1、Designation: D 4012 81(Reapproved 2002)Standard Test Method forAdenosine Triphosphate (ATP) Content of Microorganismsin Water1This standard is issued under the fixed designation D 4012; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the measurement of adenosinetriphosphate (ATP) in microorganisms in concentrat
3、ions nor-mally found in laboratory cultures, waters, wastewaters, and inplankton and periphyton samples from waters.1.2 Knowledge of the concentration ofATPcan be related toviable biomass or metabolic activity, or by utilizing an averageconcentration (or amount) of ATP per cell, an estimated countof
4、 microorganisms can be obtained in the case of unispeciescultures.1.3 This test method offers a high degree of sensitivity,rapidity, accuracy, and reproducibility. However, extreme caremust be taken at each step in the analysis to ensure meaningfuland reliable results.1.4 The analyst should be aware
5、 that the precision statementpertains only to determinations in reagent water and notnecessarily in the matrix being tested.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appr
6、o-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to Water2D 1193 Specification for Reagent Water23. Terminology3.1 DefinitionsFor definitions of terms used in this testme
7、thod, refer to Terminology D 1129.4. Summary of Test Method4.1 The biomass in the sample can be determined by directATP extraction when cell counts are greater than 20 000microorganisms per millilitre. When the cell counts are lessthan 20 000 microorganisms per millilitre, the sample may beconcentra
8、ted using either centrifugation or filtration prior toATP extraction.4.2 The ATP is extracted from the sample with boiling 0.02M tris buffer.4.3 Acarefully measured aliquot of theATPextract is mixedwith a standard quantity of buffered luciferin-luciferase reac-tion mixture and the light produced in
9、the resulting reaction ismeasured with an appropriate photometric analyzer.4.4 The data obtained from the test can be expressed interms of ATP content or biomass.5. Significance and Use5.1 A rapid and routine procedure for determining biomassof the living microorganisms in cultures, waters, wastewat
10、ers,and in plankton and periphyton samples taken from surfacewaters is frequently of vital importance. However, classicaltechniques such as direct microscope counts, turbidity, organicchemical analyses, cell tagging, and plate counts are expensive,time-consuming, or tend to underestimate total numbe
11、rs. Inaddition, some of these methods do not distinguish betweenliving and nonliving cells.5.2 The ATP firefly (luciferin-luciferase) method is a rapid,sensitive determination of viable microbial biomass.ATP is theprimary energy donor for life processes, does not exist inassociation with nonliving d
12、etrital material, and the amount ofATP per unit of biomass (expressed in weight) is relativelyconstant. (ATP per cell varies with species and physiologicalstate of the organism.)5.3 This test method can be used to:5.3.1 Estimate viable microbial biomass in cultures, waters,and wastewaters.5.3.2 Esti
13、mate the amount of total viable biomass in plank-ton and periphyton samples.5.3.3 Estimate the number of viable cells in a unispeciesculture if theATPcontent (or if the average amount ofATP) percell is known.1This test method is under the jurisdiction of Committee D19 on Water and is thedirect respo
14、nsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved May 29, 1981. Published September 1981.2Annual Book of ASTM Standards, Vol 11.01.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.3.4 Estimate and diffe
15、rentiate between zooplanktonic,phytoplanktonic, bacterial, and fungal ATP through size frac-tionation of water, and wastewater samples.5.3.5 Measure the mortality rate of microorganisms intoxicity tests in entrainment studies, and in other situationswhere populations or assemblages of microorganisms
16、 areplaced under stress.6. Interferences6.1 Reagents must be of high purity so that background lightemission is held to a minimum for the measurement of ATP.6.2 ATP-free glassware, prepared by the procedure in 7.5,isrequired for the determination of ATP.6.3 Luciferase is a protein and as such can be
17、 inhibited ordenatured by the presence of heavy metals, high salt (NaCl)concentrations, and organic solvents, in the sample. The ATPluciferase reaction is also affected by certain phosphate buffers,inorganic salts, and by high magnesium concentrations.6.4 Other energy-mediating compounds, such as ad
18、enosinediphosphate, cytidine-5-triphosphate, and inosine-5-triphosphate also react with luciferase to produce light, but ascompared to ATP they are usually present only in smallamounts and do not constitute a significant source of error.6.5 High-viscosity samples may not mix adequately withthe reage
19、nts upon injection. If this occurs, reaction rate may bereduced (reaction will go to completion, but the reaction ratewill be decreased with improper mixing) or the results may notbe reproducible.7. Apparatus7.1 ATP Photometers or Liquid ScintillationSpectrometersmay be used. The stability of the in
20、strumentshould be checked before each use with a standard light sourceavailable from the manufacturer. It is advisable to maintain arecord of the instrument response to permit detection of anyunstability or changes in response levels.7.2 Vacuum Filtration System (0.45-m membrane filters).7.3 Precisi
21、on Syringe, 50-L. A constant-rate injection at-tachment is recommended.7.4 Automatic Pipets and Disposable Tips.7.5 ATP-Free GlasswareRinse chemically clean glass-ware three times with 0.2 N HCl, rinse three times with trisbuffer (8.8), and rinse three times with low-response water(8.6).7.6 Reaction
22、 Vial, 6 by 49-mm.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where such spe
23、cifications are available.3Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.8.2 Purity of WaterUnless otherwise indicated, referencesto water shall conform to Specification D
24、1193, Type II.8.3 ATP Standard SolutionWeigh 119.3 mg of crystallineadenosine 58-triphosphate-disodium salt using ATP-free glass-ware. Dissolve the ATP in 100 mL of fresh 0.02 M tris buffercontaining 29.2 mg of EDTA (Na2H2EDTA2H2O) and 120mg of MgSO4(the resulting concentration is 1 mg of ATP/mL). T
25、he material may be dispensed in 1.0-mL aliquots andstored at 20C until required.8.4 Extraction ReagentATP can be extracted fromsamples by various reagents and procedures. The most com-monly used extracting reagent is boiling tris buffer (see 8.8).8.5 Hydrochloric Acid (17 mL/L)Add 17.0 mL of HCl (sp
26、gr 1.19) to a 1-L volumetric flask and bring to volume withwater.8.6 (LR) Water, Low-Response(Sterile ATP-free watermay be prepared by treatment in a suitable system involvingcarbon treatment with deionization, filtration glass distillation,or sterilization by autoclaving and stored under refrigerat
27、ion instoppered flasks.8.7 Luciferase/Luciferin Reaction MixtureThis material iscommercially available and should be prepared in accordancewith the suppliers instructions. Note the following whenpreparing this material:8.7.1 Clean glassware must be used.8.7.2 The luciferase/luciferin reaction mixtur
28、e must bemixed gently without shaking.8.8 Tris Buffer (0.02 M) (Tris(Hydroxymethyl)Aminomethane)Dissolve 2.5 g of the buffer crystals in 1 L ofdeionized water. Bring to pH 7.75 using HCl (pH meter).Sterilize by autoclaving for 30 min at 121C, 15 psi (103 kPa)pressure, and store refrigerated in stopp
29、ered flasks.NOTE 1Bacteria may live and multiply in the LR water and trisbuffer; this can introduce anATPinterference. The quality of the LR waterand tris buffer should be periodically tested.9. Precaution9.1 This standard may involve the use of hazardous mate-rials, operations, and equipment. It is
30、 the responsibility ofwhoever uses this standard to establish appropriate safetypractices and to determine the applicability of regulatorylimitations prior to use.10. Collection10.1 The sample sites should correspond as closely aspossible to those selected for chemical, biological, and micro-biologi
31、cal sampling, so that there is maximum correlation ofresults. The sample collection method will be determined bystudy objectives. To collect a sample, use a nonmetallic watersampling bottle. Extraction procedures should be performedimmediately after collection. The sample may be stored 2 to 3h if ne
32、cessary if the temperature and lighting conditions aremaintained; for example, do not place a warm sample from awell-lighted area into a cool, dark ice chest.3“Reagent Chemicals, American Chemical Society Specifications,” AmericanChemical Society, Washington, DC. For suggestions on the testing of re
33、agents notlisted by the American Chemical Society, see “Reagent Chemicals and Standards,”by Joseph Rosin, D. Van Nostrand Co., Inc., New York, NY, and the “United StatesPharmacopeia.”D 4012 81 (2002)211. ATP Extraction Procedures11.1 Accurate determinations of ATP require quantitativeextraction ofAT
34、P from the sample. Separate the cells from anypossible free (extracellular)ATPand other interfering materialsby filtration, centrifugation, sample washing, etc. Omit theseparation step if the sample is known to be free of solubleATPor interfering material. After separation, lyse the cell wall tofree
35、 ATP for subsequent analysis. Perform three replicate(triplicate) analyses on each sample to ensure the efficiency,reliability, and reproducibility of the method employed.11.2 Procedure ABoiling 0.02 M Tris Filtration Method:11.2.1 Filter 100 mL of sample through a 0.45-m mem-brane filter.11.2.2 Rem
36、ove the filter as soon as the filtration is complete.Do not allow the filter to dry. Break the vacuum just as the lastof the water passes through the filter and quickly transfer thefilter and place in 5.0 mL of boiling 0.02 M tris buffer.11.2.3 Heat for 5 to 10 min at 100C in a water bath.11.2.4 If
37、the analysis is not to be performed immediately,the extracted sample may be stored at 20C for a period up to6 months.11.3 Procedure BBoiling 0.02 M Tris Buffer WithoutFiltration Method:11.3.1 Add 1.0 mL of sample to approximately 35 mL of0.02 M tris buffer pH 7.75 in a 50-mLErlenmeyer flask that has
38、reached a temperature of at least 98C in a boiling water bath.11.3.2 Maintain boiling temperature for 2 to 4 min.11.3.3 Cool to room temperature.11.3.4 Analytically transfer to a 50-mL volumetric flask andbring up to volume with 0.02 M tris buffer. If the analysis is notto be performed immediately,
39、the extracted sample may bestored at 20C for a period up to 6 months.11.3.5 If marine water samples are extracted directly with-out filtrations, it is especially important to dilute the samplewith 0.02 M tris buffer to avoid inhibition of the luminescencereaction by NaCl.12. Standardization Curve12.
40、1 Pipet 1.0 mL of the standardATP solution containing 1mg of ATP/mL into a 1-L volumetric flask and bring up tovolume with 0.02 M tris buffer. Call this Solution A. SolutionA will contain 1.00 g ATP/mL. Then make the followingserial dilutions:12.1.1 1.0 mL of Solution A + 9 mL of 0.02 Mtris = Soluti
41、on B, Solution B = 1.00 3 101g ATP/mL.12.1.2 1.0 mL of SolutionA + 99 mL 0.02 M tris = SolutionC, Solution C = 1.00 3 102g ATP/mL.12.1.3 1.0 mL of Solution C + 9 mL 0.02 M tris = SolutionD, Solution D = 1.00 3 103g ATP/mL.12.2 The above concentrations should be used when prepar-ing a curve for norma
42、l laboratory samples. For oligotrophicwaters additional dilutions are required. Standards can beprepared and frozen, then thawed as needed.12.3 A minimum of three replicate determinations of eachof the standard solutions (Solutions A, B, C, and D) should beused to prepare a calibration curve. These
43、solutions should bechosen so that they contain concentrations of ATP at the lowerend, upper end, and midway in the range ofATPconcentrationsthat the analyst suspects (or knows) to be present in thesamples to be analyzed.12.4 Determine (triplicate measurements) the instrumentresponse to the reagent b
44、lank, consisting of sterile extractant.Subtract the instrument response to the blank from the responseto dilutions of the standard and plot the results versus ATPconcentration on log-log paper. If the microorganisms are notconcentrated by filtration before the ATP is extracted, test thewater carryin
45、g the microorganisms for the presence of agentsthat might interfere with the ATP-luciferase reaction. This isdone by spiking a suitable volume of filtered water, that doesnot contain soluble extracellular ATP (see 10.1) from thesamples and the unfiltered sample with a known amount ofATP to determine
46、 the percent recovery.13. ATP Measurement13.1 Rinse the microlitre syringe three times with 0.2 N HCl(8.5) by drawing acid into the entire 50 L; rinse three timeswith 0.02 M tris buffer solution to neutralize any remainingacid.13.2 Add sufficient volume of extract from 11.2 or 11.3 tothe luciferin-l
47、uciferase mixture and measure the response witha suitable ATP analyzer.NOTE 2Some systems required the luciferin-luciferase mixture to beinjected into the sample.13.3 Repeat rinse (13.1) between each sample.13.4 Convert the instrument reading to ATP units permillilitre using the standard curve.Accou
48、nt for the total samplevolume filtered or the volume actually analyzed, or both, asappropriate.14. Precision and Bias14.1 The precision data were obtained by using standardATP solutions as it was not possible to prepare a standardreference water sample forATPcontent, which would representa true envi
49、ronmental sample. Furthermore, because of theunstable nature of ATP, it was not possible to prepare and ship“unknown” standard solutions to all participating laboratories.For these reasons, the following procedure was used:14.2 Each participating laboratory prepared and calibrated aluminescence meter covering the concentration range from 0.5to 100 g/L ATP. Participating laboratories were instructed toprepare“ Pseudo Unknown” ATP samples with concentrationlevels of 0.4, 4.0, 16.0, 64.0, and 96 g/L in 0.02 M tris buffer.These solutions were prepare
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