Abstract
The culturing techniques are important and widely used in microbiological studies. However, it has been revealed in 1980's that many microbes in the environment cannot be detected by conventional culturing techniques with long history of more than 100 years. Culturing processes are time-consuming and increase the biomass of microbial cells, which shall result to increase the risk of biohazard. We regards microbes as “particles with genetic information and physiological activity”, and develop culture-independent techniques to analyze microbial cells at the single level, a cell itself, and also community level, by using fluorescent staining methods and molecular microbial ecological approaches. By using these techniques, microbial cells can be detected and enumerated rapidly and their physiological status can be estimated at the same time. Targeted cells are detected and their dynamics can be monitored accurately based on their genetic information. Culture-independent techniques are rapid and rather simple. Results can be obtained within one hour to half a day, while conventional culturing techniques require more than a day to obtain reliable results. In order to popularize these techniques, validation and automatization is important. We applied image analysis and flow cytometry for rapid analysis. We also designed a semi-automated system for enumeration of microcolony-forming cells. Compare to conventional fluorescent microscopy, applying these new approaches is more likely to bring us more rapid and quantitative results. We now apply microfluidic devices (on-chip flow cytometry) to automatize enumeration of microbial cells, which enable us to acquire data rapidly with high reproducibility and reduce the risk of biohazard by rather simple operation. These techniques should be useful in microbiological quality assurance of freshwater.
