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

Authors

  • J.P. Diaper,

    1. Department of Genetics and Microbiology, University of Liverpool, Liverpool, UK
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    • NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK.

  • C. Edwards

    Corresponding author
    1. Department of Genetics and Microbiology, University of Liverpool, Liverpool, UK
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Dr C. Edwards, Department of Genetics and Microbiology, University of Liverpool, PO Box 147, Liverpool L69 3B Y, UK.

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.

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