ASTM D4012-81(2002)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:D 4012–81(Reapproved 2002)
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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope microorganisms per millilitre. When the cell counts are less
than 20000 microorganisms per millilitre, the sample may be
1.1 This test method covers the measurement of adenosine
concentrated using either centrifugation or filtration prior to
triphosphate (ATP) in microorganisms in concentrations nor-
ATP extraction.
mally found in laboratory cultures, waters, wastewaters, and in
4.2 TheATPis extracted from the sample with boiling 0.02
plankton and periphyton samples from waters.
M tris buffer.
1.2 KnowledgeoftheconcentrationofATPcanberelatedto
4.3 AcarefullymeasuredaliquotoftheATPextractismixed
viable biomassormetabolicactivity,orbyutilizinganaverage
with a standard quantity of buffered luciferin-luciferase reac-
concentration (or amount) ofATP per cell, an estimated count
tion mixture and the light produced in the resulting reaction is
of microorganisms can be obtained in the case of unispecies
measured with an appropriate photometric analyzer.
cultures.
4.4 The data obtained from the test can be expressed in
1.3 This test method offers a high degree of sensitivity,
terms of ATP content or biomass.
rapidity, accuracy, and reproducibility. However, extreme care
mustbetakenateachstepintheanalysistoensuremeaningful
5. Significance and Use
and reliable results.
5.1 A rapid and routine procedure for determining biomass
1.4 Theanalystshouldbeawarethattheprecisionstatement
of the living microorganisms in cultures, waters, wastewaters,
pertains only to determinations in reagent water and not
and in plankton and periphyton samples taken from surface
necessarily in the matrix being tested.
waters is frequently of vital importance. However, classical
1.5 This standard does not purport to address all of the
techniques such as direct microscope counts, turbidity, organic
safety concerns, if any, associated with its use. It is the
chemicalanalyses,celltagging,andplatecountsareexpensive,
responsibility of the user of this standard to establish appro-
time-consuming, or tend to underestimate total numbers. In
priate safety and health practices and determine the applica-
addition, some of these methods do not distinguish between
bility of regulatory limitations prior to use.
living and nonliving cells.
2. Referenced Documents 5.2 The ATP firefly (luciferin-luciferase) method is a rapid,
sensitivedeterminationofviablemicrobialbiomass.ATPisthe
2.1 ASTM Standards:
2 primary energy donor for life processes, does not exist in
D 1129 Terminology Relating to Water
association with nonliving detrital material, and the amount of
D 1193 Specification for Reagent Water
ATP per unit of biomass (expressed in weight) is relatively
3. Terminology
constant. (ATP per cell varies with species and physiological
state of the organism.)
3.1 Definitions—For definitions of terms used in this test
5.3 This test method can be used to:
method, refer to Terminology D1129.
5.3.1 Estimate viable microbial biomass in cultures, waters,
4. Summary of Test Method
and wastewaters.
5.3.2 Estimate the amount of total viable biomass in plank-
4.1 The biomass in the sample can be determined by direct
ton and periphyton samples.
ATP extraction when cell counts are greater than 20000
5.3.3 Estimate the number of viable cells in a unispecies
cultureiftheATPcontent(oriftheaverageamountofATP)per
ThistestmethodisunderthejurisdictionofCommitteeD19onWaterandisthe
cell is known.
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 4012–81 (2002)
5.3.4 Estimate and differentiate between zooplanktonic, sufficiently high purity to permit its use without lessening the
phytoplanktonic, bacterial, and fungal ATP through size frac- accuracy of the determination.
tionation of water, and wastewater samples.
8.2 Purity of Water—Unlessotherwiseindicated,references
5.3.5 Measure the mortality rate of microorganisms in
to water shall conform to Specification D1193, Type II.
toxicity tests in entrainment studies, and in other situations
8.3 ATP Standard Solution—Weigh 119.3 mg of crystalline
where populations or assemblages of microorganisms are
adenosine 58-triphosphate-disodium salt usingATP-free glass-
placed under stress.
ware. Dissolve the ATP in 100 mL of fresh 0.02 M tris buffer
containing 29.2 mg of EDTA (Na H EDTA·2H O) and 120
2 2 2
6. Interferences
mg of MgSO (the resulting concentration is 1 mg of ATP/
6.1 Reagentsmustbeofhighpuritysothatbackgroundlight
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-freeglassware,preparedbytheprocedurein7.5,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)—Add17.0mLofHCl(sp
concentrations, and organic solvents, in the sample. The ATP
gr 1.19) to a 1-L volumetric flask and bring to volume with
luciferasereactionisalsoaffectedbycertainphosphatebuffers,
water.
inorganic salts, and by high magnesium concentrations.
6.4 Other energy-mediating compounds, such as adenosine 8.6 (LR) Water, Low-Response—(Sterile ATP-free water
diphosphate, cytidine-5-triphosphate, and inosine-5-
may be prepared by treatment in a suitable system involving
triphosphate also react with luciferase to produce light, but as carbon treatment with deionization, filtration glass distillation,
compared to ATP they are usually present only in small
orsterilizationbyautoclavingandstoredunderrefrigerationin
amounts and do not constitute a significant source of error.
stoppered flasks.
6.5 High-viscosity samples may not mix adequately with
8.7 Luciferase/Luciferin Reaction Mixture—Thismaterialis
thereagentsuponinjection.Ifthisoccurs,reactionratemaybe
commercially available and should be prepared in accordance
reduced (reaction will go to completion, but the reaction rate
with the supplier’s instructions. Note the following when
willbedecreasedwithimpropermixing)ortheresultsmaynot
preparing this material:
be reproducible.
8.7.1 Clean glassware must be used.
8.7.2 The luciferase/luciferin reaction mixture must be
7. Apparatus
mixed gently without shaking.
7.1 ATP Photometers or Liquid Scintillation
8.8 Tris Buffer (0.02 M) (Tris(Hydroxymethyl)
Spectrometers—may be used. The stability of the instrument
Aminomethane)—Dissolve2.5gofthebuffercrystalsin1Lof
shouldbecheckedbeforeeachusewithastandardlightsource
deionized water. Bring to pH 7.75 using HCl (pH meter).
available from the manufacturer. It is advisable to maintain a
Sterilize by autoclaving for 30 min at 121°C, 15 psi (103 kPa)
record of the instrument response to permit detection of any
pressure, and store refrigerated in stoppered flasks.
unstability or changes in response levels.
7.2 Vacuum Filtration System (0.45-µm membrane filters).
NOTE 1—Bacteria may live and multiply in the LR water and tris
7.3 Precision Syringe, 50-µL. A constant-rate injection at-
buffer;thiscanintroduceanATPinterference.ThequalityoftheLRwater
tachment is recommended.
and tris buffer should be periodically tested.
7.4 Automatic Pipets and Disposable Tips.
7.5 ATP-Free Glassware—Rinse chemically clean glass- 9. Precaution
ware three times with 0.2 N HCl, rinse three times with tris
9.1 This standard may involve the use of hazardous mate-
buffer (8.8), and rinse three times with low-response water
rials, operations, and equipment. It is the responsibility of
(8.6).
whoever uses this standard to establish appropriate safety
7.6 Reaction Vial, 6 by 49-mm.
practices and to determine the applicability of regulatory
limitations prior to use.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be
10. Collection
used in all tests. Unless otherwise indicated, it is intended that
10.1 The sample sites should correspond as closely as
all reagents shall conform to the specifications of the Commit-
possible to those selected for chemical, biological, and micro-
tee onAnalytical Reagents of theAmerican Chemical Society,
biological sampling, so that there is maximum correlation of
where such specifications are available. Other grades may be
results. The sample collection method will be determined by
used, provided it is first ascertained that the reagent is of
study obj
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