Optimization of distinction between viable and dead cells by fluorescent staining method and its application to bacterial consortia

Abstract

A technique to distinguish viable and dead cells has long been considered necessary in various fields such as sterilization, toxicity assessment, sanitary evaluation and so on. A bacterial staining method using fluorescent dye is a popular tool, although the weakness of fluorescence intensity and its fading over time constitute notable drawbacks. In the process of esterase-active bacteria staining with carboxyfluorescein diacetate (CFDA), we have reported glutaraldehyde (GTA) affected the discriminative recognition of bacteria due to prevention of fluorescence leakage from the cell. In this study, CFDA was applied to four pure bacterial strains (two Gram-negative strains and two Gram-positive strains) during the exponential growth phase and to activated sludge as an indicator of microbial viability. GTA concentration was also optimized and the effect of GTA addition was compared to the conventional method using ethylenediamine tetraacetic acid (EDTA) and the control without pretreatment. At higher concentrations of GTA, microbial viability decreased because of GTA toxicity. In the case of all conditions where CFDA staining was carried out in the assay of microbial viability, the highest viability was achieved by using of 1 g/L GTA.