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

Rapid microbiology and the detection of rare events are important challenge s in various fields of aquatic microbiology. Epifluorescence microscopy, fl ow cytometry and solid-phase cytometry are techniques used for direct metho ds in microbiology but the range of application of these different instrume nts is not clearly defined. In this study, we examined the lower limit of b acterial concentration to which each technique can be reliably used. Techni ques were compared for the enumeration of (1) fluorescent beads, (2) labele d bacteria at different ratios of labeled/non-labeled cells, (3) Escherichi a 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 cytometr y was the only technique that allowed the accurate enumeration of rare even ts (down to 1 cell) providing the same sensitivity as traditional culture m ethods. The detection sensitivity was not affected by the presence of up to 10(7) unlabeled cells on the filter. In contrast, flow cytometry was a ver y 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 accuratel y in environmental water samples in the presence of non-specific bacteria. Solid-phase cytometry combined with taxonomic probes allowed rapid and accu rate detection of a large variety of species of ecological interest in a wi de variety of aquatic environments.