Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring.

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10(6) CFU ml(-1)) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R (2) ≥ 0.97, white wine R (2) ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R (2) ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10(4) CFU ml(-1) and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.