Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10 8 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.Freshwater and drinking water quality assessment involves the specific and rapid detection of viable enteric indicators and pathogens in collected samples. Since the presence of these indicators or pathogens is often the main cause of noncompliance and subsequent boiling events, sensitive detection of bacterial contaminants in distributed water is a major goal for utilities.Currently, water quality assessment requires time-consuming, classical culture-based methods involving sample membrane filtration, incubation, and biochemical confirmation tests (24). However, depending on environmental pressures (starvation, oxidative stress, and so forth), variable proportions of enteric bacteria can be disseminated in water in viable but nonculturable (VBNC) states (for a review, see reference 9). Since drinking water is generally characterized as being highly oligotrophic and having oxidative stress, it can be assumed that distribution systems contain bacterial cells in VBNC states which cannot be detected through routine culture-based methods. Moreover, pathogens in a VBNC state could remain virulent or produce enterotoxins (20, 21). As a consequence, the occurrence of VBNC enteric pathogens or indicators in aquatic systems may change the approach to measuring health-related risks from classical culture-based methods.A large number of probes and methods enabling the physiological characterization of bacteria at the single-cell level were recently developed (for a review, see reference 11). Most of these involve fluorescence-based methods and include the direct viable count (DVC) method combined with nucleic acid staining (10,14,29), the measurement of respiratory activity with the fluorogenic dye 5-cyano-2,3-ditoyl tetrazolium chloride (13,23,27,28), the measurement of esterase activity with the ChemChrome fluorogenic substrate (7,19,22), and the measurement of membrane integrity (4, 16). However, even though most physiological probes make it possible to characterize metabolic activities or functions at the single-...
