjunction with tests for cell and spore morphology.

Outgrowth of naturally occurring Clostridium botulinum in vacuum-packaged fresh fish.

A total of 1074 test samples of commercial, domestic, vacuum-packaged fresh fish were studied to determine whether spoilage occurs before the products become toxic from naturally occurring Clostridium botulinum spores. The products were incubated for 12 days at 12 degrees C (mild abuse). After incubation, they were tested for botulinal toxin and evaluated for organoleptic acceptability. Even when only marginally acceptable to laboratory personnel, none of the 1074 test samples were positive for C. botulinum toxin. Thus, the fish either contained no C. botulinum spores, or the spores were unable to grow out and produce toxin before spoilage made the product marginally unacceptable.

An international outbreak of type E botulism due to uneviscerated fish.

In October and November 1987, eight cases of type E botulism occurred in New York City and Israel. All eight patients had eaten uneviscerated, salted, air-dried whitefish known as kapchunka. Clostridium botulinum was isolated from samples of fish, and trypsinized portions of kapchunka contained type E toxin despite levels of salt that were far in excess of those considered adequate for safety. As C. botulinum has been found in the viscera of fish from the Great Lakes, possible explanations for the outbreak include multiplication of C. botulinum and production of toxin during shipping or during processing before the fish reached inhibitory salt levels. However, there was no evidence of mishandling of the fish. More likely, the viscera provided a relatively low salt "protective" environment for organism multiplication and toxin production. A major public health campaign was initiated and regulations were passed prohibiting the processing, distribution, and sale of raw, uneviscerated, salt-cured fish products within New York City.

Enumeration of Clostridium perfringens spores in human feces: comparison of four culture media.

Enumeration of Clostridium perfringens spores was compared using 4 culture media. Duplicate 1 g portions of 35 stools (25 from C. perfringens food poisoning outbreaks and 10 from normal stools) were heat treated 20 min at 75 degrees C and tested on tryptose-sulfite-cycloserine (TSC) agar, trypticase-soy-blood (TSB) agar, lactose-sulfite (LS) medium, and iron milk (IM) medium. Dilutions were plated directly onto TSB and TSC, and a 3-tube most probable number determination was made with each specimen in LS and IM incubated at 45 degrees C. TSB was easiest to use and nonhemolytic food poisoning strains were readily differentiated from the normal hemolytic biotype on this medium. Confirmed counts on TSC and TSB were similar for all specimens, but counts of 8 of 25 outbreak specimens were 2-4 log units lower in LS and IM than on plating media; spores in specimens associated with 2 of 5 outbreaks were intolerant of the elevated temperatures. Results showed that elevated temperature MPN methods in LS and IM are inappropriate for the examination of outbreak stools.

Comparison of Selective Plating Media for Enumeration of Bacillus cereus in Foods.

Enumeration of Bacillus cereus on raw sprouts of mung beans and wheat was compared in three agars: mannitol-egg yolk-polymyxin (MYP), polymyxin pyruvate-egg yolk-mannitol-bromthymol blue, and trypticase-soy-polymyxin blood. Ten different strains of B. cereus were used to seed the sprouts. Rates of recovery for the three media were not significantly different. However, with MYP agar, B. cereus could be differentiated more easily from other microorganisms and required fewer confirmatory tests.

Effect of Low Temperatures on Growth of Nonproteolytic Clostridium botulinum Types B and F and Proteolytic Type G in Crabmeat and Broth 1.

The ability of unheated and heated spores of nonproteolytic Clostridium botulinum types B and F, and of the weakly proteolytic type G, to grow and produce toxin in crabmeat and broth at low temperatures was investigated. Sterilized crabmeat or broth was inoculated with 103 spores/g or ml and incubated anaerobically at 4, 8, 12 and 26 C for 180 days. Both heated and unheated spores of all three types grew and produced toxin at 26 C in broth and crabmeat. Types B and F grew in broth at 12, 8 and 4 C when unheated but only at 12 and 8 when heated; they did not grow in crabmeat at any of these temperatures, heated or not. Heated and unheated type G grew at 12 C in both broth and crabmeat but not at lower temperatures.

Evaluation of the Botulism Hazard from Nitrogen-Packed Sandwiches.

Clostridium botulinum was inoculated into hamburger, sausage and turkey sandwiches, which were subsequently placed in a nitrogen atmosphere. Growth of the bacterium was studied to assess the botulism hazard. Hamburgers inoculated with C. botulinum types A and B and incubated at room temperature became toxic on day 4 while remaining fully acceptable organoleptically. Sausages became toxic on day 7 while appearing marginally acceptable. In air at room temperature, all sandwiches were obviously decomposed before toxin was produced. Refrigeration under nitrogen prevented toxin production by types A and B; however, hamburgers inoculated with type E were toxic at 12 C in 30 days while appearing fully acceptable. All refrigerated sandwiches were either fully or marginally acceptable organoleptically throughout the 60-day observation period; none were obviously decomposed. Turkey sandwiches did not become toxic at any temperature or incubation time studied.

Comparison of Stomacher and Waring Blendor for homogenizing foods to be examined for Clostridium perfringens.

The Colworth Stomacher Model 400 homogenizer was compared with the Waring Blendor for preparing food homogenates to be examined for Clostridium perfringens. Forty-eight samples representing 6 different food types were inoculated with C. perfringens and examined by the AOAC official first action method for enumeration of C. perfringens in foods. Identical paired specimens of each food type were blended with the 2 devices, and plate counts were made as specified in the official first action method. The effects of frozen storage on plate counts were determined by examining 24 food samples that had been stored for 3 days at -68 degrees C and homogenized both devices. Results of a statistical analysis of the experimental data indicated no significant difference overall (P greater than 0.05) in the plate counts of homogenates prepared with the Waring Blendor or the Stomacher 400, either before or after frozen storage of the food samples. However, the overall plate count average of the 48 samples was slightly higher with the Waring Blendor than with the Stomacher 400 homogenizer.

Toxin Production by Clostridium botulinum in Shelf-Stable Pasteurized Process Cheese Spreads.

Five non-refrigerated, pasteurized process cheese spreads, considered shelf-stable, were studied for their ability to support growth and toxin production by spores of Clostridium botulinum types A and B. Based on pH and water activity (aw) Cheese with Bacon, Limburger, Cheese Whiz, Old English, and Roka Blue cheese spreads were selected for the study. The pH ranged from 5.05 to 6.32 and the aw from 0.930 to 0.953. Fifty jars of each cheese spread were inoculated with 24,000 spores each; an additional 50 jars of the Cheese with Bacon spread received 460 spores each. The inoculum consisted of five type A and five type B strains in 0.1 ml of 0.85% NaCl. At 35 C, 46 jars of Limburger and 48 jars of Cheese with Bacon spread, which received the greater inoculum, became toxic starting at 83 and 50 days, respectively. One jar of Cheese with Bacon spread which received 460 spores became toxic. The average toxicity of the Limburger was 3000 MLD/ml of extract as compared with 54 MLD/ml for the Cheese with Bacon spread. Results of this study will be considered in determining whether these cheese spread products should be treated as low-acid canned foods under the Good Manufacturing Practice Regulations of the Food and Drug Administration.

Sporulation and Toxin Production by Clostridium botulinum Type G.

A comparative study was conducted to determine the optimum conditions for sporulation and toxin production by Clostridium botulinum type G, strain 89. One solid and four liquid media were compared for their ability to promote sporulation. After being inoculated, the media were incubated at 35 C for 12 days, at 30 C for 16 days, and at 26 C for 21 days. Spores were harvested by centrifugation, washed 3 times and resuspended to give a 35 × concentration, then counted by the MPN procedure. Spores grown on the solid medium at 35 C for 16 days gave higher counts than those grown at the same temperature in the liquid media. Toxin production was studied in eight media at 35, 30 and 26 C over a 24-day period with samplings every 2 to 3 days. Three of the media contained trypsin and five were trypsinized after growth. Toxin titers were determined by intraperitoneal injection of mice and expressed as MLD/ml of culture. Higher toxin titers were obtained at 26 and 30 C in media containing 0.4% glucose.

Media for Confirming Clostridium perfringens from Food and Feces.

Several media recommended for confirming isolates of Clostridium perfringens from selective plating media were evaluated. Media for testing motility, reduction of nitrate to nitrite, fermentation of lactose, and liquefaction of gelatin were found to be the most useful. A modified motility-nitrate medium, developed during the study, and lactose-gelatin medium were the most satisfactory for doing these tests. Fermentation of salicin and raffinose in peptone-yeast extract medium was also useful for differentiating atypically reacting strains of C. perfringens from a variety of culturally similar clostridia.

Evaluation of the Botulism Hazard in Fresh Mushrooms Wrapped in Commercial Polyvinylchloride Film.

The botulism hazard in fresh mushrooms wrapped in commercial polyvinylchloride (PVC) film appears to be minimal. At the end of their normal shelf-life, 1,078 packages of PVC-wrapped mushrooms were all free of botulinum toxin. Since inoculated mushrooms were occasionally found to be toxic (14 in 250 packages) when only one 1/8-inch hole was punched in the wrapper, and none became toxic when two holes were present, it seems prudent to recommend that PVC-wrapped tills of mushrooms have two holes in the wrapper.

Collaborative study of a method for the detection of Clostridium botulinum and its toxins in foods.

The mouse toxicity and protection technique for the detection and identification of Clostridium botulinum and its toxins in foods was collaboratively studied by 11 laboratories. Each laboratory received 4 samples of cream of mushroom soup; 2 contained spores and toxin of C. botulinum type A, 1 contained spores and toxin of C. botulinum type E, and 1 contained spores of C. sporogenes. The media used were cooked meat medium (beef heart or chopped liver broth) and trypticase peptone glucose yeast extract broth with trypsin. The results indicate that this method has a high degree of repeatability and reproducibility. All 11 laboratories correctly identified the toxins and the nontoxic sample in the food and detected and identified the viable spores in the samples by means of the subsequent cultures. This method has been adopted as official first action.

Recovery of clostridia on catalase-treated plating media.

Four plating media commonly used for culturing clostridia were tested for their ability to support growth of several Clostridium species after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar rapidly became incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens agar base and Brewer anaerobic agar were less affected. Plate counts of vegetative cells of nine of the less fastidious Clostridium species on untreated LV and BHI agars, stored for 3 days at 4 degrees C, were 60 to 90% lower than counts on catalase-treated media. Counts on Shahidi Ferguson perfringens agar base were only 1 to 24% lower on untreated medium with the same species. Addition of 500 U of purified beef liver catalase to the surface of the 3-day-old agars before inoculation resulted in substantial restoration of the ability of the media to support colony formation from vegetative cells except with the most strictly anaerobic species (nonproteolytic C. botulinum types B, E, and F, and C. novyii types A and B). A similar response was obtained with spores of the less fastidious species on catalase-treated media. Our results suggest that inhibition of most Clostridium species on LV and BHI agars may be due to accumulation of peroxide during preparation, storage, and incubation of the media, and also suggest that the presence of glucose in these media is a major factor contributing to their inability to support growth. It is believed that the addition of exogenous catalase prevents the accumulation of peroxide(s), thus allowing colony formation from vegetative cells of the clostridia under what would otherwise be unsuitable cultural conditions.

Type B botulism outbreak caused by a commercial food product. West Virginia and Pennsylvania, 1973.

In the week of May 7, 1973, seven persons contracted botulism after eating together. The most common symptoms were vomiting, constipation, dry mouth, dysphagia, and dysphonia. All were treated with trivalent botulinal antitoxin, and none died. Serum specimens obtained from all seven patients were negative for botulinal toxin, but stool specimens from three patients were positive for type B toxin. Electromyographic studies performed on five patients documented the neurophysiologic abnormalities of botulism. Commercially canned peppers in oil were implicated epidemiologically, and type B toxin was identified in leftover peppers. The processor voluntarily recalled the pepper product, and no further cases were reported.

Effect of Low Temperatures on Growth of Clostridium botulinum Spores in Meat of the Blue Crab.

The ability of unheated and heated spores of Clostridium botulinum types B. E, and F to grow and produce toxin in crabmeat from the blue crab at low temperatures was investigated. Sterilized crabmeat was seeded with 103 unheated spores/g or 104 heated spores/g and incubated anaerobically at 4, 8, 12, and 26 C. Broth cultures served as controls. Both unheated and heated spores of the three strains grew and produced toxin in crabmeat at 26 C in 3 and 6 days, respectively. In addition, unheated spores of the nonproteolytic type E strain grew and produced toxin in crabmeat at 12 C in 14 days. Neither heated spores of type E nor heated or unheated spores of types B and F grew in crabmeat at any refrigerated temperature within 180 days.

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.

Incidence of Clostridium botulinum in crabmeat from the blue crab.

The incidence of Clostridium botulinum in fresh crabmeat of blue crab was six out of 986 samples; in pasteurized crabmeat one sample out of 1,000 contained the organism.