MICROBIAL ATP
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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.
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1. Principle
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.
2. Precautions
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
3.1. Samples for ATP determinations are collected in clean Niskin
bottles attached to the rosette/CTD unit.
3.2. As soon as the samples arrive on deck, water is prefiltered
through a drawing tube containing an in-line 202 µm Nitex mesh
prefilter to remove large zooplankton and particles which might
otherwise affect the precision and accuracy of microbial biomass
determinations. Samples are drawn into 4-liter polyethylene
bottles that are rinsed 3 times with approximately 100-200 ml
seawater from the appropriate depth. Filtration is begun
immediately (be sure heating block is on and has achieved a
temperature >110 °C; time required ~1 hour).
3.3. Filter triplicate samples at each depth through 47 mm GF/F filters.
Total volume required per sample will depend on depth: between
0-150 m use 1 liter per sample, below 150 m use 2 liters per sample.
3.4. As soon as the last few drops of water have passed through the
filter, remove the filter tower from the base, fold the filter
in half then in half again and plunge the folded filter into 5
ml of boiling TRIS buffer (pH 7.4; 0.02 M) which is kept partially
covered to eliminate evaporative volume loss. Try to avoid
"bumping" of the TRIS buffer (caused by superheating of the
buffer).
3.5. After a 5 minute extraction period, remove the tubes from the heating
block, allow to cool to approximately room temperature, secure the
rubber stoppers and freeze (-20 °C) in upright position.
3.6. In order to minimize sample cross-contamination it is best to start
with deepest sample, which, in most cases, contains the lowest
concentration of ATP.
4. Analysis
4.1. Prepare enough firefly lantern extract (Sigma Chemical Co., FLE-50)
to process all samples and at least ten external ATP reference
standards. Lyophilized FLE-50 should be reconstituted in 5 ml
distilled water and allowed to "age" at room temperature for at
least 6 hours (but no longer than 24 hours) in order to reduce
the background luminescence. Approximately 1 hour before
starting the assay, dilute each 50 mg vial of reconstituted
FLE-50 with 15 ml of sodium arsenate buffer (0.1 M, pH 7.4)
and 15 ml of MgSO4 (0.04 M). Immediately before use, filter
the FLE-50 mixture through a GF/F filter.
4.2. Turn on ATP photometer at least 30 minutes before use.
4.3. Prepare set of ATP reference standards ranging from 0.1-100 ng
ATP ml-1 (see Chapter 16, section 5).
4.4. Using the automatic injector and computer-assisted photometer,
analyze the peak height of light emission (0-15 seconds) for
each sample and standard.
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
where: A = absorption at 259 nm
E = ATP molar extinction coefficient (15.4 x 103)
l = path length (cm)
c = concentration of ATP in moles per liter
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
6.1. Plot ATP standard data and calculate linear regression statistics
for the standard curve. Use LOTUS spreadsheet to calculate ATP
concentration (ng l-1) from data on peak height (relative to
standards), volume of extract and volume of sample filtered.
6.2. ATP concentrations can be related to total biomass by applying
the extrapolation factor; 250 x ATP = C (Karl, 1980). This
relationship is based upon direct laboratory and field analyses
performed over the past two decades. While there is a range
in the C/ATP ratio for microorganisms grown under a variety
of environmental conditions, the relationship of C/ATP = 250
appears to be reasonable for samples collected from the
oligotrophic North Pacific Ocean (Laws et al., 1987).
7. Equipment/Supplies
Niskin bottles and rosette
sample bottles and tygon tubing
heating block and test tubes
ATP photometer (Biospherical Instrument Co.)
stainless forceps
glass fiber filters (Whatman; 47 mm GF/F)
8. Reagents
TRIS buffer (0.02 M, pH 7.4)
magnesium sulfate (0.04 M)
sodium arsenate buffer (0.1 M; pH 7.4)
firefly lantern extract (FLE-50; Sigma Chemical Co.)
reagent ATP (Sigma Chemical Co.)
9. References
Holm-Hansen, O. 1973. Determination of total microbial biomass
by measurement of adenosine triphosphate. In: Estuarine Microbial
Ecology, L. H. Stevenson and R. R. Colwell, editors, University
of South Carolina Press, Columbia, pp. 73-89.
Karl, D. M. 1980. Cellular nucleotide measurements and
applications in microbial ecology. Microbiological Reviews, 44,
739-796.
Karl, D. M. and O. Holm-Hansen. 1978. Methodology and measurement
of adenylate energy charge ratios in environmental samples.
Marine Biology, 48, 185-197.
Laws, E. A., D. G. Redalje, L. W. Haas, P. K. Bienfang, R. W.
Eppley, W. G. Harrison, D. M. Karl and J. Marra. 1984. High
phytoplankton growth and production rates in oligotrophic
Hawaiian coastal waters. Limnology and Oceanography, 29, 1161-1169.
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