A NEW FLUORESCENCE STAINING ASSAY FOR VISUALIZING LIVING MICROORGANISMS IN SOIL

5-(and 6-)Sulfofluorescein diacetate (SFDA), which is converted to a f luorescent product by intracellular esterase activity, was used to sta in 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 ep ifluorescence microscope. Bright single cells and colonies of live bac teria were observed without interference from the autofluorescence of soil minerals and detritus. Cultured Escherichia coli was killed throu gh 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 natura l soils were stained and that hyphae and spores from 1 of 28 cultured microbial strains were not stained. The SFDA method was successfully u sed to visualize and count bacteria in soil samples from Mount Shiga i n Japan.