Microscopic Counting and Adenosine 5′-Triphosphate Measurement in Determining Microbial Growth in Soils

Abstract

A microscopic technique utilizing dispersion of fungal hyphae in a Waring blender, filtration through membrane filters (Nucleopore Corp.), and counting on a fluorescence microscope was developed for counting fungal hyphal biomass. Nonfluorescent staining techniques of the soil-filter preparation did not give quantitative recoveries. Water-soluble aniline blue, which binds to the β-1,3-glucans of the fungal cell wall, made visualization of the hyphae by fluorescence possible. A range of fungi added to soil were quantitatively recovered. Adenosine 5′-triphosphate (ATP) was extracted from soil by lysis of the organisms with CHCl₃ in NaHCO₃, which prevented adsorption of the organic phosphorus to the soil colloids. Centrifugation and removal of CHCl₃ was followed by dilution with pH 7.8 tris(hydroxymethyl)aminomethane buffer. ATP concentrations were measured by using the luciferase-luciferin light reaction. Since NaHCO₃ interfered to some extent with this reaction, the standards were made up in equivalent mixtures of tris(hydroxymethyl)aminomethane buffer and NaHCO₃. Recovery of ATP was rapid and quantitative in a range of soils. Measurement of the ATP and bacterial and fungal numbers in an incubated soil showed that fungal and bacterial population increases were delayed by phosphorus deficiency. Microbial populations were not affected at a later date. The ATP content of the soil system was reduced by phosphorus deficiency throughout the incubation period. This indicated that ATP could be altered without major changes in the microbial populations.