ASTM D4012-23a

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Designation: D4012 − 23 D4012 − 23a
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content
associated with microorganisms normally found in laboratory cultures and waters in plankton and periphyton samples from waters.
1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the
concentration of ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are
converted by comparison with an ATP standard and computation to pg ATP/mL.
1.3 This method does not remove all known chemical interferences, known to either luminesce in the 530 nm 6 20 nm range, or
to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic
compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP
concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).
1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms (Appendix
X1).
1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility.
1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily
in the matrix being tested.
1.7 This test method is equally suitable for use in the laboratory or field.
1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log [pg cATP/mL]) to
4 000 000 pg cATP/mL (6.6 Log [pg cATP/mL]) in 50 mL water samples.
1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP,
although the method does not differentiate between ATP from different sources, for example, from different types of
microorganisms, such as bacteria, fungi, algae and protozoa.
1.10 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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.
Current edition approved July 1, 2023Nov. 1, 2023. Published August 2023November 2023. Originally approved in 1981. Last previous edition approved in 20152023
as D4012 – 15.D4012 – 23. DOI: 10.1520/D4012-23.10.1520/D4012-23A.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4012 − 23a
1.11 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, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.12 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1601 Test Method for Dilute Solution Viscosity of Ethylene Polymers
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration, and Reverse Osmosis Membrane Processes
D6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and
Lubricants
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample
Concentration by Filtration
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E2694 Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids
F1671 Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using
Phi-X174 Bacteriophage Penetration as a Test System
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to Terminologies D1129 and D4175.
3.1.2 adenosine monophosphate (AMP), n—molecule formed by the removal of two molecules of phosphate (one pyrophosphate
molecule) from ATP.
3.1.3 adenosine triphosphate (ATP), n—molecule comprised of a purine and three phosphate groups that serves as the primary
energy transport molecule in all biological cells.
3.1.4 aseptic, adj—sterile, free from viable microbial contamination.
3.1.5 background RLU, n—quantity of relative light units resulting from running the test method without incorporation of the
sample.
3.1.6 bioluminescence, n—production and emission of light by a living organism as the result of a chemical reaction during which
chemical energy is converted to light energy.
3.1.7 biomass, n—any matter which is or was a living organism or excreted from a microorganism. D6161
3.1.8 cellular adenosine triphosphate (cellular-ATP; c-ATP), n—ATP present in whole cells, whether they are living or dead.
3.1.8.1 Discussion—
Cellular-ATP is released upon intentional lysis of microbial cells during the sample preparation process. Microbially infected fluids
contain both cellular (cell-associated/cell-bound) and extra-cellular ATP.
3.1.9 culturable, adj—(microorganisms that are) able to proliferate as indicated by the formation of colonies on solid growth
media or the development of turbidity in liquid growth media under specific growth conditions.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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3.1.10 extra-cellular, adj—(molecules or substances that are either) excreted by living cells or released from microbial cells that
have lysed (see 3.1.14) in the sample.
3.1.10.1 Discussion—
Extra-cellular ATP is ATP that has been released from microbial cells that have either fully or partially lysed in the sample, the
upstream fluid (fuel or water phase), or both.
3.1.10.2 Discussion—
Lysis can occur due to natural life cycle process, antimicrobial treatment or a combination of these factors. Extra-cellular ATP can
under certain circumstances persist for periods greater than 24 h after cell lysis depending on physical/chemical conditions.
3.1.11 luciferase, n—general term for a class of enzymes that catalyze bioluminescent reactions.
3.1.12 luciferin, n—general term for a class of light-emitting biological pigments found in organisms capable of bioluminescence.
3.1.13 luminometer, n—instrument capable of measuring light emitted as a result of nonthermal excitation.
3.1.14 lysis, n—disintegration or destruction of whole bacterial cells. F1671
3.1.15 relative light unit (RLU), n—instrument and assay specific unit of measurement reflecting the number of photons emitted
by the Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.
3.1.15.1 Discussion—
RLU is not an SI unit, however, RLU are proportional to ATP concentration.
3.1.16 viable microbial biomass, n—metabolically active (living) microorganisms.
3.2 Acronyms:
3.2.1 AMP, n—adenosine monophosphate.
3.2.2 ATP, n—adenosine triphosphate.
3.2.3 HDPE, n—high density polyethylene.
3.2.4 PP, n—polypropylene.
-12
3.2.5 pg, n—picogram (1 × 10 g).
3.2.6 RLU, n—relative light unit.
4. Summary of Test Method
4.1 A control assay is performed using 100 μL of 1.0 ng 6 0.05 ng ATP/mL standard to produce RLU .
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4.2 A 50 mL sample of water is placed into a syringe and then pressure-filtered through a 0.7 μm, glass-fiber, in-line, depth filter.
4.3 A lysing reagent is used to release cellular-ATP from microbial cells that have been captured on the glass-fiber filter, and the
filtrate is dispensed into an unused culture tube.
4.4 The filtrate is diluted 1 to 10 with a buffer solution.
4.5 A 100 μL volume of diluted filtrate is transferred to an unused culture tube into which 100 μL of Luciferin-Luciferase reagent
has been previously dispensed.
4.6 The culture tube is placed into a luminometer and the light intensity is read as RLU .
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4.7 RLU is normalized to an actual pg ATP/mL concentration through an equation that accounts for the result of the control
obs
assay (RLU ), the volume of the sample processed, and the method dilution factor.
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NOTE 1—Optionally, for condition monitoring purposes, pg ATP/mL are converted to Log [pg ATP/mL] of sample by computation.
5. Significance and Use
5.1 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.
5.2 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells,
including bacteria, algae, protozoa, and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically
active microbial contamination in water. ATP is not associated with matter of non-biological origin.
5.3 The ATP (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.)
5.4 This test method can be used to:
5.4.1 Estimate viable microbial biomass in cultures and waters.
5.4.2 Estimate the amount of total viable biomass in plankton and periphyton samples.
5.4.3 Estimate the number of viable cells in a unispecies culture if the cATP content (or if the average amount of cATP) per cell
is known.
5.4.4 Estimate and differentiate between zooplanktonic, phytoplanktonic, bacterial, and fungal cATP through size fractionation of
water samples.
5.4.5 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.
5.5 This test method is similar to Test Methods D7687 and E2694 except for the volumes sampled, and omission of wash and
drying steps used in Test Methods D7687 and E2694 to remove interferences (1.3).
5.6 Although ATP data generally covary with culture data in water samples, different factors affect cATP concentration than those
that affect culturability.
5.6.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under
specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample may be
viable but not detected by any one culture test.
5.6.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total
bioburden (see Appendix X1).
5.6.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than
bacteria (Appendix X1).
5.6.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells, such as fungal
spores.
Sloan, W. T., Quince, C., and Curtis, T. P., “The Uncountables,” Accessing Uncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.
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5.6.2.3 The greater the total bioburden, the greater the ATP concentration in a sample.
6. Interferences
6.1 Reagents must be of high purity so that background light emission is held to a minimum for the measurement of ATP.
6.2 ATP-free glassware, prepared by the procedure in 7.5, is required for the determination of ATP.
6.3 Luciferase is a protein and as such can be inhibited or denatured through the presence of heavy metals, high salt (for example,
NaCl) concentrations, and organic solvents in the sample. Additionally, high amounts of color or turbidity in the sample can impede
light transmission, causing a negative bias. Although the method described herein is designed to mitigate such interferences in most
types of water, for samples with high amounts of such interferences consider the use of Test Methods D7687 and E2694.
6.4 Other energy-mediating compounds, such as adenosine diphosphate, cytidine-5-triphosphate, and inosine-5-triphosphate also
react with luciferase to produce light, but as compared to ATP they are usually present only in small amounts and do not constitute
a significant source of error.
7. Apparatus
7.1 Culture Tube, sterile, disposable, PP, 12 mm by 55 mm.
7.2 Culture Tube, sterile, disposable, PP, 17 mm by 100 mm with caps.
7.3 Filter, 25 mm, sterile, disposable, PP housing, in-line, 0.7 μm pore-size, glass-fiber depth-type with Luer-Lok inlet.
7.4 Luminometer, using photomultiplier tube, having a spectral range between 300 nm and 600 nm, and with a cuvette chamber
that can hold and provide an unobstructed line of sight to the reactants in a 12 mm by 55 mm test culture tube (7.1), providing
a ratio of RLU /RLU (refer to Section 10 and Appendix X5) ≤0.01 and optimally having five decades of linearity (refer
background ctrl
to Appendix X2).
NOTE 2—It is the responsibility of the user to ensure that the luminometer selected for use meets the criteria listed in 7.4 and to consult with the
luminometer manufacturer to ensure that use of the luminometer with the apparatus, reagents and materials described in Sections 6 and 7 does not result
in the inability of the instrument manufacturer to provide technical support or loss of instrument warranty.
NOTE 3—The preliminary interlaboratory study and data presented in Table X4.2, respectively, were developed using a Kikkoman Lumitester C-110,
which provides nominally a 5000 RLU and 50 RLU . Although this test method is optimized to function on this luminometer, users may
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examine the use of other luminometers according to key performance criteria, including linear measurement range (Appendix X2) and RLU level
background
(Appendix X5).
7.5 Macropipeter, adjustable, 1.0 mL to 5.0 mL.
7.6 Micropipeter, adjustable, 100 μL to 1000 μL.
7.7 Pipet Tips, sterile, disposable, PP, 100 μL to 1000 μL.
7.8 Pipet Tips, sterile, disposable, PP, 1.0 mL to 5.0 mL.
7.9 Sample Collection Container, sterile, wide-mouth bottle, PP or HDPE, 100 mL.
NOTE 4—ATP can adsorb onto glass surfaces. Consequently, PP or HDPE containers are strongly preferred.
The sole source of supply of the Kikkoman Lumitester C-110 apparatus known to the committee at this time is Hach Company, Colorado. If you are aware of alternative
suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical
committee, which you may attend.
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7.10 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.
7.11 Test Tube Rack, 12 mm.
7.12 Test Tube Rack, 17 mm.
7.13 Waste Receptacle, any container suitable for receiving and retaining filtrate fluid for ultimate disposal.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
8.2 Purity of Water—Unless otherwise indicated, references to water shall conform to Specification D1193, Type II.
8.3 ATP Standard Solution, 1.0 ng 6 0.05 ng ATP/mL—Weigh 119.3 mg of crystalline adenosine 5'-triphosphate-disodium salt
using ATP-free glassware. Dissolve the ATP in 100 mL of fresh 0.02 M tris buffer containing 29.2 mg of EDTA (Na H
2 2
EDTA·2H O) and 120 mg of MgSO (the resulting concentration is 1 mg of ATP/mL). The material may be dispensed in 1.0-mL
2 4
aliquots and stored at –20 °C until required.
8.4 ATP Extract Dilution Buffer, (proprietary).
8.5 ATP Extraction Reagent, (proprietary).
8.6 Luciferin-Luciferase Reagent, (proprietary). Store between –20 °C and 4 °C; allow to equilibrate to ambient temperature
before using.
NOTE 5—Follow manufacturers’ instructions regarding product storage and shelf life.
9. Hazards
9.1 Warning—Not all luminometers are explosion proof. Luminometers that are not explosion-proof should not be operated in
explosive atmospheres or in locations where there may be explosive fumes, as it cannot be grounded. Consult the manufacturer’s
guidelines for further information.
10. Precaution
10.1 This standard may involve the use of hazardous materials, operations, and equipment. It is the responsibility of whoever uses
this standard to establish appropriate safety practices and to determine the applicability of regulatory limitations prior to use.
11. Sample Collection
11.1 The sample sites should correspond as closely as possible to those selected for chemical, biological, and microbiological
sampling, so that there is maximum correlation of results. The sample collection method will be determined by study objectives.
To collect a sample, use a nonmetallic water sampling bottle. Extraction procedures should be performed immediately after
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
The sole source of supply of the proprietary ATP dilution buffer, ATP extraction reagent, filter wash reagent, and Luciferin-Luciferase reagent, is LuminUltra Technologies
Ltd., Fredericton, New Brunswick, Canada, http://www.luminultra.com. If you are aware of alternative suppliers, please provide this information to ASTM International
Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
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collection. The sample may be stored 2 h to 3 h if necessary if the temperature and lighting conditions are maintained; for example,
do not place a warm sample from a well-lighted area into a cool, dark ice chest.
12. Calibration and Standardization
12.1 It is necessary to perform only one calibration during each set of tests performed on the same day, using the same reagent
batches, under the same temperature, using the same materials and the same luminometer. In general, one calibration per day of
testing is sufficient so long as the aforementioned variables are kept the same.
NOTE 6—Although multi-point calibration is not required, guidance on performing a multi-point calibration is provided in Appendix X2.
12.2 Turn on power to luminometer (6.4) and allow instrument to equilibrate, in accordance with manufacturer’s recommenda-
tions.
12.2.1 It is possible, that during prolonged use, over the course of several hours, some luminometer models will heat up
sufficiently to affect the temperature of the reactants (13.25) to affect the test result (13.25). Excessive on-time can also stimulate
the photomultiplier tube to produce increased instrument background noise. Consequently, it is advisable to repeat 12.6 – 12.9 after
every 3 h of continuous operation.
12.3 Ensure that all reagents have equilibrated to ambient temperature (22 °C 6 3 °C) before running any tests.
NOTE 7—The kinetics of the enzymatic reaction measured by this procedure are affected by temperature.
12.3.1 It is important to ensure that the assay is performed at constant temperature (T 6 3 °C), that is, the instrument and all
reagents must remain at same temperature during the measurement series.
12.3.2 If the measurement results are to be compared to a standard curve or control samples these must be measured at the same
conditions and temperature as the test sample.
12.3.3 Changes in the instrument, diluted sample (13.19) ATP standard (8.3) or Luciferin-Luciferase reagent (8.6) temperature
while testing multiple samples will affect the RLU and consequently introduce a bias to the test results.
12.4 Use a micropipeter (7.6) with a new 100 μL to 1000 μL tip (7.7) to dispense 100 μL Luciferin-Luciferase reagent (8.6) to an
unused 12 mm by 55 mm culture tube (7.1).
12.5 Replace the micropipeter tip with a fresh 100 μL to 1000 μL tip.
12.6 Dispense 100 μL of 1 ng ATP/mL standard solution (8.3) into the culture tube.
12.7 Swirl gently for five times.
12.8 Place the culture tube into the luminometer.
12.9 Read and record RLU (RLU ).
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13. ATP Measurement Procedure
13.1 Use aseptic procedure while performing this test method; cellular-ATP from analyst’s hands, sputum, etc., can contaminate
the sample with cellular-ATP from sources other than the sample itself.
13.2 Remove plunger from a new 60 mL syringe (7.10) and place onto 17 mm test tube rack so that plunger tip does not contact
any surfaces so as to avoid contamination with ATP.
13.3 Affix filter (7.3) onto the 60 mL syringe.
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13.4 Place a fresh 1.0 mL to 5.0 mL tip (7.8) onto the macropipeter (7.5).
13.5 Shake sample for 15 s to ensure homogeneity.
13.6 With minimal delay, remove lid from sample container and, using the macropipeter, transfer ten volumes of 5.0 mL (50 mL
total) water sample, or one to the 60 mL syringe barrel.
13.6.1 If desired, consult Appendix X3 for guidelines on adjusting method detection range by modifying the standard sample
volume.
13.7 While holding the barrel over the waste receptacle (7.13), replace the plunger into the 60 mL syringe.
13.8 Apply even pressure to the 60 mL syringe plunger to pressure filter the sample, having filtrate discharge into the waste
receptacle.
13.8.1 For samples having high particulate loads, it may not be possible to filter the full subsample. When this occurs, record the
actual volume filtered and substitute this value for “V” in Eq 1 (14.1).
13.9 Remove filter from the 60 mL syringe and place onto 17 mm test tube rack (7.12) so that filter outlet does not contact any
surfaces.
13.10 Remove plunger from the 60 mL syringe and place onto 17 mm test tube rack so that plunger tip does not contact any
surfaces so as to avoid contamination with ATP.
13.11 Replace filter onto the end of the 60 mL syringe barrel.
13.12 Place an unused 17 mm by 100 mm culture tube (7.2) into 17 mm test tube rack.
13.13 Place a fresh 100 μL to 1000 μL pipet tip onto the micropipeter.
13.14 Use micropipeter to dispense 1.0 mL of ATP extraction reagent (8.5) into the 60 mL syringe barrel.
13.15 While holding the barrel over the 17 mm by 100 mm culture tube (13.12), replace the 60 mL syringe plunger.
13.16 Apply even pressure to the 60 mL syringe plunger, to dispense ATP Extraction Reagent and extracted ATP into the 17 mm
by 100 mm culture tube.
NOTE 8—At this point in the protocol, this ATP extract may be stored for up to seven days at 2 °C to 8 °C prior to completing the test.
13.17 If not already performed (12.2), turn power on luminometer (7.4) and allow instrument to equilibrate, in accordance with
manufacturer’s recommendations.
13.18 Place a fresh 1.0 mL to 5.0 mL pipet tip onto the macropipeter.
13.19 Use macropipeter to dispense two 4.5 mL portions (9.0 mL total) of ATP extract dilution buffer (8.4) into the culture tube
to prepare diluted ATP extract.
13.20 Place cap on culture tube and invert three times to mix well.
NOTE 9—Diluted ATP extract is stable for up to 4 h at room temperature (20 °C 6 2 °C).
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