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To establish rapid counting methods of living Salmonella in biological fluids, 4 different detection methods were evaluated. A new flow cytometer able to count particles bigger than 0.25 micromite4 was successfuIly applied. for rapicl viable bacterial counting. When cell numbers were bigger than 5000 cfu/mL, this method worked. The method was also able to dffirentiate living and clead bacterial population in fluid within 30 min. l-aser scanning microscopic counting method needetl high concentration of bacteria more than 106/mI to effectively find bacteria under a microscope. The laser scanning microscopy y,tas also used to analyze different popalation within a single culture. S. typhi cells were separated into two dffirent populations after 8 hr culture in LB broth but cells of S. typhimurium remained uniform even after 24 hr culture. This dffirence of cell population in a single strain was easily visualizerJ by this methocl. Photon counting VIM camera was most sensitive but reduction of background was biggest problems for actual application for clinical samples. Quantitative PCR was also applied for bacterial counting. Rapid cycling PCR preparecl from V, ftagella, and rJb sequences could detect organisms around 20-100 cfii'/ml. During amplification process, amplicon was sequentially monitoretl by fLuorescence and the result was obtained within 20 min.

Direct counting and detection of living Salmonella in biological fluid