Kinetics of germination of wet-heat-treated individual spores of Bacillus species, monitored by Raman spectroscopy and differential interference contrast microscopy.

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca²+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.