Flow cytometry (FC) can be considered a useful method for microbiological quality control in wineries, and for the investigation of the growth dynamics of microorganisms in wine.
It offers a number of advantages, among them a high speed (and a concomitant detection of thousands of individual cells), high precision, simultaneous measurements of multiple cellular parameters, possibility of detecting the presence of heterogeneous populations (e.g. different species simultaneously), preservation of cell viability and cellular functions, and easiness to use.
In this work, we have adapted previous fluorescence in situ hybridization (FISH) protocols for yeasts to FC, optimizing at the same time a liquid hybridization protocol.
For this, we used specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in wine, both spoilage and beneficial.
To optimize the hybridization protocol, we tested different parameters such as yeast growth, fixation and permeabilization of cells, hybridization parameters, and others. Then, the potential of using FC in combination with fluorescent probes for rapidly detection and identification of yeasts in laboratory media, musts and wines (reds and whites) was confirmed.
One important advantage is that the hybridized yeasts in liquid can be now examined under a microscope (FISH) and/or by FC. Hybridization with fluorescent probes is extremely specific, which facilitates the identification and counting of the different species of yeasts in the same wine with a single analysis.
Poster presented at Enoforum 2013, 7-9 May, Arezzo (Italy)
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