Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat

To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. 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. 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. 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.