Improving rapidity of microbial counting with dark-field illumination and polarized light.

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The purpose of this research is enumerating viable bacteria rapidly. In order to realize rapid enumeration, microbial colonies should be found while colonies are still small. But thickness of an agar disturbs precise focusing to take high resolution images. So, one of the best way to detect colonies early is to get high contrast images. Tb get high contrast ratio, dark-sight illumination is effective, and polarized light and long wavelength light are also used to improve it First, the best requirements of dark-sight illumination are considered to get higher contrast ratio and less unevenness of brightness over all the media plate. Second, contrast ratios of acquired images with E.coli are compared using dark-field illumination with using common bright-field illumination, the former is about 2 to 3 times higher than the latter. Next, as the effect of using either p-polarized light or long wavelength light together with dark-field illumination, each contrast ratio is improved to about 12 to 1.3 times and about 1.5 to 1.8 times higher than that of without them. And then, in experiments to take images of media plate containing E.coli with a CCD camera at regular intervals from the initial stage of culturing, colony counting time with those methods are compared to one another As a result, measuring time is about 3 hours shorter with dark-field illumination than with the previous method, and further 1 hour is shortened by using p-polarized light. In conclusion, the previously needed about 12 hours of measuring time is shortened to 8 hours by this research.