The objective of this research is the rapid detection of Oenococcus oeni in wine during malolactic fermentation (MLF) using antibodies and flow cytometry. Detection of O. oeni in wine has and continues to be a time-consuming, laborious process: wine samples must be diluted and plated on special agar plates and incubated for 3-7 days to obtain countable colonies. By the time such results are obtained, correcting MLF problems has often been delayed too long.
A flow cytometer, an instrument still mostly used in hospitals for white cell counts, can count 1000?s of cells in a liquid sample in seconds. We have adapted this technology to the counting of yeast and, now, bacterial, cells in wine. Cells of interest can be separated and ?gated? from other cells on a graph of forward versus side light scattering, a function of cell size and surface smoothness. Cells can also be ?gated? base on fluorescence. To make cells of interest fluorescent, an antibody specific for that species is employed to bind the cells. In the second step, a second fluorescently tagged antibody is used to bind to the primary antibody. The cells? fluorescence separates them from other microbial cells and grape material in the sample, allowing them to be gated and counted.
Other fluorescent stains can be used to determine if the cells of interest are dead or alive. We have used propidium iodide (PI) to stain for dead yeast cells. PI only gets into a cell if its membrane is compromised, an indication of incipient, if not, actual death. In this study, PI was adding to wine samples post-ML inoculation, and demonstrated dead yeast but not dead ML bacteria.
Real-time monitoring of Oenococcus oeni in wine, accomplished in approximately 45 min after a sample is taken, provides the winemaker with a tool to evaluate MLF as soon as the first day of inoculation. Viable and non-viable cell numbers are readily determined and given an immediate indication of the health of the of the ML culture and, by implication, the malo-lactic fermentation. The control provided to the winemaker by this procedure will help increase, or at least maintain, the quality of the wine.