Resistance of Clostridium perfringens Type A Spores to y - Radiation

MIDURA, T. F. (The University of Michigan, Ann Arbor), L. L. KEMPE, J. T. GRAIKOSKI, AND N. A. MILONE. Resistance of Clostridium perfringens type A spores to --radiation. Appl. Microbiol. 13:244-247. 1965.-The radiation resistance of the spores of a classical strain and of an atypical, heat-resistant strain of Clostridium perfringens was determined. Spores were produced in Ellner's and in a Trypticase broth medium. Approximately 106 viable spores per milliliter were suspended in 0.06 M phosphate buffer and irradiated with -y rays from cobalt-60; the survivors were counted in Tryptone-yeast extract-agar by the Prickett-tube technique. Radiation D values for spores of the atypical strain in phosphate buffer and in cooked-meat broth were 0.23 and 0.30 Mrad, respectively, and the D value of the classical strain was 0.25 Mrad in phosphate buffer. Spores of the classical and atypical strains of C. perfringens type A are characterized by differences in heat resistance; yet, all strains tested demonstrated similar radiation resistance. Also, the spores were more resistant to ionizing radiation in cooked-meat broth than in phosphate buffer. Foodborne enterotoxemia and dysentery in animals have been associated with four Clostridium perfringens serotypes, namely, B, C, D, and E. Type F has been established as the causative agent of necrotic enteritis in humans (Zeissler and Rassfelt-Sternberg, 1949), but type A is perhaps the most widely known of the group, even though it was not associated with foodborne disease in humans until 1945 (McClung, 1945). A few years later, atypical type A strains were identified with food poisoning in humans (Hobbs et al., 1953); these strains were considered atypical because of the high heat resistance of their spores. Classical strains have also been found to cause food poisoning (Collee, Knowlden, and Hobbs, 1961; Hall et al., 1963). Since foods can be processed with ionizing radiations, the resistance of C. perfringens to such radiations must be known. Midura et al. (1963) reported on the resistance of C. perfringens type A, Hobbs serotype 2, to y rays with the spores suspended in phosphate buffer. Recently Matsuyama, Thornley, and Ingram (1964) evaluated the resistance of C. perfringens type A, Hobbs serotype 1, to these radiations in a cooked-meat medium. Radiation resistances of classical and atypical strains of C. perfringens are compared in the present paper. MATERIALS AND METHODS Two C. perfringens type A strains were selected. The classical type A strain G47, isolated, from a wound infection, was obtained from T. F. Wetzler, The University of Michigan, Ann Arbor; the Hobbs "heat resistant" strain, E2, isolated from a food-poisoning event, was obtained from H. Hall, of the Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio. Stock cultures were maintained in modified Robertson's chopped-heart medium (Wetzler, Marshall, and Cardella, 1956). Hydrogen was used for all work done under anaerobic gaseous environments. Suspensions of spores were prepared in Ellner's (1956) medium and in a Trypticase broth medium. Spore yields in Ellner's medium varied with different subcultures of the same strain in the same batch of medium; for example, yields of approximately 105 per milliliter were obtained with strain G47, in marked contrast to strain E2 which either did not sporulate or produced spores in very limited numbers. The following procedure consistently encouraged the sporulation of strain E2. Stock cultures were grown in tubes of modified Robertson's chopped-heart medium at 37 C. These tubes were then refrigerated at 4 C. For use, one tube was placed in a water bath at 80 C for 15 min; then 0.5 ml of the culture was inoculated into a freshly steamed and cooled tube of modified Robertson's chopped-heart medium which was incubated for 24 hr at 37 C. Next, approximately 45 ml of freshly prepared Trypticase Soy Broth in 25 by 200 mm test tubes (Kimble Glass Division, Owens-Illinois Glass Co., Toledo, Ohio) were inoculated with 1 to 2 ml of the chopped-heart culture. The concentration of Trypticase (BBL) in this broth was increased to 4%, and the medium was designated 244 on A uust 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom 1-RADIATION OF C. PERFRINGENS SPORES as TSB-4. The inoculated tubes were incubated for 48 hr at 37 C. Spore crops were harvested by pooling the contents of 5 tubes in one bottle, centrifuging at 3,000 rev/min for 30 min at 0 C, and washing three times with distilled water. After removal of the last supernant fraction, sterile distilled water was added in the least amount sufficient to facilitate pipetting the spore suspension into screw-cap test tubes. Viable spores were counted after heating at 80 C for 15 min. The spore suspension was stored at 4 C. Five media were tested to select the most suitable medium for determining survival of C. perfringens spores subjected to -y-radiation and heating at 80 or 100 C. These media were: pork-pea infusion agar (Andersen, 1951), Tryptone-yeast extract-agar (Mossel et al., 1956), reinforced clostridial agar (Oxoid), iron-sulfite-agar (Oxoid), and sulfite-polymyxin-sulfadiazine-agar (Angelotti et al., 1962). The number of viable spores was finally determined by dilution counts with the use of MillerPrickett tubes (Miller, Garrett, and Prickett, 1939) and the basal medium of Mossel et al. (1956). The latter contains 1.5% Tryptone (Difco), 1%c yeast extract (Difco), and 1.5% agar (Difco). For the irradiation studies, approximately 106 spores per milliliter were suspended in 0.06 M phosphate buffer at pH 7.0, or in the supernatant liquid of cooked-meat medium (Difco) at pH 6.8. Samples (4-ml) of these suspensions were dispensed into 5-ml ampoules (Kimble Glass Division). The ampoules were heat-sealed and immersed in ice water, and the spore suspensions were irradiated with -y rays at a rate of 72,000 or 74,000 rad per hour. The cobalt-60 source at the Phoenix Radiation Facility of The University of Michigan was