Kinetics of Germination of Individual Spores of as Measured by Raman Spectroscopy and Differential Interference Contrast Microscopy

Abstract

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (Tlag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at Trelease, and ΔTrelease (Trelease – Tlag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing Tlag values. 4) Values of Tlag and Trelease were heterogeneous among individual spores, but ΔTrelease values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average Tlag values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species.