To provide information on the time dependent behavior 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 (S. cerevisiae) and typical dyes, including DAPI 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 one second 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 second with increasing DAPI concentration while it remained constant for 2-3 seconds with increasing Auramine-O concentration, e.g., at a yeast concentration of 100 µg mL .For the conditions of yeast> 10 µg mL , DAPI> 1 µg mL and Auramine-O> 0·1 µg mL , τ could be adjusted to below 5 seconds to achieve a rapid and stable staining.Design and operating conditions for rapid and online measurements of microbes can be optimized.