Investigations described in this paper are related entirely to the recovery of viable spores of PA-3679 and Clostridium botulinum type B. Spores of the respective test organisms were subjected to thermal treatment and gamma radiation independently. Questionnaires were distributed in January, 1957, to 26 individuals at various laboratories whose personnel have been interested in spore recovery after exposure to either heat and/or radiation. Information relative to recovery media used in counting anaerobes was requested. The cooperation of the recipients was appreciated as it permitted the study of a rather broad spectrum of media.A widespread interest in counting anaerobic spores, particularly thermally treated spores, has been manifested since the early 1930's. At processing conferences held in conjunction with the annual convention of the National Canners Association, the subject has been regularly discussed. Periodic attempts have been made to procure uniform procedures for thermal death time studies, and spore counting has been one of the several facets.In a restricted National Canners Association Report (Z), data were presented based on the cooperative work of 7 laboratories. The type of media employed was only one of several variables investigated. The Institute of Food Technologists has for some years been interested in this phase along with other problems related to thermal death time work. I n 1952 the Western Utilization Research Branch, USDA, conducted a survey on spore counting media with Clostridiztin botuliizziiiz and the results were distributed in private communications.Frank and Campbell (1) reported that the recovery media employed in a study with PA-3679 had an effect on the thermal resistance values. Six media with modifications were studied.Replies to our questionnaire listed 20 different media. It was indicated that they are used under varying conditions, such as spore counts on processed and non-processed foods, as well as thermal death time work. Several were used for Most Probable Number determinations, others for colony counting, and others merely for the development of growth as indicated by gas formation with odor.

Determination of the radiation dose required to destroy spores of the toxin producing organism, Cl. botulinum, and produce a safe, sterile product is essential before irradiated foods can be introduced to consumers. The magnitude of cost and of such problems as changes in flavor, odor, texture, and nutrients, of course, depends very largely upon the radiation dose required for sterilization.Six-hundred cans each of peas, cream of chicken soup, and parboiled pork were inoculated with approximately 1,000,000 spores of CZ. botulinunz per can and subjected, in the frozen state, to various levels of gamma radiation from mixed fission products in spent fuel elements.Counts were made of surviving organisms in representative samplcs after irradiation and the remaining samples placed in incubation at 86" F. (30" C.) for one year.Counts of surviving spores, as well as tests for botulinum toxin, were made on flat cans surviving the year of incubation. E X P E R I M E N T A L P R O C E D U R EInoculum. Spore suspensions used throughout consisted of a mixture of 5 strains of C1. botzilinzm. Type A, and a mixture of 5 strains of Type B, all of which were grown separately on Wheaton's beef heart casein medium (7), concentrated, suspendcd in water, and heat shocked 10 minutes at 170" F. (77" C.) The 2 mixtures were provided by C. T.Townsend of the Western Laboratory of the National Canners Association. Dilutions of the original suspensions were made to permit inoculation of approximately 5,000 spores of cach type per gram of product.Peas for this pack, Perfection variety garden run, were blanched 3 minutes in boiling water, cooled, and frozen until ready for use. Sixty-one grams of peas were weighed into each 202 x 202 can and the mixed spore inoculum added by pipette. Cans were hrined with 36 ml. cold brine (1.4% sodium chloride, 4.8% sucrose) and close,d at 58-65°F. (14-18°C.) under 25 inches of mechanical vacuum.Chicken soup. Concentrated cream of chicken soup was removed from a commercial line without thermal processing and was returned frozen to Barrington for inoculation. Ninety-eight grams were filled into each 202 x 202 can and mixed spore inoculum added by pipette. Contents were stirred to insure distribution of spores, and the cans were closed at 5558°F. (13-14°C.) under 25 inches of mechanical vacuum.Peas.

SUMMARY Survival curves were obtained for massive concentrations of spores of Clostridium botulinum strain 12885A, when subjected to a wide range of gamma radiation doses under various conditions. The spore concentrations ranged from 37,000 to 64,800,000,000 per ampoule, with no recovery at 5 Mrad or more. Only at lower doses were observed survival counts compatible with classical hit theory. More than one hit is apparently required to inactivate the Cl. botulinurn spore by gamma radiation. The number of hits required for inactivation is estimated at somewhere near 13. Freeze‐dried spores exhibited less radioresistance in the dry state than when resuspended in neutral phosphate buffer. Spore suspensions in neutral phosphate buffer exhibited less radioresistance when irradiated under air than when irradiated under nitrogen. Spores exhibited less radioresistance suspended in phosphate buffer than in a nutrient medium (pork‐pea infusion). Changes in concentration of spores over a range of 37 × 103 to 53 × 109 did not significantly affect the percent survival for a given dose except in the tail area. Regardless of spore concentration, a so‐called “tailing off” of surviving spores was observed at the higher dose levels employed for varying spore concentrations. The “tailing off” phenomenon did not appear to follow the classical hit theory.

SUMMARY Subculture counts using 16 different media representing three general types (fresh‐meat infusion, formulated media, and dehydrated) were compared for the recovery of severely heated spores of two strains of PA3679. Comparative tests with these same media were made on the recovery of heated spores of PA3679, which had been produced in four sporogenic media. In general, the subculture media prepared from fresh‐meat infusions showed the highest recovery counts for all spore suspensions in all experiments. Dehydrated media showed the poorest recovery. The intermediate counts with the formulated media overlapped the counts with the other two groups of media. Pork‐pea infusion extract and beef infusion extract consistently yielded the highest counts and were appreciably higher than any of the dehydrated media. Differences were noted between the response of the two strains of PA3679 to the various subculture media. In addition, differences were noted between the responses to various subculture media on a single strain when the spores were grown on different substrates. Spores of a single strain of PA3679 grown in different substrates showed marked differences in heat resistance.

SUMMARY Spore recovery counts made on five strains of Clostridium botulinum indicated striking differences in radioresistance. Strain 12885A was most resistant, and strain 32B least resistant. A difference was noted in the survival of spores in five food products (green beans, chicken, codfish, pork, and beef), with green beans providing the most destruction. It was also observed that a given substrate was found to allow a greater per cent survival of one strain than another, so that two strains compared in one food do not always show the same relationship as when compared in another food. Some evidence indicates a modest recovery of viability of the irradiated spores during frozen storage prior to culturing. Since the character of the food determines in part the dose required for the destruction of Cl. botulinum, no single dose would be best for all food products.