1、Designation: D4012 15Standard Test Method forAdenosine Triphosphate (ATP) Content of Microorganismsin Water1This standard is issued under the fixed designation D4012; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、 revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a protocol for capturing, ex-tracting and quantifying the cellular adenosine triphosphate(cATP) co
3、ntent associated with microorganisms normallyfound in laboratory cultures, waters, wastewaters, and inplankton and periphyton samples from waters.1.2 The ATP is measured using a bioluminescence enzymeassay, whereby light is generated in amounts proportional tothe concentration of ATP in the samples.
4、 The light is producedand measured quantitatively as relative light units (RLU)which are converted by comparison with an ATP standard andcomputation to pg ATP/mL.1.3 This method does not remove all known chemicalinterferences, known to either luminesce in the 530 6 20 nmrange, or to quench light emi
5、tted in that range. It should not beused to determine ATP concentrations in samples with dis-solved organic compounds, heavy metals or 10 000 ppm totaldissolved solids. Alternative methods have been developed fordetermining ATP concentrations in fluids samples likely tocontain such interferences (Te
6、st Methods D7687 and E2694).1.4 Knowledge of the concentration ofATPcan be related toviable biomass or metabolic activity of microorganisms (Ap-pendix 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 precisi
7、on statementpertains only to determinations in reagent water and notnecessarily in the matrix being tested.1.7 This test method is equally suitable for use in thelaboratory or field.1.8 The method normally detects cATPconcentrations in therange of 0.1 pg cATP/mL (1.0Log10pg cATP/mL) to 4 000000 pg c
8、ATP/mL (6.6 Log10pg cATP/mL) in 50 mL watersamples.1.9 Providing interferences can be overcome, biolumines-cence is a reliable and proven method for qualifying andquantifying ATP, although the method does not differentiatebetween ATP from different sources, for example, from differ-ent types of micr
9、oorganisms, such as bacteria, fungi, algae andprotozoa.1.10 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.11 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresp
10、onsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD1601 Test Method f
11、or Dilute Solution Viscosity of Ethyl-ene PolymersD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD5847 Practice for Writing Quality Control Specificationsfor Standard Test Methods for Water AnalysisD6161 Terminology Used for Microfiltration, Ultrafiltration,Nanofiltration
12、and Reverse Osmosis Membrane ProcessesD6300 Practice for Determination of Precision and BiasData for Use in Test Methods for Petroleum Products andLubricantsD7687 Test Method for Measurement of Cellular AdenosineTriphosphate in Fuel, Fuel/Water Mixtures, and Fuel-Associated Water with Sample Concent
13、ration by FiltrationE2694 Test Method for Measurement of Adenosine Triphos-phate in Water-Miscible Metalworking FluidsF1671 Test Method for Resistance of Materials Used in1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24
14、 on Water Microbiology.Current edition approved July 1, 2015. Published September 2015. Originallyapproved in 1981. Last previous edition approved in 2009 as D4012 81 (2009).DOI: 10.1520/D4012-15.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at
15、 serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Protective Clothing to Penetration by Blood-Borne P
16、atho-gens Using Phi-X174 Bacteriophage Penetration as a TestSystem3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this standard, refer toTerminologies D1129 and D4175.3.1.2 adenosine monophosphate (AMP), nmoleculeformed by the removal of two molecules of phosphate (onepyrophosph
17、ate molecule) from ATP.3.1.3 adenosine triphosphate (ATP), nmolecule com-prised of a purine and three phosphate groups that serves as theprimary energy transport molecule in all biological cells.3.1.4 aseptic, adjsterile, free from viable microbial con-tamination.3.1.5 background RLU, nquantity of r
18、elative light unitsresulting from running the test method without incorporation ofthe sample.3.1.6 bioluminescence, nproduction and emission of lightby a living organism as the result of a chemical reaction duringwhich chemical energy is converted to light energy.3.1.7 biomass, nany matter which is
19、or was a livingorganism or excreted from a microorganism. D61613.1.8 cellular adenosine triphosphate (cellular-ATP;c-ATP), nATP present in whole cells, whether they are livingor dead.3.1.8.1 DiscussionCellular-ATP is released upon inten-tional lysis of microbial cells during the sample preparationpr
20、ocess. Microbially infected fluids contain both cellular (cell-associated/cell-bound) and extra-cellular ATP.3.1.9 culturable, adj(microorganisms that are) able toproliferate as indicated by the formation of colonies on solidgrowth media or the development of turbidity in liquid growthmedia under sp
21、ecific growth conditions.3.1.10 extra-cellular, adj(molecules or substances that areeither) excreted by living cells or released from microbial cellsthat have lysed (see 3.1.14) in the sample.3.1.10.1 DiscussionExtra-cellular ATP is ATP that hasbeen released from microbial cells that have either ful
22、ly orpartially lysed in the sample, the upstream fluid (fuel or waterphase), or both.3.1.10.2 DiscussionLysis can occur due to natural lifecycle process, antimicrobial treatment or a combination ofthese factors. Extra-cellular ATP can under certain circum-stances persist for periods greater than 24
23、h after cell lysisdepending on physical/chemical conditions.3.1.11 luciferase, ngeneral term for a class of enzymesthat catalyze bioluminescent reactions.3.1.12 luciferin, ngeneral term for a class of light-emittingbiological pigments found in organisms capable of biolumi-nescence.3.1.13 luminometer
24、, ninstrument capable of measuringlight emitted as a result of nonthermal excitation.3.1.14 lysis, ndisintegration or destruction of whole bac-terial cells. F16713.1.15 relative light unit (RLU), ninstrument and assayspecific unit of measurement reflecting the number of photonsemitted by the Lucifer
25、in-Luciferase driven hydrolysis of ATPto AMP plus pyrophosphate.3.1.15.1 DiscussionRLU is not an SI unit, however, RLUare proportional to ATP concentration.3.1.16 viable microbial biomass, nmetabolically active(living) microorganisms.3.2 Acronyms:3.2.1 AMP, nadenosine monophosphate.3.2.2 ATP, nadeno
26、sine triphosphate.3.2.3 HDPE, nhigh density polyethylene.3.2.4 PP, npolypropylene.3.2.5 pg, npicogram (1 10-12g).3.2.6 RLU, nrelative light unit.4. Summary of Test Method4.1 Acontrol assay is performed using 100 Lof 1.0 6 0.05ng ATP/mL standard to produce RLUctrl.4.2 A 50 mL sample of water is place
27、d into a syringe andthen pressure-filtered through a 0.7 m, glass-fiber, in-line,depth filter.4.3 A lysing reagent is used to release cellular-ATP frommicrobial 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 dilut
28、ed 1 to 10 with a buffer solution.4.5 A 100 L volume of diluted filtrate is transferred to anunused culture tube into which 100 L of Luciferin-Luciferasereagent has been previously dispensed.4.6 The culture tube is placed into a luminometer and thelight intensity is read as RLUobs.4.7 RLUobsis norma
29、lized to an actual pg ATP/mL concen-tration through an equation that accounts for the result of thecontrol assay (RLUctrl), the volume of the sample processed,and the method dilution factor.NOTE 1Optionally, for condition monitoring purposes, pgATP/mLareconverted to Log10pg ATP/mL of sample by compu
30、tation.5. Significance and Use5.1 A rapid and routine procedure for determining biomassof the living microorganisms in cultures, waters, wastewaters,and in plankton and periphyton samples taken from surfacewaters is frequently of vital importance. However, classicaltechniques such as direct microsco
31、pe counts, turbidity, organicchemical analyses, cell tagging, and plate counts are expensive,time-consuming, or tend to underestimate total numbers. Inaddition, some of these methods do not distinguish betweenliving and nonliving cells.5.2 This test method measures the concentration of cellular-ATP
32、present in the sample. ATP is a constituent of all livingcells, including bacteria, algae, protozoa, and fungi.Consequently, the presence of cellular-ATP is an indicator ofD4012 152total metabolically active microbial contamination in water.ATP is not associated with matter of non-biological origin.
33、5.3 The ATP (luciferin-luciferase) method is a rapid, sensi-tive determination of viable microbial biomass. ATP is theprimary energy donor for life processes, does not exist inassociation with nonliving detrital material, and the amount ofATP per unit of biomass (expressed in weight) is relativelyco
34、nstant. (ATP per cell varies with species and physiologicalstate of the organism.)5.4 This test method can be used to:5.4.1 Estimate viable microbial biomass in cultures, waters,and wastewaters.5.4.2 Estimate the amount of total viable biomass in plank-ton and periphyton samples.5.4.3 Estimate the n
35、umber of viable cells in a unispeciesculture 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 sizefractionation of water, and wastewater samples.5.4.5 Measure the mortali
36、ty rate of microorganisms intoxicity tests in entrainment studies, and in other situationswhere populations or assemblages of microorganisms areplaced under stress.5.5 This test method is similar to Test Methods D7687 andE2694 except for the volumes sampled, and omission of washand drying steps used
37、 in Test Methods D7687 and E2694 toremove interferences (1.3).5.6 AlthoughATPdata generally covary with culture data inwater samples, different factors affect cATP concentration thanthose that affect culturability.5.6.1 Culturability is affected primarily by the ability ofcaptured microbes to prolif
38、erate on the growth mediumprovided, under specific growth conditions. Consequently, aproportion of the active or inactive microbial populationpresent in a sample may be viable but not detected by any oneculture test.35.6.2 ATP concentration is affected by: the microbial spe-cies present, the physiol
39、ogical states of those species, and thetotal bioburden (see Appendix X1).5.6.2.1 One example of the species effect is that the amountof ATP per cell is substantially greater for active fungal cellsthan bacteria (Appendix X1).5.6.2.2 Within a species, cells that are more metabolicallyactive will have
40、 more ATP per cell than dormant cells, such asfungal spores.5.6.2.3 The greater the total bioburden, the greater the ATPconcentration in a sample.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 glasswar
41、e, prepared by the procedure in 7.5,isrequired for the determination of ATP.6.3 Luciferase is a protein and as such can be inhibited ordenatured through the presence of heavy metals, high salt (forexample, NaCl) concentrations, and organic solvents in thesample. Additionally, high amounts of color o
42、r turbidity in thesample can impede light transmission, causing a negative bias.Although the method described herein is designed to mitigatesuch interferences in most types of water, for samples withhigh amounts of such interferences consider the use of TestMethods D7687 and E2694.6.4 Other energy-m
43、ediating compounds, such as adenosinediphosphate, 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.7. Apparatus7.1 Culture Tube, ste
44、rile, disposable, PP, 12 by 55 mm.7.2 Culture Tube, sterile, disposable, PP, 17 by 100 mm withcaps.7.3 Filter, 25 mm, sterile, disposable, PP housing, in-line,0.7 m pore-size, glass-fiber depth-type with Luer-Lok inlet.7.4 Luminometer, using photomultiplier tube, having aspectral range between 300 a
45、nd 600 nm, and with a cuvettechamber that can hold and provide an unobstructed line of sightto the reactants in a 12 by 55 mm test culture tube (7.1),providing a ratio of RLUbackground/RLUctrl(refer to Section 10and Appendix X5) 0.01 and optimally having five decades oflinearity (refer to Appendix X
46、2).NOTE 2It is the responsibility of the user to ensure that theluminometer selected for use meets the criteria listed in 7.4 and to consultwith the luminometer manufacturer to ensure that use of the luminometerwith the apparatus, reagents and materials described in Sections 6 and 7does not result i
47、n the inability of the instrument manufacturer to providetechnical support or loss of instrument warranty.NOTE 3The preliminary interlaboratory study and data presented inAppendix X6 and Table X4.2, respectively, were developed using aKikkoman Lumitester C-110,4which provides nominally a 5000 RLUctr
48、land 50 RLUbackground. Although this test method is optimized to functionon this luminometer, users may examine the use of other luminometersaccording to key performance criteria, including linear measurementrange (Appendix X2) and RLUbackgroundlevel (Appendix X5).7.5 Macropipeter, adjustable, 1.0 t
49、o 5.0 mL.7.6 Micropipeter, adjustable, 100 to 1000 L.7.7 Pipet Tips, sterile, disposable, PP, 100 to 1000 L.7.8 Pipet Tips, sterile, disposable, PP, 1.0 to 5.0 mL.7.9 Sample Collection Container, sterile, wide-mouthbottle, PP or HDPE, 100 mL.NOTE 4ATP can adsorb onto glass surfaces. Consequently, PP orHDPE containers are strongly preferred.7.10 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.7.11 Test Tube Rack,12mm.3Sloan, W. T., Quince, C., and Curtis, T. P., “The Uncountables,” AccessingUncultivated Microorganisms, ASM Press, Washing
