In situ enumeration methods utilizing fluorescent probes were used to estimate the number of physiologically active bacteria in river water. Two fluorogenic compounds, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) and 6-carboxy fluorescein diacetate (6CFDA), were chosen for direct epifluorescent microscopic detection of active bacteria. CTC is a soluble redox indicator which is reduced by respiring bacteria to fluorescent CTC-formazan crystals. 6CFDA is hydrolyzed by nonspecific esterases to produce the fluorescent compound, 6-carboxy fluorescein. Estimates of the number of active bacteria identified by these fluorescent probes were compared with those obtained with the plate count method. Bacteria with respiratory activity, as determined by CTC reduction, accounted for approximately 10% of the total bacteria at oligotrophic sites and 15 to 20% at eutrophic sites. These values exceeded those obtained with the plate count method. Estimates of enzymatically active bacteria, as determined by 6CFDA hydrolysis, were also higher than those obtained with the plate count method, constituting 40 to 50% of the total bacteria at both oligotrophic and eutrophic sites. These results indicate that some non-culturable bacteria retain physiologic activity and may play an important role in the ecosystem.
the Japanese Society of Microbial Ecology (JSME)