Killing of bacterial spores by H202 at elevated but sublethal temperatures and neutral pH occurred without lysis. However, with prolonged exposure or higher concentrations of the agent, secondary lytic processes caused major damage successively to the coat, cortex, and protoplast, as evidenced by electron and phase contrast microscopy. These processes were also reflected in changes in differential scanning calorimetric profiles for H202-treated spores. Endothermic transitions in the profiles occurred at lower temperatures than usual as a result of H202 damage. Thus, H202 sensitized the cells to heat damage. Longer exposure to H202 resulted in total disappearance of the transitions, indicative of major disruptions of cell structure. Spores but not vegetative cells were protected against the lethal action of H202 by the transition metal cations Cu+, Cu21, Co2+, Co3+, Fe2+, Fe3+, Mn2+, Ti3+, and Ti4+. The metal chelator EDTA was also somewhat protective, while o-phenanthroline, citrate, deferoxamine, and ethanehydroxydiphosphonate were only marginally so. Superoxide dismutase and a variety of other free-radical scavengers were not protective. In contrast, reducing agents such as sulfhydryl compounds and ascorbate at concentrations of 20 to 50 mM were highly protective. Decoating or demineralization of the spores had only minor effects. The marked dependence of H202 sporicidal activity on moderately elevated temperature and the known low reactivity of H202 itself suggest that radicals are involved in its killing action. However, the protective effects of a variety of oxidized or reduced transition metal ions indicate that H202 killing of spores is markedly different from that of vegetative cells.

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