Direct microscopic methods using several fluorescent staining were applied to estimate the proportion of physiologically active bacteria in the water environment and evaluate the efficacy of disinfection with chlorine. 4′,6-diamidino-2-phenylindole (DAPI) was used to determine total bacterial numbers, and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was chosen for direct detection of respiring bacteria. BacLightTM kit was used to assess bacterial membrane integrity. Bacteria with growth potential were enumerated using the DVC method and microcolony technique. The total bacterial number in river was 8 × 106∼3 × 1010 cells/mL, and colony forming units on R2A medium were 1 × 104∼4 × 105 cfu/mL. In the case of wastewater treatment plant, 1 ∼ 10% of total bacterial cells could form colonies. Physiologically active bacteria in river and wastewater treatment plant determined by fluorescent staining were much higher than those obtained by plate counting. The effect of chlorine on the physiological viability of Escherichia coli was also investigated. Microscopic viable bacteria were even more chlorine resistant than culturable bacteria. The inactivation rate coefficients of direct viable bacteria were one-second to third those of culturable bacteria.

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