Purpose: The aim of this study is to follow by flow cytometry the fate of spores after specific heat-treatments and recovery in sub-optimal conditions. Especially, the dynamic of the physiological states leading the spore to give a vegetative cell was studied.
Methods : The spores of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 were obtained at optimal and suboptimal growth temperatures. After heat-treatments allowing a ten-fold reduction, spore recovery in nutritive broth was investigated for different pHs and temperatures distributed along the growth domain (5-40°C for B. weihenstephanensis, 15-60°C for B. licheniformis and pH 4.0-8.0 for both strains). The germination and growth recovery were monitored over time using flow cytometry, taking into account cell size and permeability (Syto9 staining) and respiratory activity (CTC staining).
Results: Different physiological stages were efficiently evidenced: refractive spores, germinated spores, outgrowing cells and vegetative cells. In optimal conditions, most cells evolved rapidly towards multiplication. Recovery of heat-treated spores was slower at suboptimal pH and temperature than at optimal pH and optimal temperature, and a significantly lower proportion of spores successfully formed vegetative cells. Although large proportion of spores germinated and showed some early signs of potential growth after a heat-treatment, only a few were actually able to form a colony on agar plates. Monitoring spore recovery using flow cytometry is a powerful method, allowing an exploration of the individual evolution of cells among populations. The number of analysed cells is potentially high (over 200 000 in the present work), offering an accurate estimation of variability in individual cell development within populations.
Significance: A mathematical probabilistic model has been developed to describe the dynamic aspects of heat-treated spore germination and growth restoration and allow the estimation of the proportion of each physiological stage regarding incubation temperature and pH, and the duration of incubation. This can lead to a better prediction of spore forming bacteria development in foods.