The use of fluorogenic esters to detect viable bacteria by flow cytometry

Abstract

The ability of flow cytometry (FCM) to detect viable bacteria after staining with a range of fluorogenic esters was investigated with several bacterial species. The dyes studied were the fluorescein diacetate (FDA) derivatives carboxyfluorescein diacetate, 2′,7′‐bis‐(2‐carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester and calcein acetoxymethyl ester, as well as ChemChrome B, a commercially‐available stain for the detection of viable bacteria in suspension. No one dye was found to be universal but ChemChrome B dye stained the widest number of Gram‐positive and Gram‐negative species, whereas the FDA derivatives preferentially stained Gram‐positive bacteria. The use of ChemChrome B to detect viable bacteria in environmental samples was investigated further by studying the survival of Klebsiella pneumoniae in lakewater. During survival studies, a higher number of viable bacteria were detected both by direct viable counts and FCM after staining with rhodamine 123 and ChemChrome B than by colony‐forming units, suggesting the presence of viable but nonculturable cells. These results demonstrate the potential use of FCM to enumerate viable bacteria in natural waters.