Optimal fluorescent-dye staining time for the real-time detection of microbes: a study of Saccharomyces cerevisiae

To provide information on the time-dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis.

For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4',6-diamidino-2-phenylindole) and Auramine-O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2-3 s with increasing Auramine-O concentration, for example at a yeast concentration of 100 µg ml^-1 .

For the conditions of yeast >10 µg ml^-1 , DAPI >1 µg ml^-1 and Auramine-O >0·1 µg ml^-1 , τ could be adjusted to below 5 s to achieve a rapid and stable staining.

Design and operating conditions for rapid and online measurements of microbes can be optimized.