Monitoring changes in germination and permeability of Bacillus cereus endospores following chemical, heat and enzymatic treatments using flow cytometry

The germination inhibition of Bacillus cereus endospores and the removal of various structural components were undertaken using a number of standard treatments. Concomitantly, the changes in the entry and retention of the fluorescent nucleic acid dyes, SYTO 9, propidium iodide (PI) or Hoechst 33342, or hydrolysis of carboxyfluorescein diacetate (CFDA), were analyzed using flow cytometry (FCM) and epifluorescence microscopy. SYTO 9 and PI staining followed by FCM analysis detected six discrete subpopulations of endospores across all treatments, while CFDA/Hoechst 33342 staining allowed the identification of subpopulations of permeabilized endospores possessing intracellular esterase activity. Differential permeability to PI and Hoechst 33342 as revealed using FCM appeared related to the germination stage and the absence of specific structural components. Additionally, endospores halted at specific points during germination had characteristic FCM fluorescence profiles. This study highlighted the potential of FCM as a useful technique for rapidly studying endospore germination and structural alterations following treatment with sporistatic/sporicidal physicochemical treatments.