ABSTRACT 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.PRACTICAL APPLICATIONSThe flow cytometry methods described in this report could be used to differentiate between endospores halted at various stages of germination, e.g., those that have lost dipicolinic acid only versus those that have also begun to degrade their cortex. Furthermore, the methods could simultaneously supply information on the mechanism of action of germination inhibitors and whether or not enzyme activity (nonspecific intracellular esterase) was present in treated endospores. The method also allows the discrimination between endospores with a specific structural component removed, e.g., endospores lacking coat (highly propidium iodide [PI] permeable) versus intact endospores (impermeable to PI). An extension of this methodology would be the quantification of the percentage of endospores exhibiting structural damage, e.g., damage to the exosporium, following treatment with sporistatic/sporicidal physicochemical treatments. Indeed, using these methods, the direct examination of food, environmental or clinical samples to rapidly evaluate the viability/structural integrity of contaminating endospores is possible.