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Freshwater environments and natural water parks are important as recreation areas; however, people enjoying recreation at the river- or lake-side are sometimes infected with pathogenic microbes. Microbiological monitoring is fundamental for the routine evaluation of water quality. Fluorescent staining techniques are regarded as among the most useful rapid microbiological methods; however, preparation of samples for fluorescence microscopy is often labor-intensive, and one usually has to take the samples to a laboratory for measurement, which often alters the culturability of bacteria in the samples. These factors have created demand for a rapid and simple method of bacterial quantification in freshwater that can be performed on-site. In this study, we applied our microfluidic device, which was originally designed for on-chip fluorescent staining and semi-automated counting of target microbial cells with fluorescent antibody-staining, to enumerate bacterial cells in freshwater. This was combined with a self-made portable system for rapid on-site monitoring of the bacterial cells. Numbers of both esterase-active bacteria and total bacteria in pond water samples could be successfully determined by on-chip staining with 6-carboxyfluorescein diacetate and SYBR Green II, respectively, using the portable microfluidic counting system. The counting was completed within 1 h (30 min for pre-filtration of freshwater and 30 min for on-chip staining and counting). These results indicate that rapid and accurate counting of bacterial cells in freshwater can be performed and this technique could be applied for "on-site first screening" purposes in microbial quality control of freshwater.

Rapid On-Site Monitoring of Bacteria in Freshwater Environments Using a Portable Microfluidic Counting System