Four fluorescent dyes were compared: red fluorescent dyes Propidium iodide and TO3CN and green fluorescent dyes TO and SOFIA GREEN. Their absorptione spectra were observed. Their emission spectra before and after binding to DNA were compared. The excitation and emission peak for each dye was determined. The rate of emission enhancement after binding to DNA was established. The dyes were combined and applied for live/dead staining of Saccharomyces cerevisiae and monitored by microscopic imaging. A double staining procedure was carried out. Propidium iodide (only dead cells staining) was used in combination with TO (both dead and live cells staining) and SOFIA GREEN (only dead cells staining) was used in combination with TO3CN (both dead and live cells staining). The optimal concentration of the dyes for the cell staining was determined. The cell culture was inoculated in YPD medium and grown for 48 hours. Cell suspensions in concentration of 3.0 x 107 in phosphate buffer, pH 7.4, was prepared and washed twice with centrifugation at 220 x g. A part from the cell suspension was treated at 70 ° C for 15 min. After that it was mixed with the untreated culture to have 50 % dead and 50 % live cells. After the addition of the fluorescent dyes the samples were incubated at room temperature. The incubation period varied from 5 to 60 minutes. A drop from the suspension was applied on a slide and was observed under a fluorescent microscope. At least three repetitions were performed at every experimental stage. The use of such combination of fluorochromes that selectively permeate in live or dead cells could lead to the development of rapid procedure for yeast viability investigation during fermentation in food processing industry.

Application of four nucleic acid-binding fluorescent dyes: Propidium iodide, TO3CN, TO and SOFIA GREEN in Saccharomyces cerevisiae live/dead staining



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