Bacillus cereus endospores were challenged by heat treatments simulating typical domestic/industrial cooking regimes and the resulting effects on germination, viability and sub-lethal heat damage determined using differential plate counting on a rich versus selective medium, flow cytometry (FCM), beta-D-glucuronidase (GUD) activity and OD600 measurement. Additionally, these techniques were used to investigate the effect on endospores of storage in a non-nutrient medium at 4 degrees C for I month. Plate counting revealed that heating generated sub-populations of sub-lethally damaged endospores, with the more severe heat treatments generating larger proportions of sub-lethally damaged endospores. These findings were also reflected in FCM analyses, which detected large amounts of heterogeneity among the populations of heat-treated endospores and uncovered differences in the proportions of membrane-damaged endospores and those displaying esterase activity pre- and post-treatment. Plate count data suggested that both the control and heat-treated endospores lost viability during storage, with FCM data indicating that the proportion of membrane-damaged endospores increased and those displaying the esterase activity decreased. The FCM, GUD and OD600 data suggested that germination rates decreased with the increasing severity of heat treatment. This study demonstrates that a combination of plate counting and FCM can be used to detect heterogeneity in the response of endospores to insults. (C) 2007 Elsevier Ltd. All rights reserved.