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.

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.

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