Clostridium Botulinum in Honey, Syrups and Dry Infant Cereals.

A total of 150 honey, 43 syrup and 40 dry cereal samples were analyzed for Clostridium botulinum spores, each in triplicate quantities of 25 g. The foods were sampled randomly, except for two lots of honey which were potentially associated with illness. Botulinal spores were detected in a sample of honey associated with infant botulism and in a single sample of rice cereal.

Toxin Production by Clostridium botulinum and Organoleptic Changes in Vacuum-Packaged Raw Beef.

Vacuum-packaged beef, inoculated with Clostridium botulinum spores of types A and B and incubated at 25°C, was monitored for the production of toxin and for changes in appearance and odor. Toxin was detected first after 6 d of incubation and was always accompanied by significant organoleptic changes when compared to meat stored at 4°C. Uninoculated meat samples stored at 25°C remained non-toxic, whereas their sensory scores, in particular with respect to appearance, were similar to those of the inoculated samples.

A Modified Method for Ascertaining Water Activities Within Defined Limits.

The salt crystal method of Northolt and Heuvelman (J. Food Prot. 45:537-540, 1982) for testing the water activity of foods was modified to facilitate the recognition of crystal liquefaction. The proposed device is assembled from basic laboratory ware, i.e., an Erlenmeyer flask, a rubber stopper and a test tube.

Prevalence of Clostridium botulinum in Commercial Liver Sausage.

Samples of 75 g of commercial liver sausage were cultured, with and without prior heating, for the presence of viable Clostridium botulinum . Three of 276 heated cultures and 2 of 276 unheated cultures produced botulinal toxin, all of type A. The most probable number of botulinal spores was estimated at 0.15/kg. The estimate for "total" C. botulinum , based on 5 toxic cultures in 276 heated and unheated pairs, was 0.24/kg. The 99% confidence limits were 0.02 to 0.53 per kg and 0.05 to 0.69 per kg, respectively .

Contribution of Nitrite to the Control of Clostridium botulinum in Liver Sausage.

Liver sausage was formulated with different brine and nitrite concentrations, challenged with a mixture of spores of five strains each of Clostridium botulinum , types A and B at 10-fold increasing concentrations, temperature-abused at 27°C, and assayed for botulinal toxin after various periods. From the number of toxic sausages and initial spore concentrations, the probability (P) of a single spore to give rise to toxin within a given period of abuse was estimated. At moderate brine concentrations (3.8-4.2% salt), 50 or 100 ppm of nitrite had little or no effect on toxigenesis; the estimated P values for one week at 27°C were from 10-2 to 10-4 with 0 and 50 ppm of nitrite, and from 10-2 to 10-5 with 100 ppm. With 150 ppm, however, P was consistently <2 × 10-6 At a higher brine concentration, an appreciable delay in toxigenesis was also obtained with 50 or 100 ppm of nitrite. Randomly typed extracts from 44 toxic sausages all contained C. botulinum type A toxin only. At toxin levels ≥100 mouse MLD/g the sausages had a putrid odor, but sausages with less toxin often appeared organoleptically acceptable. Storage of sausages at 8°C for 6 weeks before incubation at 27°C resulted in nearly complete disappearance of detectable nitrite, but did not diminish the inhibitory effect of the initial nitrite. Increased processing temperature and/or prolonged time of processing reduced the inhibitory effect of nitrite.

Incidence of Clostridium botulinum in Commercial Bacon.

Of 416 75-g samples of commercial bacon cultured with or without prior heating. only one gave rise to formation of botulinal toxin. The most probable number of Clostridium botulinum was estimated at 0.064 per kg, with a 99% confidence limit of 0.0004 to 0.478 per kg.

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods.

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.

Effect of Salt Content and pH on Toxigenesis by Clostridium botulinum in Caviar.

Bottled lumpfish caviar was prepared with different salt (NaCl) concentrations and pH, and injected with spores of Clostridium botulinum . Under abusive storage conditions (30 C), outgrowth and toxigenesis occurred at combinations of ≤ 3.95% salt in the water phase and pH ≥ 5.2, and of ≤ 4.67% salt and pH ≥ 5.6. No toxin was formed at salt concentrations of ≥ 5.56% or at pH ≤ 5.0. A survey of commercial caviar products showed that most of these had salt-pH combinations which would effectively inhibit C. botulinum at abusive temperatures during storage.

Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure.

Colonies of Clostridium botulinum could be easily distinguished from meat particles by supplementing Wynne agar with 0.4% egg yolk. The pour-plate method was suitable for enumeration of C. botulinum, provided the medium was covered with a layer of agar containing 0.01% dithiothreitol. Viable counts of heat-treated spores were consistently higher in Wynne agar supplemented with egg yolk (Wynne-EY agar) than in Wynne agar alone.

Enumeration of food-borne Clostridium perfringens in egg yolk-free tryptose-sulfite-cycloserine agar.

The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes.

Enumeration of fecal Clostridium perfringens spores in egg yolk-free tryptose-sulfite-cycloserine agar.

The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin.

Evaluation and modifications of media for enumeration of Clostridium perfringens.

The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose.

Preparation of 14C-labeled tuberculin purified protein derivative.

(14)C-labeled tuberculin purified protein derivative ((14)C-tuberculin PPD) has been prepared from culture filtrates of Mycobacterium tuberculosis var. hominis grown in a culture medium containing uniformly labeled (14)C-amino acids. With a mixture of (14)C-amino acids (an acid hydrolysate of (14)C-Chlorella protein) in the medium, the recoveries of (14)C in the final product were higher than with (14)C-labeled-l-glutamic acid. (14)C-tuberculin PPD was separated into tuberculoprotein and nucleic acid by paper electrophoresis. The specific radioactivity of tuberculoprotein was substantially greater than that of the nucleic acid. (14)C-tuberculin PPD is advocated as a means to measure the adsorption of tuberculin to glass or other surfaces. It could also prove useful as a means to study the structure and mode of action of tuberculin.

Adsorption of tuberculin PPD to glass and plastic surfaces.

For some time it has been known that the adsorption of tuberculin to glass is a source of practical difficulties in tuberculin testing; for example, it leads to a loss of potency in diluted tuberculin PPD preparations used in the intracutaneous method of skin testing. The authors have correlated decreasing biological potency with decreasing radioactivity in solutions of tuberculin PPD labelled with (14)C.The decrease in radioactivity is due to adsorption of PPD-(14)C to the glass or plastic surface of containers; it can be prevented by the addition of 0.0005% Tween 80. The extent of the decrease is affected by the type and size of the containers, the volume of solution used and the storage temperature. It is the same in the presence of 0.3% phenol or 0.01% Chinosol used as preservatives. The concentration of Tween 80 does not affect the size of the tuberculin skin reactions in BCG-sensitized guinea-pigs.It is recommended that an anti-adsorption agent be added to all dilute solutions of tuberculin PPD; in solutions for intracutaneous use containing 50 TU per ml, Tween 80 at a concentration of 0.0005% is satisfactory.