Aims:  To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results:  Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions:  Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study:  This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.

Aims: To isolate and characterize spores superdormant (SD) for germination with either Ca 2+ -dipicolinic acid (CaDPA) or dodecylamine. Methods and Results: Bacillus subtilis spores were germinated three times with either CaDPA or dodecylamine and germinated spores removed after each germination treatment, yielding 0Á9% (CaDPA-SD spores) or 0Á4% (dodecylamine-SD spores) of initial dormant spores. Compared to dormant spores, CaDPA-SD spores germinated poorly with CaDPA and better with dodecylamine and nutrient germinants, although release of DPA from individual CaDPA-SD spores was slow during nutrient germination, and this germination was strongly inhibited by TbCl 3 . The CaDPA-SD spores were sensitive to hypochlorite and had elevated levels of nutrient germinant receptors (GRs) relative to levels in dormant spores. Dodecylamine-SD spores' germination with dodecylamine and nutrients was similar to that of dormant spores, their germination with Ca-DPA was slower than that of dormant spores, and these SD spores' GR levels were lower than in dormant spores. However, dodecylamine-SD spores were not sensitive to hypochlorite, and the nutrient germination of these SD spores was only partially inhibited by TbCl 3 . Conclusions: CaDPA-SD spores appear to have a coat defect and accompanying low levels of the cortex-lytic enzyme CwlJ. The defect in dodecylamine-SD spores, however, is not clear. Significance and Impact of the Study: The results suggest that triggering germination by non-GR-dependent germinants is a potential strategy for efficient spore inactivation.