1、Designation: D7463 161Standard Test Method forAdenosine Triphosphate (ATP) Content of Microorganismsin Fuel, Fuel/Water Mixtures, and Fuel Associated Water1This standard is issued under the fixed designation D7463; the number immediately following the designation indicates the year oforiginal adopti
2、on or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTESubsection 12.4 was corrected editorially in October 2016.1. Scope*1.1 This test
3、method provides a protocol for capturing,concentrating, and testing the adenosine triphosphate (ATP)present in a fuel system sub-sample (that is, test specimen)associated with:1.1.1 Microorganisms and hydrophilic particles found inliquid fuels as described in Table X6.1,or1.1.2 Microorganisms and hy
4、drophilic particles found inmixture of fuel and associated bottom water or just associatedbottom water.1.1.3 ATP detected by this bioluminescence test can bederived from cellular ATP, extra-cellular ATP, or some combi-nation of both.1.1.4 Cellular and extra-cellular ATP utilized to performATP biolum
5、inescence are captured and concentrated from afuel system sample into an aqueous test specimen (that is,sub-sample) for testing. For example, for a fuel system samplethat does not contain any visible fuel associated bottom water,the aqueous test specimen is the capture solution itself de-scribed in
6、8.2.1.1. For fuel system samples that are a mixtureof fuel and associated bottom water (that is, free water), the testspecimen is an aliquant of the capture solution and associatedbottom water.1.2 The ATP is measured using a patented bioluminescenceenzyme assay, whereby light is generated in amounts
7、 propor-tional to the concentration of ATP in the sample. The light isproduced and measured quantitatively using dedicatedATPtestpens2and a dedicated luminometer2and reported in (instru-ment specific) Relative Light Units.1.3 This test method is equally suitable for use in thelaboratory or field.1.4
8、 Although bioluminescence is a reliable and proventechnology, this method does not differentiate ATP frombacteria or fungi.1.5 For water or capture solution samples, the concentrationrange of ATP detectable by this test method is11011Mto3108M which is equivalent to11014moles/mL to 3 1011moles/mL for
9、 water samples or capture solution. Assum-ing testing on fuel phase is performed on a 500 mL volume offuel the equivalent concentrations is fuel would be:61011Mto21014M.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6.1 T
10、here is one exceptionRelative Light Unit (RLU) asdefined in 3.1.19.1.7 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 appro-priate safety and health practices and determine the ap
11、plica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D396 Specification for Fuel OilsD975 Specification for Diesel Fuel OilsD1655 Specification for Aviation Turbine FuelsD2880 Specification for Gas Turbine Fuel OilsD4012 Test Method forAdenosine Triphosphate
12、 (ATP) Con-tent of Microorganisms in WaterD4175 Terminology Relating to Petroleum Products, LiquidFuels, and LubricantsD6300 Practice for Determination of Precision and Bias1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lubricants and is the
13、 direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved June 1, 2016. Published June 2016. Originallyapproved in 2008. Last previous edition approved in 2015 as D7463 15. DOI:10.1520/D7463-16E01.2The sole source of supply, repair, recertific
14、ation, and technical support of theapparatus or test pen known to the committee at this time is Merck KGaA, 64271Darmstadt, Germany (Worldwide) or Fuel Quality Services, Inc., 4584 Cantrell Rd.,Flowery Branch, GA30542 (USA). If you are aware of alternative suppliers, pleaseprovide this information t
15、o ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of AS
16、TMStandards volume information, refer to the standards Document Summary page onthe ASTM website.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Data for Use in Test Meth
17、ods for Petroleum Products andLubricantsD7464 Practice for Manual Sampling of Liquid Fuels, As-sociated Materials and Fuel System Components forMicrobiological TestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend(B6 to B20)3. Terminology3.1 Definitions:3.1.1 For definition of terms used i
18、n this test method, referto Terminology D4175.3.1.2 adenosine triphosphate, nmolecule comprised of apurine and three phosphate groups, that serves as the primaryenergy transport molecule in all biological cells.3.1.3 adenosine monophosphate, nmolecule formed bythe removal of two (2) molecules of pho
19、sphate (one pyrophos-phate molecule) from ATP.3.1.4 aseptic, adjsterile, free from viable microbiologicalcontamination.3.1.5 bioluminescence, nproduction and emission of lightby a living organism as the result of a chemical reaction duringwhich chemical energy is converted to light energy.3.1.6 biom
20、ass, nbiological material including any mate-rial other than fossil fuels which is or was a living organism orcomponent or product of a living organism.3.1.7 capture solution, naqueous solution of proprietarycomposition used to capture and concentrate hydrophilic com-pounds and particles from liquid
21、 fuels.3.1.8 cellular adenosine triphosphate (cellular-ATP),nATPpresent in whole cells, whether they are living or dead.3.1.8.1 DiscussionCellular-ATP is released upon inten-tional lysis (rupturing) of microbial cells during the samplepreparation process. Microbially infected fluids contain bothcell
22、ular (cell-associated/cell-bound) and extra-cellular ATP.3.1.9 culturable, adjmicroorganisms that proliferate asindicated by the formation of colonies in or on solid growthmedia, or the development of turbidity in liquid growth mediaunder specified growth conditions.3.1.10 extracellular ATP, nATP th
23、at is not containedinside a cell.3.1.10.1 DiscussionATP is released into the environmentwhen cells die and break open (lyse), for example, as whenthey are killed by exposure to some microbicides.ATPreleasedinto the environment can persist for several days after a cell hasbeen lysed. Consequently ext
24、racellularATP must be subtractedfrom total ATP to determine the concentration of viablecell-associated (biomass associated) ATP. However, extracel-lular ATP can also be an indicator of “distant” biomass, forexample, biofilm in the system.3.1.11 free water, nundissolved water present in a hydro-phobi
25、c material.3.1.11.1 DiscussionFree water in fuel such as hydrocar-bon diesel fuel can be present as a suspended haze, as dropletson the walls of the vessel, or as a separate layer on the bottomof the vessel.3.1.12 fungus, (pl. fungi), nsingle cell (yeasts) or filamen-tous (molds) microorganisms that
26、 share the property of havingthe true intracellular membranes (organelles) that characterizeall higher life forms (Eukaryotes).3.1.13 hydrophilic particles, ncompounds such as ATP,NAD+, NADP+, NADH, NADPH, enzymes, free fatty acids,preservatives, biocides, salts, as well as microorganisms orother ar
27、ticles are often dispersed or distributed in hydrophobicliquid matrices such as crude oil, vegetable oil, petrol, andkerosine.3.1.14 invert emulsion layer, ninterface between the waterphase and fuel phase of a fuel water sample which consists ofwater micelles dispersed in the fuel.3.1.15 luciferase,
28、 ngeneral term for a class of enzymesthat catalyze bioluminescent reactions.3.1.16 luciferin, ngeneral term for a class of light-emittingbiological pigments found in organisms capable of biolumi-nescence.3.1.17 luminometer, ninstrument capable of measuringlight emitted as a result of non-thermal exc
29、itation.3.1.18 pyrogen free, nfree of substances which can inducefever.3.1.19 relative light unit (RLU), ninstrument-specific unitof measurement reflecting the number of photons emitted bythe Luciferin-Luciferase driven hydrolysis ofATPtoAMPpluspyrophosphate.3.1.19.1 DiscussionRLU is not an SI unit,
30、 however, RLUare proportional to ATP concentration.3.1.20 test specimen, na representative piece of a sample.3.1.20.1 DiscussionFor this test method, the test specimenis an aqueous sub-sample drawn from the fuel system samplethat is tested for the presence of cellular and/or extra-cellularATP. In th
31、e case of a fuel system sample that is fuel only in theabsence of associated bottom water, the test specimen is thecapture solution (3.1.7). For fuel system samples that containassociated bottom water, the test specimen is an aliquant of thecapture solution and associated bottom water (3.1.11).3.1.2
32、1 viable microbial biomass, nmetabolically active(living) micro-organisms3.2 Abbreviations:3.2.1 AMPadenosine monophosphate3.2.2 ATPadenosine triphosphate3.2.3 HDPEhigh density polyethylene3.2.4 NAD+nicotinamide adenine dinucleotide, oxidizedform3.2.5 NADHnicotinamide adenine dinucleotide, reducedfo
33、rm3.2.6 NADP+nicotinamide adenine dinucleotidephosphate, oxidized form3.2.7 NADPHnicotinamide adenine dinucleotidephosphate, reduced form3.2.8 PPpolypropylene3.2.9 RLUrelative light unitsD7463 16124. Summary of Test Method4.1 A fuel system sample is obtained either for conditionmonitoring or for dia
34、gnostic testing, for example, fuel from afuel system that is exhibiting problems such as sedimentformation or filter plugging where the presence of micro-organisms is suspected.4.2 Microbial ATP is captured from the fuel system sample,concentrated into a test specimen, and tested using a biolumi-nes
35、cence reaction. The light generated by the luminescencereaction is proportional to the amount ofATPpresent in the testspecimen as measured in a luminometer.24.3 Test results should be documented for evaluation andtrending.4.4 Specialized test methods for fuel samples, watersamples, extracellular det
36、ermination, or resolving potentialmatrix interference in bottom water samples are described inAppendix X4 and Appendix X5.5. Significance and Use5.1 This test method measures the concentration of ATPpresent in the sample. ATP is a constituent of all living cellsincluding bacteria and fungi. Conseque
37、ntly, the presence ofATP is a reliable indicator of microbial contamination in fuelsystems. ATP is not associated with matter of non-biologicalorigin.5.2 This test method differs from Test Method D4012 asfollows:5.2.1 By providing for the rapid determination of ATPpresent in a fuel (petroleum) sampl
38、e, a fuel and water mixturesample, fuel-associated bottom water sample, and extracellularATP freely available in the fuel or aqueous sample matrix;5.2.2 By providing for a method to capture, extract, andquantify ATP using self-contained test device and luminom-eter;5.2.3 By providing a method of qua
39、ntifying ATP present infuel or water matrices in generally less than 10 min; and5.2.4 By providing for the rapid separation of the ATP fromchemical interferences that have previously prevented the useof ATP determinations in complex fluids containing hydrocar-bons and other organic molecules.5.3 Thi
40、s test method does not require the use of hazardousmaterials and does not generate biohazard waste.5.4 This test method can be used to estimate viable micro-bial biomass, to evaluate the efficacy of antimicrobialpesticides, and to monitor microbial contamination in fuelstorage and distribution syste
41、ms.6. Interferences6.1 Sample containers and sampling devices shall be cleanand free of both ATP and microbial contamination.6.2 Ensure that the sampling stick on theATPTest Pen doesnot come into contact with any contaminating surfaces. Con-tact with a surface or substance can cause contamination wi
42、thhigh levels of ATP, giving erroneous results.6.3 Luciferase is an enzyme, which can be inhibited ordenatured by high temperatures, the presence of heavy metals,and high salt concentrations in the sample. These conditionsare unlikely to occur except in samples containing largevolumes of bottom-wate
43、r samples from storage tanks andsimilar systems.6.3.1 For samples in which inhibition is suspected or likelyto occur, testing of a dilution of the sample is described inAppendix X4.7. Apparatus7.1 An example of the luminometer2is shown as a diagramin Fig. 1.7.2 WarningThe apparatus is not explosion-
44、proof. Theinstrument should not be operated in explosive atmospheres orin locations where there may be explosive fumes, as it cannotbe grounded.7.3 Sample bottle, round wide-mouth, nominal capacity500 mL or 1000 mL, HDPE (High Density Poly Ethylene) orequivalent. There shall be sufficient excess vol
45、ume in thesample bottle so that there is at least 10 % head space inaddition to the 500 mL or 1000 mL sample volume to facilitatethe shearing and mixing of the capture solution.7.3.1 Sample bottles may be reused provided they arecleaned and dried correctly. Refer to test suppliers informationregardi
46、ng recommended cleaning procedure.7.4 Pipettors, fixed volume or adjustable, capable of provid-ing discrete volumes of bottom water to determine the presenceof matrix interference as described in Appendix X4. Examplepipettor volumes include 10 L, 50 L, and 100 L.FIG. 1 LuminometerD7463 16138. Reagen
47、ts and Materials8.1 Reagents:8.1.1 ATP di-sodium salt.8.1.2 Water, Pyrogen free.8.2 Materials:8.2.1 ATP test pens:28.2.1.1 HY-LiTE4Fuel Test Pen, as shown in Fig. 2.8.2.1.2 HY-LiTE4Free ATP Pen, as shown in Fig. 2.8.2.2 Pasteur pipettes, sterile, disposable, polyethylene,1.0 mL.8.2.3 Pasteur pipette
48、s sterile, disposable, polyethylene,10.0 mL.9. Sampling, Test Specimens, and Test Units9.1 Samples shall be drawn in accordance with PracticeD7464 and dispensed into a clean 500 mL sample bottle (7.3).9.2 Aircraft fuel systems shall be drawn in accordance withthe applicableAircraft Maintenance Manua
49、l and dispensed intoa clean 1000 mL sample bottle (7.3).9.3 To reduce the risk of accidental contamination, samplesintended for microbiological testing shall not be used for othertests until after they are no longer needed for microbiologicaltesting.9.4 It may be possible to accidentally cross contaminate thesample under field conditions. To reduce risk of potentialcross-contamination, rinse the sample device(s) and samplecontainer(s) with a 70 % alcohol (isopropyl alcohol or ethanol)and water solution and let air dry. All d
