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AssigneeMd Invention Oeno
InventorMatthieu DUBERNET
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The invention relates to a method for detecting, quantifying and differentiating by flow cytometry Brettanomyces spp yeast cells contained in an organic liquid substrate which contains fermentable sugars, whereby a sample of said substrate is taken, optionally diluted, at least a first fluorochrome capable of binding to the DNA of dead and/or live cells is added to said optionally diluted substrate, said sample is irradiated so as to obtain the fluorescence emission of said first fluorochrome and said sample is also irradiated so as to obtain a fluorescence emission of said sample at 670 nm, a biparametric histogram is plotted giving for each point the fluorescence intensity due to the first fluorochrome coupled with the fluorescence intensity emitted at 670 nm, at least a first point cloud is thus obtained corresponding to a greater fluorescence intensity emitted and detected at 670 nm than that detected for the other points, it is inferred therefrom that the points of said first cloud correspond to the Brettanomyces spp cells.
A method for detecting, quantifying and differentiating by flow cytometry Brettanomyces spp yeast cells contained in an organic liquid substrate which contains fermentable sugars, whereby a sample of the substrate is taken, optionally diluted, at least a first fluorochrome capable of binding to the DNA of dead and/or live cells is added to the optionally diluted substrate, the sample is irradiated so as to obtain the fluorescence emission of the first fluorochrome and the sample is also irradiated so as to obtain a fluorescence emission of the sample at 670 nm, a biparametric histogram is plotted giving for each point the fluorescence intensity due to the first fluorochrome and the fluorescence intensity emitted at 670 nm, at least a first point cloud is thus-obtained corresponding to a greater fluorescence intensity emitted and detected at 670 nm than that detected for the other points, wherein the points of the first cloud correspond to the Brettanomyces spp cells and the number of Brettanomyces spp cells is optionally enumerated by counting the points of the first cloud.
A fluorochromic mixture containing or consisting of a solvent and a first fluorochrome selected from fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 652 nm and a maximum fluorescence emission wavelength of 676 nm and a fluorescence quantum yield on DNA of 0.27, fluorochromes capable of binding to DNA and having a maximum fluorescence absorption wavelength of 657 nm and a maximum fluorescence emission wavelength of 673 nm and a fluorescence quantum yield on DNA of 0.17 and mixtures thereof, a second fluorochrome selected from fluorochromes having a maximum fluorescence absorption wavelength of 547 nm and a maximum fluorescence emission wavelength of 570 nm and a fluorescence quantum yield on DNA of 0.9 and a third fluorochrome the third fluorochrome is selected from 5-carboxyfluorescein diacetate, 6-carboxyfluorescein diacetate, mixtures of 5-carboxyfluorescein diacetate and 6-carboxyfluorescein diacetate and 5,6 carboxylate fluorescein diacetate succinimidyl ester of the following general formula (1):
Dependencies are inferred from the claim text.