Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, D-glucose, or L-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15 degrees C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.