A new fluorescence staining assay for visualizing living microorganisms in soil.

5- (and 6-)Sulfofluorescein diacetate (SFDA), which is converted to a fluorescent product by intracellular esterase activity, was used to stain living microorganisms, including bacteria, Saccharomyces cerevisiae, and fungi, in soil. SFDA (1 mM) dissolved in ethyl alcohol was added to an intact soil sample, and the preparation was examined with an epifluorescence microscope. Bright single cells and colonies of live bacteria were observed without interference from the autofluorescence of soil minerals and detritus. Cultured Escherichia coli was killed through heat treatment; thus, SFDA was concluded to stain only living cells. Microbial colonies obtained from natural soils and various cultured strains were tested. It was found that 151 of 154 colonies from natural soils were stained and that hyphae and spores from 1 of 28 cultured microbial strains were not stained. The SFDA method was successfully used to visualize and count bacteria in soil samples from Mount Shiga in Japan.