Application of flow cytometry and capillary electrophoresis to assess the viability of stressed Oenococcus oeni strains.

The aim of this study was to assess the different physiological states of Oenococcus (O.) oeni strains exposed to ethanol stress conditions. A nucleic acid double-staining by using two fluorochromes [SYBR Green II RNA gel stain (SYBR-II) and propidium iodide (PI)] was developed to distinguish viable from damaged and dead cells of O. oeni strains after exposure to 7, 12 and 13% ethanol concentration treatments. Cells were discriminated in density plots of green fluorescence versus red fluorescence. Cytograms showed that dual staining differentiated cell populations in different physiological states: alive cells with intact membranes, an intermediate physiological state that corresponded to damaged cells, still alive but with injured membranes, so with even a recovery ability, and dead cells killed because of a permanent membrane damage. This study evidenced that because of the complexity of the physiological status and heterogeneity of bacterial cells in a culture, especially after stress, multiparameter analysis is preferable. Moreover, as bacteria carry charged or chargeable groups on their outer surface, the changes in the charging rates under optimal and stress conditions were also evaluated by capillary electrophoresis analysis. Electropherograms showed significant differences among the cells grown in optimal conditions and those obtained after ethanol stresses at various concentrations and also among the different O. oeni strains tested. This approach allowed to achieve information about the dynamics and physiological heterogeneity of microbial populations and to monitoring changes in bacterial behaviour after exposure to different stresses. This novel assay has potential for physiological research on lactic acid bacteria and for application in the food industry.