酵母真菌に対する高圧滅菌および消毒薬処理効果のLive/Dead染色を用いたフローサイトメトリ法による解析

摘要

Flow cytometric analysis using live/dead staining dyes was applied to discriminate yeast cells treated with autoclave and various disinfectants. One fluorogenic dye, propidium iodide cannot leak into live cells with intact membranes, whereas another fluorogenic dye, thiazole orange is permeant and can enter all cells, live and dead. Thus a combination of these two dyes can classify cell populations into live, injured and dead cells. When the yeast cells were autoclaved at 121C for 5 min, almost all the cells were classified as being dead, but some yeast strains contained a little injured cells (up to 8.5%). It was also demonstrated that none of the yeast cells autoclaved could produce a visible colony on the agar plate. The 70% ethylalcohol was the most effective disinfectant tested. Almost all the yeast cells treated were classified as being dead, and no visible colony appeared after agar plate incubation. The cell suspensions of yeast treated with 0.5% chlorhexidine contained injured cells with >95%, but no visible colony appeared. Whereas, the numbers of live yeast cells after treatment with 0.025% benzalkonium chloride were less than detectable counts (< 1.0%) by flow cytometry, however, visible colonies appeared on the agar plate with 101 to 3.6 X10~3 colony forming units/ml.With these results, it became apparent that the combination with fluorogenic dyes and flow cytometry can provide a reliable test method to discriminate dead and/or injured cells from the reproductive live yeast cells, and is applicable for further studies. However, it should be noted that minimum detectable levels are sometimes far different between the standard colony forming assay and the flow cytometric determination.