Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment

The respiratory and enzymatic activity of bacteria in polluted and unpolluted river water was investigated by flow cytometry. Double staining with 6CFDA (6-carboxy fluorescein diacetate) and PI (propidium iodide) was used to examine bacterial esterase activity. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was employed as the indicator of bacterial respiration. The ratios of colony-forming units to total bacterial number were less than 2%, except highly polluted sites. The number of respiring bacteria determined by flow cytometry amounted to 10–15% of the total bacterial number at both unpolluted and polluted sampling stations, while it was 30% at the highly polluted station. Almost 50% of total bacteria demonstrated esterase activity in the unpolluted areas, whereas 70–90% of total bacteria retained this enzymatic activity in the polluted areas. Thus, many of the non-culturable bacteria in the natural aquatic environment have enzymatic activity regardless of the pollution level, and 6CFDA was more superior in the sensitivity for the evaluation of physiological activity of bacteria in the natural environment. The ratio of bacteria with esterase activity increased in direct proportion to the pollution level of river water and suggested that this ratio would be a useful indicator in evaluating the pollution levels in an aquatic environment.