Fluorescent staining of microbes for total direct counts

Abstract

Bacteria and fungi are the primary decomposers of dead organic matter and play an important role in food webs and nutrient cycling. In studies into the effects of management and pollution on the structure and functioning of ecosystems, reliable estimates of microbial numbers, biomass and activity are needed. Since 1970 epifluorescence microscopy has become the major technique for direct enumeration of microbes in water and soil. In principle, a known amount of water or homogenized soil suspension is placed on a known area of a microscopic slide, the microorganisms are stained with a fluorescent dye, and numbers are tallied with a microscope. Biovolumes and biomass can be estimated from lengths and widths. The frequency of dividing cells (FDC), i.e., the percentage of cells showing an invagination, can be used as an index of the in situ specific growth rate of bacteria in water [17] and in soil [8]. Metabolically active fungal hyphae can be estimated after staining with fluorescein diacetate (FDA) which becomes fluorescent when it is enzymatically hydrolyzed [32]. The FDA method has been used also for bacteria. However, not all bacteria are able to take up FDA. The same limitation applies to other viability probes for bacteria [10].