The measurement of adenosine triphosphate in pure algal cultures and natural aquatic samples

Three methods for the extraction of adenosine triphosphate (ATP) neutral dimethyl sulfoxide (DMSO), boiling tris buffer, and butanol-octanol extraction were equally effective on the alga Chlorella vulgwia. Dilution of extracted ATP samples was linear. Filtration of different volumes of samples resulted in proportional values for ATP in the extracts. Measurement of activity by either peak height or integration of the area under the peak were equally sensitive and reproducible. The assay of ATP sample was inhibited by mercuric chloride > cadmium chloride > calcium chloride > potassium or sodium phosphate, and by high concentrations of the extractant DMSO. Analysis of ATP in aquatic environments led to the problem of transferring a sample from the field to the laboratory without obtaining a change in ATP concentration. Membrane filtration of the sample followed by chilling at 4°C, slow freezing at 20° C, or freezing on dry ice were ineffective in maintaining a constant level of ATP. Chilling caused a marked increase in ATP, whereas slow freezing caused a significant loss of ATP. Freezing on dry ice was variable but generally resulted in large losses of ATP. Quick freezing by immersion of filter and algae in liquid nitrogen and storage on dry ice maintained a constant ATP level. Field extraction of the ATP followed by quick freezing in an acetone-dry-ice bath maintained the ATP in a convenient and stable form.