1、Designation: D7463 14Standard 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 adoption
2、 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.1. Scope*1.1 This test method provides a protocol for capturing,extracting and quantifyi
3、ng the adenosine triphosphate (ATP)content associated with:1.1.1 Microorganisms found in conventional liquid fuelswith kinematic viscosities (at 40C) of 8mm2s1asdescribed in Table X6.1,1.1.2 Microorganisms found in fuel-associated bottomwater, and1.1.3 Extracellular (non-cellular) ATP present in the
4、 samplematrix.1.2 The ATP is measured using a patented bioluminescenceenzyme assay, whereby light is generated in amounts propor-tional to the concentration of ATP in the sample. The light isproduced and measured quantitatively using dedicatedATPtestpens2and a dedicated luminometer2and reported in (
5、instru-ment specific) Relative Light Units.1.3 This test method is equally suitable for use in thelaboratory or field.1.4 Although bioluminescence is a reliable and provenmethod for qualifying and quantifying ATP, this method doesnot differentiate between ATP from different sources, forexample, from
6、 different types of microorganism such asbacteria 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 water samples or capture solution. Assum-ing testing on fuel ph
7、ase 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.1.7 This standard does not purport to address all of thesafety concerns
8、, 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 applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D396 Specification for Fuel OilsD975 Specif
9、ication for Diesel Fuel OilsD1129 Terminology Relating to WaterD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels (Withdrawn 2003)4D2880 Specification for Gas Turbine Fuel OilsD3699 Specification for KerosineD4012 Test Method forAdenosine Triphosphate (ATP) Con-tent o
10、f Microorganisms in WaterD4057 Practice for Manual Sampling of Petroleum andPetroleum ProductsD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD6161 Terminology Used for Microfiltration, Ultrafiltration,Nanofiltration and Reverse Osmosis Membrane ProcessesD6469 Guide for Mic
11、robial Contamination in Fuels and FuelSystemsD6751 Specification for Biodiesel Fuel Blend Stock (B100)for Middle Distillate Fuels3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this test method, referto Terminologies D1129 and D4175, and Guide D6469.1This test method is under the
12、 jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved May 1, 2014. Published May 2014. Originallyapproved in 2008. Last previous edition appro
13、ved in 2008 as D7463 08. DOI:10.1520/D7463-14.2The sole source of supply of the apparatus known to the committee at this timeis Merck KGaA, 64271 Darmstadt, Germany. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquarters.Your comments will receiv
14、e careful consideration at a meeting of the responsibletechnical 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 ASTMStandards volume information, refer to the standards Docum
15、ent Summary page onthe ASTM website.4The last approved version of this historical standard is referenced onwww.astm.org.*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 States13
16、.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 phosphate (one pyrophos-phate molecule) from ATP.3
17、.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 biomass, nany matter which is or was a livingorgani
18、sm or excreted from a microorganism. D61613.1.7 capture solution, naqueous solution of proprietarycomposition used to capture and concentrate hydrophilic com-pounds and particles from liquid fuels.3.1.8 culturable, adjmicroorganisms that proliferate asindicated by the formation of colonies in or on
19、solid growthmedia, or the development of turbidity in liquid growth mediaunder specified growth conditions.3.1.9 extracellular ATP, nATP that is not contained insidea cell.3.1.9.1 DiscussionATP is released into the environmentwhen cells die and break open (lyse), for example, as whenthey are killed
20、by exposure to some microbicides.ATPreleasedinto the environment can persist for several days after a cell hasbeen lysed. Consequently extracellularATP must be subtractedfrom total ATP to determine the concentration of viablecell-associated (biomass associated) ATP. However, extracel-lular ATP can a
21、lso be an indicator of “distant” biomass, forexample, biofilm in the system.3.1.10 fungus, (pl. fungi), nsingle cell (yeasts) or filamen-tous (molds) microorganisms that share the property of havingthe true intracellular membranes (organelles) that characterizeall higher life forms (Eukaryotes).3.1.
22、11 invert emulsion layer, ninterface between the waterphase and fuel phase of a fuel water sample which consists ofwater micelles dispersed in the fuel.3.1.12 luciferase, ngeneral term for a class of enzymesthat catalyze bioluminescent reactions.3.1.13 luciferin, ngeneral term for a class of light-e
23、mittingbiological pigments found in organisms capable of biolumi-nescence.3.1.14 luminometer, ninstrument capable of measuringlight emitted as a result of non-thermal excitation.3.1.15 pyrogen free, nfree of substances which can inducefever.3.1.16 relative light unit (RLU), ninstrument-specific unit
24、of measurement reflecting the number of photons emitted bythe Luciferin-Luciferase driven hydrolysis ofATPtoAMPpluspyrophosphate.3.1.16.1 DiscussionRLU is not an SI unit, however, RLUare proportional to ATP concentration.3.1.17 viable microbial biomass, nmetabolically active(living) micro-organisms3
25、.2 Abbreviations:3.2.1 AMPadenosine monophosphate3.2.2 ATPadenosine triphosphate3.2.3 HDPEhigh density polyethylene3.2.4 PPpolypropylene3.2.5 RLUrelative light units4. Summary of Test Method4.1 A fuel sample is obtained either for condition monitor-ing or for diagnostic testing, for example, fuel fr
26、om a fuelsystem that is exhibiting problems such as sediment formationor filter plugging where the presence of micro-organisms issuspected.4.2 Microbial ATP is captured, extracted, and quantifiedusing a bioluminescence reaction. The light generated by theluminescence reaction is measured in a lumino
27、meter.24.3 Specialized test methods for fuel samples, watersamples, extracellular determination or resolving potentialmatrix interference in bottom water samples are described inthe appendixes.5. Significance and Use5.1 This test method measures the concentration of ATPpresent in the sample. ATP is
28、a constituent of all living cellsincluding bacteria and fungi. Consequently, 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 providin
29、g for the rapid determination of ATPpresent in a fuel (petroleum) sample, 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-cont
30、ained test device and luminom-eter;5.2.3 By providing a method of quantifying 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 comple
31、x fluids containing hydrocar-bons and other organic molecules.5.3 This 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
32、monitor microbial contamination in fuelstorage and distribution systems.6. Interferences6.1 Sample containers and sampling devices shall be cleanand free of both ATP and microbial contamination.D7463 1426.2 Ensure that the sampling stick on theATPTest Pen doesnot come into contact with any contamina
33、ting surfaces. Con-tact with a surface or substance can cause contamination withhigh levels of ATP, giving erroneous results.6.3 Luciferase is an enzyme, which can be inhibited ordenatured by high temperatures, the presence of heavy metalsand high salt concentrations in the sample. These conditionsa
34、re unlikely to occur except in samples containing largevolumes of bottom-water 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 luminome
35、ter2is shown as a diagramin Fig. 1.7.2 WarningThe apparatus is not explosion 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 capacity 500mL, HDPE (High Density
36、 Poly Ethylene) or equivalent. Thereshall be sufficient excess volume in the sample bottle so thatthere is at least 10 % head space in addition to the 500 mLsample volume to facilitate the shearing and mixing of thecapture solution.7.3.1 Sample bottles may be reused provided they arecleaned and drie
37、d correctly. Refer to test suppliers informationregarding recommended cleaning procedure.7.4 Pipettors, fixed volume or adjustable capable of provid-ing discreet volumes of bottom water to determine the presenceof matrix interference as described in Appendix X4. Examplepipettor volumes include 10 L,
38、 50 L, and 100 L.8. Reagents 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-LiTE5Fuel Test Pen, as shown in Fig. 2.8.2.1.2 HY-LiTE5Free ATP Pen, as shown in Fig. 2.8.2.2 Pasteur pipettes, sterile, disposable, polyethylene, 1
39、.0mL.8.2.3 Pasteur pipettes sterile, disposable, polyethylene, 10.0mL.9. Sampling, Test Specimens, and Test Units9.1 Samples shall be drawn in accordance with PracticeD4057 as amplified by Hill.69.2 To reduce the risk of accidental contamination, samplesintended for microbiological testing shall not
40、 be used for othertests until after they are no longer needed for microbiologicaltesting.9.3 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 (i
41、sopropyl alcohol or ethanol)and water solution and let air dry. All devices (except factorynew, clean bottles) should be disinfected is this manner tominimize the likelihood of cross-contamination. Use care tonot touch the interior of the freshly decontaminated sampledevices or sample bottles. Remov
42、e the container lid immedi-ately before dispensing the sample into the container andreplace the lid on the container as soon as possible.9.4 Microbial contaminant populations are dynamic. Mi-crobes within the sample can proliferate or die during theinterval between collection and testing. Consequent
43、ly, samplesshall be processed within 24 h after collection.9.5 If samples are to be tested later than 4 h after collection,store the samples either on ice, or refrigerated at 0 to 5C untiltested.Avoid freezing samples.Allow samples to equilibrate toroom temperature before testing.10. Calibration and
44、 Standardization10.1 The luminometer2which is specific to this test isfactory calibrated and temperature compensated to give a linear5Registered trademark of Merck KGaA, 64271 Darmstadt, Germany.6Hill, G., “Sampling Methods for Detecting Microbial Contamination in Fuelsand Fuel Systems,” Fuels and F
45、uel Systems Microbiology: Fundamentals,Diagnosis, and Contamination Control, ASTM MNL 47, ASTM International, WestConshohocken, PA, 2002, p. 14.FIG. 1 LuminometerD7463 143response from 0-99,000 RLU at temperatures between 41 and95F (5 and 35C). No calibration is necessary becausecalibrations checks
46、are performed automatically during start-up.10.2 RLU data may be converted to ATP concentration byinterpolating from a standard curve as described in AppendixX5.10.3 1 RLU is equivalent to approximately51015gramsATP.11. Procedure11.1 Analysis of Fuel and Combined Fuel and WaterSamples:11.1.1 Collect
47、 sample according to 9.1.11.1.1.1 If sample is 500 mL, collect dispense directly intoclean sample bottle (7.3).11.1.1.2 If total sample is 500 mL, transfer 500 mL to aclean sample bottle (7.3).11.1.2 Obtain the small pipette from the fuel test kit.11.1.3 Using a clean implement (for example, scissor
48、s orknife), cut the protective plastic sleeve open at the bulb-endand remove the pipette. Do not touch the tip and lower stem ofthe pipette by hand or against any surfaces.11.1.4 Using the small sterile pipette, transfer the capturesolution from the fuel test pen reservoir into the bottle thatcontai
49、ns the sample.11.1.5 Rinse the interior of the pipette with sample to ensuremaximum transfer of the capture solution to the sample.11.1.6 Dispose of the pipette as solid (fuel-contaminated)waste according to local regulations.11.1.7 Close the lid securely on the sample vessel.11.1.8 Shake the sample vigorously for 30 s.11.1.9 Place the sample vessel on level surface and let standfor 5 min.NOTE 1The capture solution will readily dissolve into the free waterassociated with the fuel sample or those samples that contain only water.Th
