Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55°C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.

Spores of Clostridium botulinum type 62A were germinated in a chemically defined medium (8 mM L-cysteine, 11.9 mm sodium bicarbonate, 4.4 mm sodium thioglycolate; buffered with 100 mm TES, pH 7.0). The rate and extent of germination were increased when an aqueous spore suspension was heated sublethally (80 C, 60 min) before addition to the germination medium. Neither sublethal nor lethal doses of gamma radiation had any marked effect on subsequent germination. Maximum germination (> 90% in 2 hr) in the defined medium occurred in the pH range of 6.5 to 7.5, at 30 to 37 C, with an L-cysteine level of 8 mM. Increasing L-cysteine to 32 mM increased the rate (over that with 8 mM L-cysteine) but not the extent of germination. The rate and extent of germination increased with NaHCO3 addition to 8.3

Spores of Bacillus megaterium QM B1551 treated with thioglycolate (0.4 M, pH 2.6) at 50 C for 30 min remained refractile, but they became stainable, lysozymesensitive, and nonviable, and they lost dipicolinic acid (DPA). The loss of DPA and of viability were functions of the time and temperature of exposure to thioglycolate. Spores treated with thioglycolate at a lower temperature and for a shorter time (30 C, 5 min) retained DPA, viability, and nonstainability. Although these spores also retained their resistance to y radiation and to lysozyme, they lost thermoresistance. Their percentage of germination over a 2-hr period in glucose was markedly reduced. Germinability and heat resistance were restored by exogenous cations, suggesting that the thioglycolate treatment (30 C, 5 min) resulted in the loss of spore ions essential for normal germination in glucose and for heat resistance.

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