Development of fluorescence microscopic image analyzing system for rapid detection of bacterial cells

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In order to prevent epidemics of infectious diseases and to maintain microbiological safety in pharmaceutical and food industry, we developed a system to detect microbes easily and rapidly. We optimized a direct count method with fluorescent staining, a fluorescent antibody method and a fluorescent in situ hybridization method to detect specific bacteria under fluorescent microscopy For the detection of physiologically active bacteria, double staining by CTC and DAPI were used.This system was applied to specific detection of Escherichia coli O157 with physiological activity. Staining O157 first by CTC and have it reacted with anti O157 antibody labeled with FITC next allowed specific detection of physiologically active O157. E.coli with O157 antigen were also specifically detected by double staining with Cy3 labeled gene probe and with anti O157 antibody labeled with FITC.In order to apply the system routinely in the practical scenes, automation is essential. We developed software to analyze images acquired by a cooled CCD camera. Bacterial cells were automatically discriminated from other particles by image analysis with smoothing, secondary differential filing and edge detection. Bacterial number, morphological character, and fluorescent intensity of individual cells were obtained at the same time using this system. A user interface to enable the software to function as a comprehensive system easily used in everyday analysis was established.