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

Published on Jun 1, 2020in Journal of Applied Microbiology3.066
· DOI :10.1111/JAM.14577
Suthida Piriyakarnsakul1
Estimated H-index: 1
(Kanazawa University),
K. Takarada (Kanazawa University)+ 3 AuthorsMasami Furuuchi19
Estimated H-index: 19
(Kanazawa University)
Sources
Abstract
AIMS: 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. METHODS AND RESULTS: 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 tau, 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 microg ml(-1) . CONCLUSIONS: For the conditions of yeast >10 microg ml(-1) , DAPI >1 microg ml(-1) and Auramine-O >0.1 microg ml(-1) , tau could be adjusted to below 5 s to achieve a rapid and stable staining. SIGNIFICANCE AND IMPACT OF THE STUDY: Design and operating conditions for rapid and online measurements of microbes can be optimized.
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