Determination of Microbial Cell Numbers in Subsurface Samples

Abstract

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.

Organic phosphorus in marine sediments : chemical structure, diagenetic alteration, and mechanisms of preservation

“…Since the range of intracellular ATP concentrations considered here is calculated based on a C:ATP ratio, this imposes a similar uncertainty of the intracellular ATP concentration estimate. However, our assumptions are supported by the direct measurements of intracellular ATP concentration reported by Webster et al (1985). Their measured value of 1.7 ng ATP per 10 7 cells, or 3.3 x 10-13 !tmol ATP per cell, is often cited as the "canonical" value and is in excellent agreement with the range of intracellular ATP concentrations considered here (Table 3-2).…”

“…Nevertheless, the good correlation between sediment ATP concentration and direct cell counts (Webster et at., 1985) indicates that sedimentary ATP concentration is a reliable proxy for biomass abundance.…”

“…Such nutrient-starved conditions are not found in the surface sediments of the Santa Barbara Basin. A C:ATP ratio of 250 is often assumed (Holm-Hansen, 1973;Webster et al, 1985), based on extensive monoculture studies. For example, in culture studies of marine algae (Holm-Hansen, 1970) and marine bacteria (Hamilton & Holm-Hansen, 1967), cellular C:ATP ratios of 287 and 250, respectively, were observed in a wide number of species.…”

Environmental assessment of the potential effects of aquifer thermal energy storage systems on microorganisms in groundwater

“…1984; Ghiorse and Bal kwill 1984; Bal kwi 11 Smith et al 1986;Hirsch and Rades-Rohkohl 1984;Beeman and Sufl ita 1987;Wi 11 is et a1 . 1975;Webster et al 1985;Ehrlich et al 1984;Wilson et al 1983). The highest numbers of microorganisms are usually detected in sandy aquifer sediments, while most unsaturated zones and clay aquifer sediments harbor low populations of microorganisms.…”

“…However, the viability of microorganisms detected in the subsurface is considerably more variable. A tetrazolium dye reduction variation of the direct counting procedure has been used to estimate the proportion of active microorganisms in subsurface environments (Webster et al 1985). Using this technique, viable cell counts have been shown to range from essentially zero to values as high as the total microscopic counts (Webster et al 1985).…”