Flow cytometric assessment of viability of lactic acid bacteria

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics, We investigated the usefulness of Bow cyt ometry (FCM) for viability: assessment of lactic acid bacteria. The esteras e substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA b inding probes propidium iodide (PI) and TOTO-1 were tested for live/dead di scrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count exper iments were performed to validate the results of the FCM assays. The result s showed that c-FDA was an accurate stain for live cells; in exponential-ph ase cultures almost all cells were labeled, while 70 degreesC heat-killed c ultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discriminat ion between live and dead cells. The combination of cFDA and TOTO-1 gave th e best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent pla ting on agar medium provided direct evidence that cFDA labels the culturabl e subpopulation and that TOTO-1 labels the nonculturable subpopulation. App lied to cultures exposed to deconjugated bile salts or to acid, cFDA and TO TO-1 proved to be accurate indicators of culturability. Our experiments wit h lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.