Rapid enumeration of Oenococcus oeni during malolactic fermentation by flow cytometry

Abstract

The aim of this study was to provide a method to rapidly enumerate Oenococcus oeni cells during malolactic fermentation (MLF). To keep MLF under control, it is important to monitor the growth of the bacteria O. oeni. However, the enumeration of O. oeni using the plate count technique requires a very long incubation time of about 10 days or more, which is not adapted to monitoring MLF in real time. Flow cytometry (FCM), in combination with several fluorescent probes, is a rapid method for counting large numbers of bacterial cells. However, probes based on fluorescein [FDA, carboxyfluorescein diacetate (cFDA)] did not give good results for O. oeni. For the first time, we propose using the BacLight™ kit for enumeration of O. oeni, and we compare the results with three methods: plate count and FCM, in combination with either fluorescein probes or the BacLight™kit. The last method provides a perfect correlation with the plate count method. FCM coupled with the Baclight™ kit makes it possible to count O. oeni cells during MLF with a perfect correlation with the plate count method. The result is obtained in 20 min vs 10 days with the reference method which will be very useful for wine microbiologists. Moreover, it should be emphasized that FDA/cFDA staining is not recommended because it can lead to an erroneous count during the latency period or at the end of growth due to the variation of intracellular pH in the O. oeni cells during growth.