In many cases, toxic effects of chemical or physical factors do not lead directly to cell death. Such factors may cause a number of morphological, intracellular, or metabolic alterations that will result in the inability of a cell to divide, yet the cell itself may still be alive. The method for assessing cell viability provide information only on alive and dead cells in the whole population, but do not provide information about the number of vitality cells. In this aspect, cell vitality defining the physiological capabilities of the cell is a very important parameter. Measurement of cell vitality is advisable for conducting effective control of the fermentation process at a significant concentration of inhibitory substances that are produced during the process. It is very important for controlling the metabolic activity of yeast cells in fermentation in the brewing industry, in the production of bioethanol, in the bakery industry, etc. The Yeast Vitality Method is based on the determination of the enzyme esterase activity by means of the esterase substrate 5-carboxyfluorescein diacetate (CFDA). In parallel with this analysis, it is important to determine the total number of cells in the yeast suspension. This can be achieved by using the Sofia-Green fluorescent dye, which only stains the dead cells. To determine the total number of cells, it is necessary to treat the cells so that the membrane of the living cells becomes permeable to Sofia- Green. This is achieved by mixing the cell suspension with a lysis reagent for 10 minutes. The combination of these two analyzes results in an effective fermentation control and an increase in the yield and quality of the bio product obtained
The 5-carboxyfluorescein diacetate (CFDA) esterase substrate is used to determine the vitality of yeast. CFDA penetrates the cell membranes of living cells, the diacetate groups are cleaved from non-specific esterases and a fluorescent, charged substrate form (carboxyfluorescein) is obtained that stains the cells in green and flows out of the cells very slowly. In parallel, in a separate sample with yeast cell suspension, the total number of yeast cells was determined using the fluorescent dye Sofia Green. To determine the total number of cells, it is necessary for the membrane of living cells to become permeable to Sofia Green. This is achieved by mixing the cell suspension with a lysis reagent for 10 minutes. Sofia Green penetrates through damaged cell membranes, binds to core nucleus, and cells with damaged membranes turn green.
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