ASTM D4012-81(1997)

Designation: D 4012 – 81(Reapproved 1997)
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
in Water
This standard is issued under the fixed designation D 4012; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4.2 The ATP is extracted from the sample with boiling 0.02
M tris buffer.
1.1 This test method covers the measurement of adenosine
4.3 A carefully measured aliquot of the ATP extract is mixed
triphosphate (ATP) in microorganisms in concentrations nor-
with a standard quantity of buffered luciferin-luciferase reac-
mally found in laboratory cultures, waters, wastewaters, and in
tion mixture and the light produced in the resulting reaction is
plankton and periphyton samples from waters.
measured with an appropriate photometric analyzer.
1.2 Knowledge of the concentration of ATP can be related to
4.4 The data obtained from the test can be expressed in
viable biomass or metabolic activity, or by utilizing an average
terms of ATP content or biomass.
concentration (or amount) of ATP per cell, an estimated count
of microorganisms can be obtained in the case of unispecies
5. Significance and Use
cultures.
5.1 A rapid and routine procedure for determining biomass
1.3 This test method offers a high degree of sensitivity,
of the living microorganisms in cultures, waters, wastewaters,
rapidity, accuracy, and reproducibility. However, extreme care
and in plankton and periphyton samples taken from surface
must be taken at each step in the analysis to ensure meaningful
waters is frequently of vital importance. However, classical
and reliable results.
techniques such as direct microscope counts, turbidity, organic
1.4 The analyst should be aware that the precision statement
chemical analyses, cell tagging, and plate counts are expensive,
pertains only to determinations in reagent water and not
time-consuming, or tend to underestimate total numbers. In
necessarily in the matrix being tested.
addition, some of these methods do not distinguish between
1.5 This standard does not purport to address all of the
living and nonliving cells.
safety concerns, if any, associated with its use. It is the
5.2 The ATP firefly (luciferin-luciferase) method is a rapid,
responsibility of the user of this standard to establish appro-
sensitive determination of viable microbial biomass. ATP is the
priate safety and health practices and determine the applica-
primary energy donor for life processes, does not exist in
bility of regulatory limitations prior to use.
association with nonliving detrital material, and the amount of
2. Referenced Documents ATP per unit of biomass (expressed in weight) is relatively
constant. (ATP per cell varies with species and physiological
2.1 ASTM Standards:
state of the organism.)
D 1129 Terminology Relating to Water
5.3 This test method can be used to:
D 1193 Specification for Reagent Water
5.3.1 Estimate viable microbial biomass in cultures, waters,
3. Terminology
and wastewaters.
5.3.2 Estimate the amount of total viable biomass in plank-
3.1 Definitions—For definitions of terms used in this test
ton and periphyton samples.
method, refer to Terminology D 1129.
5.3.3 Estimate the number of viable cells in a unispecies
4. Summary of Test Method
culture if the ATP content (or if the average amount of ATP) per
cell is known.
4.1 The biomass in the sample can be determined by direct
5.3.4 Estimate and differentiate between zooplanktonic,
ATP extraction when cell counts are greater than 20 000
phytoplanktonic, bacterial, and fungal ATP through size frac-
microorganisms per millilitre. When the cell counts are less
tionation of water, and wastewater samples.
than 20 000 microorganisms per millilitre, the sample may be
5.3.5 Measure the mortality rate of microorganisms in
concentrated using either centrifugation or filtration prior to
toxicity tests in entrainment studies, and in other situations
ATP extraction.
where populations or assemblages of microorganisms are
placed under stress.
This test method is under the jurisdiction of Committee D-19 on Water and is
the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved May 29, 1981. Published September 1981.
Annual Book of ASTM Standards, Vol 11.01.
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D 4012
6. Interferences containing 29.2 mg of EDTA (Na H EDTA·2H O) and 120
2 2 2
mg of MgSO (the resulting concentration is 1 mg of ATP/
6.1 Reagents must be of high purity so that background light
mL). The material may be dispensed in 1.0-mL aliquots and
emission is held to a minimum for the measurement of ATP.
stored at − 20°C until required.
6.2 ATP-free glassware, prepared by the procedure in 7.5, is
8.4 Extraction Reagent—ATP can be extracted from
required for the determination of ATP.
samples by various reagents and procedures. The most com-
6.3 Luciferase is a protein and as such can be inhibited or
monly used extracting reagent is boiling tris buffer (see 8.8).
denatured by the presence of heavy metals, high salt (NaCl)
8.5 Hydrochloric Acid (17 mL/L)—Add 17.0 mL of HCl (sp
concentrations, and organic solvents, in the sample. The ATP
gr 1.19) to a 1-L volumetric flask and bring to volume with
luciferase reaction is also affected by certain phosphate buffers,
water.
inorganic salts, and by high magnesium concentrations.
8.6 (LR) Water, Low-Response—(Sterile ATP-free water
6.4 Other energy-mediating compounds, such as adenosine
may be prepared by treatment in a suitable system involving
diphosphate, cytidine-5-triphosphate, and inosine-5-
carbon treatment with deionization, filtration glass distillation,
triphosphate also react with luciferase to produce light, but as
or sterilization by autoclaving and stored under refrigeration in
compared to ATP they are usually present only in small
stoppered flasks.
amounts and do not constitute a significant source of error.
8.7 Luciferase/Luciferin Reaction Mixture—This material is
6.5 High-viscosity samples may not mix adequately with
commercially available and should be prepared in accordance
the reagents upon injection. If this occurs, reaction rate may be
with the supplier’s instructions. Note the following when
reduced (reaction will go to completion, but the reaction rate
preparing this material:
will be decreased with improper mixing) or the results may not
8.7.1 Clean glassware must be used.
be reproducible.
8.7.2 The luciferase/luciferin reaction mixture must be
mixed gently without shaking.
7. Apparatus
8.8 Tris Buffer (0.02 M) (Tris(Hydroxymethyl)
7.1 ATP Photometers or Liquid Scintillation
Aminomethane)—Dissolve 2.5 g of the buffer crystals in 1 L of
Spectrometers—may be used. The stability of the instrument
deionized water. Bring to pH 7.75 using HCl (pH meter).
should be checked before each use with a standard light source
Sterilize by autoclaving for 30 min at 121°C, 15 psi (103 kPa)
available from the manufacturer. It is advisable to maintain a
pressure, and store refrigerated in stoppered flasks.
record of the instrument response to permit detection of any
unstability or changes in response levels. NOTE 1—Bacteria may live and multiply in the LR water and tris
buffer; this can introduce an ATP interference. The quality of the LR water
7.2 Vacuum Filtration System (0.45-μm membrane filters).
and tris buffer should be periodically tested.
7.3 Precision Syringe, 50-μL. A constant-rate injection at-
tachment is recommended.
9. Precaution
7.4 Automatic Pipets and Disposable Tips.
9.1 This standard may involve the use of hazardous mate-
7.5 ATP-Free Glassware—Rinse chemically clean glass-
rials, operations, and equipment. It is the responsibility of
ware three times with 0.2 N HCl, rinse three times with tris
whoever uses this standard to establish appropriate safety
buffer (8.8), and rinse three times with low-response water
practices and to determine the applicability of regulatory
(8.6).
limitations prior to use.
7.6 Reaction Vial, 6 by 49-mm.
10. Collection
8. Reagents and Materials
10.1 The sample sites should correspond as closely as
8.1 Purity of Reagents—Reagent grade chemicals shall be
possible to those selected for chemical, biological, and micro-
used in all tests. Unless otherwise indicated, it is intended that
biological sampling, so that there is maximum correlation of
all reagents shall conform to the specifications of the Commit-
results. The sample collection method will be determined by
tee on Analytical Reagents of the American Chemical Society,
study objectives. To collect a sample, use a nonmetallic water
where such specifications are available. Other grades may be
sampling bottle. Extraction procedures should be performed
used, provided it is first ascertained that the reagent is of
immediately after collection. The sample may be stored 2 to 3
sufficiently high purity to permit its use without lessening the
h if necessary if the temperature and lighting conditions are
accuracy of the determination.
maintained; for example, do not place a warm sample from a
8.2 Purity of Water—Unless otherwise indicated, references
well-lighted area into a cool, d
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