ASTM D4012-15

5.1 A rapid and routine procedure for determining biomass of the living microorganisms in cultures, waters, wastewaters, and in plankton and periphyton samples taken from surface waters is frequently of vital importance. However, classical techniques such as direct microscope counts, turbidity, organic chemical analyses, cell tagging, and plate counts are expensive, time-consuming, or tend to underestimate total numbers. In addition, some of these methods do not distinguish between living and nonliving cells.

5.2 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells, including bacteria, algae, protozoa, and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically active microbial contamination in water. ATP is not associated with matter of non-biological origin.

5.3 The ATP (luciferin-luciferase) method is a rapid, sensitive determination of viable microbial biomass. ATP is the primary energy donor for life processes, does not exist in association with nonliving detrital material, and the amount of ATP per unit of biomass (expressed in weight) is relatively constant. (ATP per cell varies with species and physiological state of the organism.)

5.4 This test method can be used to:

5.5 This test method is similar to Test Methods D7687 and E2694 except for the volumes sampled, and omission of wash and drying steps used in Test Methods D7687 and E2694 to remove interferences (1.3).

5.6 Although ATP data generally covary with culture data in water samples, different factors affect cATP concentration than those that affect culturability.

1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content associated with microorganisms normally found in laboratory cultures, waters, wastewaters, and in plankton and periphyton samples from waters.

1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are converted by comparison with an ATP standard and computation to pg ATP/mL.

1.3 This method does not remove all known chemical interferences, known to either luminesce in the 530 ± 20 nm range, or to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).

1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms (Appendix X1).

1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility.

1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily in the matrix being tested.

1.7 This test method is equally suitable for use in the laboratory or field.

1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log_{1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP, although the method does not differentiate between ATP from different sources, for example, from different types of microorganisms, such as bacteria, fungi, algae and protozoa.}

1.10 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.11 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.