APPLICATION OF FLOW CYTOMETRY TO ASSESS THE VIABILITY OF STRESSED LACTOBACILLUS SAKEI

The aim of this study was to develop a rapid assay based on a nucleic acid double-staining by using two fluorochromes [SYBR Green II RNA gel stain (SYBR-II) and propidium iodide (PI)] (Gregori et al., 2001) to distinguish viable from damaged and dead cells of Lactobacillus sakei strains after exposure to heat and acid stresses. Cells were discriminated in density plots of green fluorescence versus red fluorescence. After single labeling firstly with SYBR-II and then with PI, cytograms showed two bacterial clusters, live and dead cells. Moreover, dual staining differentiated cell populations in different physiological states, alive cells with intact membranes, an intermediate physiological state that corresponded to damaged cells that are still alive but with injured membranes, so with even a recovery ability, and dead cells killed because of a permanent membrane damage. This application was validated against the classical plate-counting method and the comparison of two methods produced interesting results. In the traditional method, the removal of the stress and the incubation of the cells in favourable growth conditions caused an increase of colony counts that corresponding to the recovery ability, attributed to damaged cells of the cytofluorimetric analyses. This study evidenced that, due to the complexity of the physiological status and heterogeneity of bacterial cells in a culture, especially after stress, multiparameter analysis is preferable. 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 is the first report about the use of double staining cytofluorimetric analysis on an important food bacteria as Lactobacillus sakei. This novel assay has potential for physiological research on lactic acid bacteria and for application in the food industry. References Gregori G., Citterio S., Ghiani A., Labra M., Sgorbati S., Brown S., Denis M. 2001. Freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl Environ Microbiol 67: 4662-4670.