Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i
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SUMMARY: ATP, an obligate constituent of all living organisms, is extracted from viable microorganisms in boiling TRIS buffer following sample concentration by vacuum filtration. The extracted ATP is analyzed in a photometer by the firefly bioluminescence reaction, and the ATP content is related to total living (biomass) microbial carbon by the application of a laboratory- derived extrapolation factor.
In field studies it is often desirable to determine the total amount of living cellular material (biomass). Conventional methods (i.e., fresh or dry weight determinations, rate of increase of cell numbers, etc.) usually cannot be used owing to (a) lack of sensitivity in the analytical procedures, (b) the presence of a heterogeneous assemblage of organisms, (c) the presence of dead cells and (d) the presence of detrital (non-living) organic material which is not associated with the living cells. Estimation of cellular biomass by measurement of adenosine triphosphate (ATP) is not limited by any of these considerations.
The rationale for using ATP to estimate biomass is the ubiquitous distribution of ATP in all living cells, the rapid loss of ATP from dead cells and the fairly uniform concentration of ATP in the protoplasm of all microbial cells. Data on ATP concentrations can thus be extrapolated to biomass parameters, such as cellular organic carbon or dry or fresh weight (Holm-Hansen, 1973). ATP is extracted from cells using boiling TRIS buffer and is stored frozen (-20 °C) prior to analysis by firefly bioluminescence.
ATP samples must be processed as rapidly as possible, because the ATP content of microorganisms can change rapidly when cells are stressed. Furthermore, a phenomenon referred to as the "filtration effect" causes a loss of ATP when cells are exposed to dessicating conditions immediately after the water is drawn through the filter pad (Karl and Holm-Hansen, 1978). For this reason it is very important that the samples are filtered immediately upon sampling and extracted immediately upon filtration; any delay will cause a decline in ATP content. Because the firefly bioluminescence assay is inhibited by metals, it is also important to use clean stainless steel forceps when handling the filters. It is also essential that the extraction buffer (TRIS) is boiling (100 °C), as inefficient extraction results at temperatures (<95 °C). TRIS buffer boiling must be confirmed before starting the filtration process.
3. Sampling, Filtration, Extraction and Storage
5. Preparation of ATP Standards
A primary (reference) ATP standard is prepared by dissolving exactly 10 mg of high purity (99.9%) ATP (sodium salt) into exactly 10 ml of sterile distilled water. Be sure to weigh out an amount equivalent to 10 mg of free acid form of ATP (not 10 mg of hydrated Na-ATP) taking into account the cation contribution and average hydration state of the molecule. This latter information is supplied by the manufacturer. Immediately prepare 1/10 and 1/100 dilutions of the primary stock, place a portion of each solution into a 1 cm quartz spectrophotometercuvette and measure the absorbance at 259 nm. Calculate the exact concentration as follows:
A = Elc
After calculating the precise concentration of ATP in the primary standard, dilute the stock (gravimetrically) into sterile-filtered (0.2 µm) 0.02 M TRIS buffer pH 7.4 to yield an ATP standard solution of exactly 1 µg ml-1. Compare with "old" standard and if different by >|1%|, repeat the dilution step. Store frozen in 1 ml aliquots.
Working standards, ranging from 0.1-100 ng ATP ml-1, are prepared by diluting a vial of the primary standard with freshly-prepared TRIS buffer (0.02 M) just prior to use. These working standards are discarded at the end of the day.
6. Data Reduction and Calculations