Combined effects of ionizing-irradiation and different environments on Clostridium botulinum type E spores

We examined the combined effects of γ-radiation (24 °C) on spores of Clostridium botulinum-type Eklund strain suspended in different gas-saturated Na-phosphate buffer in absence or presence of protectors or sensitizers. Response surface methodology (RSM) was also used to ascertain the effects of radiation on the recovery of spores using a medium containing various levels of NaCl or Na-thioglycollate. The former (<0.5%) decreased viable spore counts, but the latter (0.15%) did not. Irradiation inactivation of Eklund spores was most effective in air-saturated buffers compared to N2O and N2 gas. The Na2–EDTA (0.01 M) was the most efficient radioprotector of spores due to its reactivity toward hydroxy radicals, followed by t-butanol (0.1 M) in NO2 or N2-saturated buffers, respectively. Catalase (10.0 mg ml−1) and dl-cysteine (0.1 mM) sensitized the spores during irradiated N2O or N2-saturated buffers, and NaCl (0.01 M) only sensitized spores in N2 environment. Spores frozen at −75 °C for 30 days and thawed prior to use were more sensitive to radiation damage compared to freshly prepared spores. Glycerol (15%), in Na-phosphate buffer (pH 7.0, 0.06 M), protected Eklund spores and increased the number of spores from 106 to 1011 colony forming unit (CFU) ml−1, and enhanced their radiosensitivities. Seven strains of C. botulinum type E were screened for plasmids and strain BL764 had two plasmids (15.8 and 46.8 mDa), BL4028 also had two (4.4 and 13.2 mDa), BL4850 contained only one (4.9 mDa), whereas EQA, BL211, Eklund, and Beluga had none. γ-Radiation (10 kGy, absorbed dose) cured the 15.8-mDa plasmid in strain BL764, but its absence yielded no changes in toxigenicity.