The yeast analysis provides information about the total number of yeast cells in suspension, the number of vital, viable and dead cells expressed in number of cells in mL and the viability and vitality of yeast cells expressed in percentages. The analysis is performed using EASYCOUNTER YC fluorescence image cytometer. The instrument counts the individual cells into yeast suspension by detecting the fluorescence of stained DNA in the cell nuclei with SOFIA GREEN fluorescent dye.

Determination of Viability of yeast cells

Yeast viability analysis is the most commonly used test to determine the purity, quantity and metabolic status of yeast population before and after fermentation. It is used for prediction of the condition of yeast population, so that the fermentation performance can be optimised.

By using image cytometry for counting individual cells in yeast suspension, EASYCOUNTER YC calculates total yeast cells and dead cells. The embedded software and the average result are calculated by using the formula from ISO 4833.

The instrument detects the fluorescence of stained with SOFIA GREEN fluorescent dye DNA in the cell nuclei. Though, the cell membrane of viable cells is practically impermeable to SOFIA GREEN, whereas non-viable, dead and, to some extent, apoptotic cells are permeable to the dye. Thus, the staining with SOFIA GREEN cell nuclei in the analyzed sample give results on the number of non-viable cells in the sample. Determining the total number of cells requires membranes of viable cells to become permeable for SOFIA GREEN. This is achieved by mixing a cell suspension with a lysing reagent and SOFIA GREEN dye previously dried into a microcentrifuge tube.

EASYCOUNTER YC enables the user to obtain a total number of yeast cells and determine the viability of the yeast in the sample.The system considers individual cells in suspension using image cytometry.

EASYCOUNTER YC counts the individual cells into suspension by detecting the fluorescent signals of stained DNA in the cell nuclei with SOFIA GREEN fluorescent dye. The cell membrane of viable cells is practically impermeable to SOFIA GREEN, whereas non-viable, dead and, to some extent, apoptotic cells are permeable to the dye. Thus staining with SOFIA GREEN on a sample of the cell suspension gives results on the number of non-viable cells in the sample. Determining the total number of cells requires membranes of viable cells to become permeable for SOFIA GREEN. This is achieved by mixing a cell suspension with a lysing reagent and SOFIA GREEN dye previously dried into a microcentrifuge.

The lysing reagent penetrates the cell walls, thus enabling all the cells in the suspension to stain. 8 μL of the ready sample with all stained cells is required to be pipetted directly in a camera A or camera C of single CELLCHIP. The other 8 μL from the sample with only dead stained cells is required to be pipetted in camera B or camera D of the same CELLCHIP. Then the chip is loaded into the image cytometer. The analysis takes place over a period of 40 seconds to 2 minutes, the length of which depends on the number of fields captured. The EASYCOUNTER YC system automatically focuses on the chip and the colored cells are captured by the sensitive CMOS camera. The algorithm for digital imaging analysis determines the number of fluorescent cells and calculates their concentration, size and viability. The result is automatically displayed on the screen.

Determination of Vitality of yeast cells

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 analysis, performed with EASYCOUNTER YC, 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. 8 μL of the ready sample with all stained cells is required to be pipetted directly in a camera A or camera C of single CELLCHIP. The other 8 μL from the sample with only vital stained cells is required to be pipetted in camera B or camera D of the same CELLCHIP. Then the chip is loaded into the image cytometer. The analysis takes place over a period of 40 seconds to 2 minutes, the length of which depends on the number of fields captured. The EASYCOUNTER YC system automatically focuses on the chip and the colored cells are captured by the sensitive CMOS camera. The algorithm for digital imaging analysis determines the number of fluorescent cells and calculates their concentration, size and vitality. The result is automatically displayed on the screen.

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.