An uncontaminated subsurface aquifer sediment contains a sparse microbial community consisting primarily of coccobacillary bacteria of relatively uniform size which can be counted directly with appropriate staining. The morphological simplicity and the relatively decreased cell numbers, when compared with surface soils and sediments, make the subsurface an ideal natural community with which to compare the utility of chemical measures of microbial biomass to direct microscopic counts. The membrane phospholipids (estimated as the polar lipid fatty acids, the lipid phosphate, and phosopholipid glycerol phosphate), lipopolysaccharide lipid A (estimated as the LPS hydroxy fatty acids), cell walls (estimated as the muramic acid), and adenosine triphosphate all give essentially identical estimates of cell numbers and dry weight as the direct counts, using conversion factors determined on subsurface microorganism monocultures. Assays of microbial cell components are thus validated by comparison with the classical direct count in at least one soil/sediment.

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Ground‐water pollution by organic compounds has become a major environmental concern. Because the transport and fate of the organic pollutants may be influenced by microorganisms present in subsurface material, reliable measurements of the number of organisms in subsurface samples and their metabolic activity are needed. A special drilling rig and aseptic procedures have been developed by the Robert S. Kerr Environmental Research Laboratory of the United States Environmental Protection Agency to yield uncontaminated subsurface samples. The number of bacteria in subsurface samples has been determined by microscopic counting after acridine orange staining; the proportion of cells capable of respiration was determined by INT reduction. An independent measure of metabolic activity was obtained by measuring ATP extracted from the samples. A procedure and extradant for the extraction of ATP from subsurface material have been developed. The extractant contains reagents to reduce the loss of the extracted ATP. Subsurface samples from Oklahoma and Texas contain 106‐107 cells per g of subsurface material (depths of 2–9 m). Both methods show that usually between 1 and 10% of the cells were metabolically active. Thus, significant numbers of metabolically active bacteria exist in subsurface material with the potential to modify pollutants.