AME 25:301-309 (2001)  -  doi:10.3354/ame025301

Comparative assessment of epifluorescence microscopy, flow cytometry and solid-phase cytometry used in the enumeration of specific bacteria in water

Karine Lemarchand, Nathalie Parthuisot, Philippe Catala, Philippe Lebaron*

Observatoire Océanologique, Laboratoire ARAGO, Université Pierre et Marie Curie (Paris 6), Institut National des Sciences de l¹Univers (INSU) CNRS-UMR-7621, BP 44, 66651 Banyuls-sur-Mer Cedex, France
*Corresponding author. E-mail:

ABSTRACT: Rapid microbiology and the detection of rare events are important challenges in various fields of aquatic microbiology. Epifluorescence microscopy, flow cytometry and solid-phase cytometry are techniques used for direct methods in microbiology but the range of application of these different instruments is not clearly defined. In this study, we examined the lower limit of bacterial concentration to which each technique can be reliably used. Techniques were compared for the enumeration of (1) fluorescent beads, (2) labeled bacteria at different ratios of labeled/non-labeled cells, (3) Escherichia coli O157:H7 cells inoculated at different densities in tap water, and (4) E. coli O157:H7 cells in artificially contaminated natural seawater. The different methods gave results that correlated well despite the presence of a significant background of unlabeled cells. However, solid-phase cytometry was the only technique that allowed the accurate enumeration of rare events (down to 1 cell) providing the same sensitivity as traditional culture methods. The detection sensitivity was not affected by the presence of up to 107 unlabeled cells on the filter. In contrast, flow cytometry was a very rapid and accurate method but it could not be applied to the detection of rare events. E. coli O157:H7 cells could be detected rapidly and accurately in environmental water samples in the presence of non-specific bacteria. Solid-phase cytometry combined with taxonomic probes allowed rapid and accurate detection of a large variety of species of ecological interest in a wide variety of aquatic environments.

KEY WORDS: Range of application · Epifluorescence microscopy · Flow cytometry · Rare events · Solid-phase cytometry · Specific bacteria

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