ASTM D4012-81(2009)
(Test Method)Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
SIGNIFICANCE AND USE
A rapid and routine procedure for determining biomass of the living microorganisms in cultures, waters, wastewaters, and in plankton and periphyton samples taken from surface waters is frequently of vital importance. However, classical techniques such as direct microscope counts, turbidity, organic chemical analyses, cell tagging, and plate counts are expensive, time-consuming, or tend to underestimate total numbers. In addition, some of these methods do not distinguish between living and nonliving cells.
The ATP firefly (luciferin-luciferase) method is a rapid, sensitive determination of viable microbial biomass. ATP is the primary energy donor for life processes, does not exist in association with nonliving detrital material, and the amount of ATP per unit of biomass (expressed in weight) is relatively constant. (ATP per cell varies with species and physiological state of the organism.)
This test method can be used to:
Estimate viable microbial biomass in cultures, waters, and wastewaters.
Estimate the amount of total viable biomass in plankton and periphyton samples.
Estimate the number of viable cells in a unispecies culture if the ATP content (or if the average amount of ATP) per cell is known.
Estimate and differentiate between zooplanktonic, phytoplanktonic, bacterial, and fungal ATP through size fractionation of water, and wastewater samples.
Measure the mortality rate of microorganisms in toxicity tests in entrainment studies, and in other situations where populations or assemblages of microorganisms are placed under stress.
SCOPE
1.1 This test method covers the measurement of adenosine triphosphate (ATP) in microorganisms in concentrations normally found in laboratory cultures, waters, wastewaters, and in plankton and periphyton samples from waters.
1.2 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity, or by utilizing an average concentration (or amount) of ATP per cell, an estimated count of microorganisms can be obtained in the case of unispecies cultures.
1.3 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility. However, extreme care must be taken at each step in the analysis to ensure meaningful and reliable results.
1.4 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily in the matrix being tested.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D4012 − 81(Reapproved 2009)
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
in Water
This standard is issued under the fixed designation D4012; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
3.1 Definitions—For definitions of terms used in this test
1.1 This test method covers the measurement of adenosine
method, refer to Terminology D1129.
triphosphate (ATP) in microorganisms in concentrations nor-
mally found in laboratory cultures, waters, wastewaters, and in
4. Summary of Test Method
plankton and periphyton samples from waters.
4.1 The biomass in the sample can be determined by direct
1.2 KnowledgeoftheconcentrationofATPcanberelatedto
ATP extraction when cell counts are greater than 20000
viable biomassormetabolicactivity,orbyutilizinganaverage
microorganisms per millilitre. When the cell counts are less
concentration (or amount) ofATP per cell, an estimated count
than 20000 microorganisms per millilitre, the sample may be
of microorganisms can be obtained in the case of unispecies
concentrated using either centrifugation or filtration prior to
cultures.
ATP extraction.
1.3 This test method offers a high degree of sensitivity,
4.2 TheATPis extracted from the sample with boiling 0.02
rapidity, accuracy, and reproducibility. However, extreme care
M tris buffer.
mustbetakenateachstepintheanalysistoensuremeaningful
and reliable results. 4.3 AcarefullymeasuredaliquotoftheATPextractismixed
with a standard quantity of buffered luciferin-luciferase reac-
1.4 Theanalystshouldbeawarethattheprecisionstatement
tion mixture and the light produced in the resulting reaction is
pertains only to determinations in reagent water and not
measured with an appropriate photometric analyzer.
necessarily in the matrix being tested.
4.4 The data obtained from the test can be expressed in
1.5 The values stated in SI units are to be regarded as
terms of ATP content or biomass.
standard. No other units of measurement are included in this
standard.
5. Significance and Use
1.6 This standard does not purport to address all of the
5.1 A rapid and routine procedure for determining biomass
safety concerns, if any, associated with its use. It is the
of the living microorganisms in cultures, waters, wastewaters,
responsibility of the user of this standard to establish appro-
and in plankton and periphyton samples taken from surface
priate safety and health practices and determine the applica-
waters is frequently of vital importance. However, classical
bility of regulatory limitations prior to use.
techniques such as direct microscope counts, turbidity, organic
chemicalanalyses,celltagging,andplatecountsareexpensive,
2. Referenced Documents
time-consuming, or tend to underestimate total numbers. In
addition, some of these methods do not distinguish between
2.1 ASTM Standards:
living and nonliving cells.
D1129Terminology Relating to Water
D1193Specification for Reagent Water
5.2 TheATP firefly (luciferin-luciferase) method is a rapid,
sensitivedeterminationofviablemicrobialbiomass.ATPisthe
primary energy donor for life processes, does not exist in
association with nonliving detrital material, and the amount of
This test method is under the jurisdiction of ASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
ATP per unit of biomass (expressed in weight) is relatively
Current edition approved May 1, 2009. Published June 2009. Originally
constant. (ATP per cell varies with species and physiological
approved in 1981. Last previous edition approved in 2002 as D4012–81 (2002).
state of the organism.)
DOI: 10.1520/D4012-81R09.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
5.3 This test method can be used to:
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
5.3.1 Estimate viable microbial biomass in cultures, waters,
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. and wastewaters.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4012 − 81 (2009)
5.3.2 Estimate the amount of total viable biomass in plank- tee onAnalytical Reagents of theAmerican Chemical Society,
ton and periphyton samples. where such specifications are available. Other grades may be
5.3.3 Estimate the number of viable cells in a unispecies used, provided it is first ascertained that the reagent is of
cultureiftheATPcontent(oriftheaverageamountofATP)per sufficiently high purity to permit its use without lessening the
cell is known. accuracy of the determination.
5.3.4 Estimate and differentiate between zooplanktonic,
8.2 Purity of Water—Unless otherwise indicated, references
phytoplanktonic, bacterial, and fungal ATP through size frac-
to water shall conform to Specification D1193, Type II.
tionation of water, and wastewater samples.
5.3.5 Measure the mortality rate of microorganisms in 8.3 ATP Standard Solution—Weigh 119.3 mg of crystalline
toxicity tests in entrainment studies, and in other situations adenosine 5`-triphosphate-disodium salt usingATP-free glass-
where populations or assemblages of microorganisms are ware. Dissolve the ATP in 100 mL of fresh 0.02 M tris buffer
placed under stress. 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. Interferences
mL). The material may be dispensed in 1.0-mL aliquots and
stored at−20°C until required.
6.1 Reagentsmustbeofhighpuritysothatbackgroundlight
emission is held to a minimum for the measurement of ATP.
8.4 Extraction Reagent—ATP can be extracted from
samples by various reagents and procedures. The most com-
6.2 ATP-freeglassware,preparedbytheprocedurein7.5,is
monly used extracting reagent is boiling tris buffer (see 8.8).
required for the determination of ATP.
6.3 Luciferase is a protein and as such can be inhibited or
8.5 HydrochloricAcid(17mL/L)—Add17.0mLofHCl(sp
denatured by the presence of heavy metals, high salt (NaCl)
gr 1.19) to a 1-L volumetric flask and bring to volume with
concentrations, and organic solvents, in the sample. The ATP
water.
luciferasereactionisalsoaffectedbycertainphosphatebuffers,
8.6 (LR) Water, Low-Response—(Sterile ATP-free water
inorganic salts, and by high magnesium concentrations.
may be prepared by treatment in a suitable system involving
6.4 Other energy-mediating compounds, such as adenosine
carbon treatment with deionization, filtration glass distillation,
diphosphate, cytidine-5-triphosphate, and inosine-5-
orsterilizationbyautoclavingandstoredunderrefrigerationin
triphosphate also react with luciferase to produce light, but as
stoppered flasks.
compared to ATP they are usually present only in small
8.7 Luciferase/Luciferin Reaction Mixture—This material is
amounts and do not constitute a significant source of error.
commercially available and should be prepared in accordance
6.5 High-viscosity samples may not mix adequately with
with the supplier’s instructions. Note the following when
thereagentsuponinjection.Ifthisoccurs,reactionratemaybe
preparing this material:
reduced (reaction will go to completion, but the reaction rate
8.7.1 Clean glassware must be used.
willbedecreasedwithimpropermixing)ortheresultsmaynot
8.7.2 The luciferase/luciferin reaction mixture must be
be reproducible.
mixed gently without shaking.
7. Apparatus
8.8 Tris Buffer (0.02 M) (Tris(Hydroxymethyl)
7.1 ATP Photometers or Liquid Scintillation Aminomethane)—Dissolve2.5gofthebuffercrystalsin1Lof
deionized water. Bring to pH 7.75 using HCl (pH meter).
Spectrometers—may be used. The stability of the instrument
shouldbecheckedbeforeeachusewithastandardlightsource Sterilize by autoclaving for 30 min at 121°C, 15 psi (103 kPa)
pressure, and store refrigerated in stoppered flasks.
available from the manufacturer. It is advisable to maintain a
record of the instrument response to permit detection of any
NOTE1—BacteriamayliveandmultiplyintheLRwaterandtrisbuffer;
unstability or changes in response levels.
this can introduce an ATP interference. The quality of the LR water and
tris buffer should be periodically tested.
7.2 Vacuum Filtration System (0.45-µm membrane filters).
7.3 Precision Syringe, 50-µL. A constant-rate injection at-
9. Precaution
tachment is recommended.
9.1 This standard may involve the use of hazardous mate-
7.4 Automatic Pipets and Disposable Tips.
rials, operations, and equipment. It is the responsibility of
7.5 ATP-Free Glassware—Rinse chemically clean glass-
whoever uses this standard to establish appropriate safety
ware three times with 0.2 N HCl, rinse three times with tris
practices and to determine the applicability of regulatory
buffer (8.8), and rinse three times with low-response water
limitations prior to use.
(8.6).
7.6 Reaction Vial, 6 by 49-mm.
Reagent Chemicals, American Chemical Society Specifications, American
8. Reagents and Materials
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
listed by the American Chemical Society, see Annual Standards for Laboratory
8.1 Purity of Reagents—Reagent grade chemicals shall be
Chemicals, BDH Ltd., Poole
...
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