1、Designation: D7687 11Standard Test Method forMeasurement of Cellular Adenosine Triphosphate in Fuel,Fuel/Water Mixtures, and Fuel-Associated Water withSample Concentration by Filtration1This standard is issued under the fixed designation D7687; the number immediately following the designation indica
2、tes the year oforiginal adoption 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. Scope1.1 This test method covers a protocol for captu
3、ring, ex-tracting and quantifying the cellular adenosine triphosphate(cellular-ATP) content associated with microorganisms foundin fuels, fuel/water mixtures and fuel-associated water.1.2 The ATP is measured using a bioluminescence enzymeassay, whereby light is generated in amounts proportional toth
4、e concentration of cellular-ATP in the samples. The light isproduced and measured quantitatively as relative light units(RLU) which are converted by comparison with an ATPstandard, computation to pg ATP/mL and optional furthertransformation to Log10pg ATP/mL.1.3 This test method is equally suitable
5、for use as alaboratory or portable method.1.4 This test method is limited to fuels with a nominalviscosity # 75cSt at test temperature.1.5 This test method detectsATPconcentrations in the rangeof 5.0 pg ATP/mL (0.699 log10pg ATP/mL) to 100 000 pgATP/mL (5.000 log10pg ATP/mL) for 20 mL samples of fue
6、lor fuel/water mixtures, and 20 pg ATP/mL (1.301 log10pgATP/mL) to 400 000 pg ATP/mL (5.602 log10pg ATP/mL)for 5 mL samples of fuel-associated water.NOTE 1These ranges were calculated with the formula for calculatingsample ATP in pg/mL provided in 12.1 based on the minimum recom-mended RLU fora1ngAT
7、P/mL standard when using the reagentsspecified in Section 7 and the luminometer specified in 6.4 and correctedwith a reagent-method blank as determined in Appendix X5.1.6 Providing interferences can be overcome, biolumines-cence is a reliable and proven method for qualifying andquantifying ATP. This
8、 test method does not differentiatebetween ATP from different sources, for example: from differ-ent types of microorganisms, such as bacteria and fungi.1.7 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 This standard does n
9、ot 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 applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standar
10、ds:2D396 Specification for Fuel OilsD975 Specification for Diesel Fuel OilsD1129 Terminology Relating to WaterD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels3D2880 Specification for Gas Turbine Fuel OilsD3699 Specification for KerosineD4012 Test Method for Adenosin
11、e Triphosphate (ATP)Content of Microorganisms in WaterD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD6161 Terminology Used for Microfiltration, Ultrafiltra-tion, Nanofiltration and Reverse Osmosis Membrane Pro-cessesD6300 Practice for Determination of Precision and BiasDa
12、ta for Use in Test Methods for Petroleum Products andLubricantsD6751 Specification for Biodiesel Fuel Blend Stock (B100)for Middle Distillate FuelsD7463 Test Method for Adenosine Triphosphate (ATP)Content of Microorganisms in Fuel, Fuel/Water Mixturesand Fuel Associated WaterD7464 Practice for Manua
13、l Sampling of Liquid Fuels,Associated Materials and Fuel System Components forMicrobiological TestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend(B6 to B20)1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products and Lubricants and is the direct responsibili
14、ty of SubcommitteeD02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved April 1, 2011. Published April 2011. DOI:10.1520/D768711.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMSta
15、ndards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn. The last approved version of this historical standard is referencedon www.astm.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States
16、.E2523 Terminology for Metalworking Fluids and Opera-tionsE2694 Test Method for Measurement of AdenosineTriphosphate in Water-Miscible Metalworking FluidsF1671 Test Method for Resistance of Materials Used inProtective Clothing to Penetration by Blood-Borne Patho-gens Using Phi-X174 Bacteriophage Pen
17、etration as a TestSystem3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this test method, referto Terminology D1129, D4175, D6161, and E2523.3.1.2 adenosine monophosphate (AMP), nmoleculeformed by the removal of two molecules of phosphate (onepyrophosphate molecule) from ATP.3.1.
18、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 relative light unitsresultin
19、g 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 or was a livingorganism or
20、excreted from a microorganism. D61613.1.8 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 of microbial cells during the sample preparationprocess. Microbially infected fluids
21、 contain both cellular (cell-associated/ cell-bound) and extra-cellular ATP.3.1.9 culturable, adjmicroorganisms that proliferate asindicated by the formation of colonies on solid growth mediaor the development of turbidity in liquid growth media underspecific growth conditions.3.1.10 extra-cellular,
22、 adjmolecules 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 fully orpartially lysed in the sample, the upstream
23、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 h after cell lysisdepending on physical/chemical
24、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-emitting biological pigments found in organisms capable ofbioluminescence.3.1.13 luminometer, ninstrument capable of measuringlight emitted as
25、 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 Luciferin-Luciferase driven hydrolysis of ATPto AMP plus
26、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 AMPadenosine monophosphate.3.2.2 ATP adenosine triphosphate.3.2.3 HDPEhigh density polyethylene.3
27、.2.4 PPpolypropylene.3.2.5 RLUrelative light unit.4. Summary of Test Method4.1 Acontrol assay is performed using 100 L of 1.0 6 0.05ng ATP/mL standard to produce RLUctrl.4.2 A 20 mL sample of fuel or fuel/water mixture or 5.0 mLbottom-water is placed into a syringe and then pressure-filteredthrough
28、a 0.7 m, glass-fiber, in-line, depth filter.4.3 The retentate is then washed with a reagent to removeextra-cellular ATP and non-ATP contaminants that might oth-erwise interfere with the cellular-ATP assay.4.4 The filter is air-dried.4.5 A lysing reagent is used to release cellular-ATP frommicrobial
29、cells that have been captured on the glass-fiber filter,and the filtrate is dispensed into an unused culture tube.4.6 The filtrate is diluted 1 to 10 with a buffer solution.4.7 A 100 L volume of diluted filtrate is transferred to anunused culture tube into which 100 L of Luciferin-Luciferasereagent
30、has been previously dispensed.4.8 The culture tube is placed into a luminometer and thelight intensity is read as RLUobs.4.9 RLUobsis normalized 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,an
31、d the method dilution factor.NOTE 2Optionally, for condition monitoring purposes, pg ATP/mLare converted to Log10pg ATP/mL of sample by computation.5. Significance and Use5.1 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all livingcells, i
32、ncluding bacteria and fungi. Consequently, the presenceof cellular-ATP is an indicator of total metabolically activemicrobial contamination in fuels. ATP is not associated withmatter of non-biological origin.5.2 This test method is similar to Test Method E2694 exceptfor the volumes sampled.5.3 This
33、test method differs from Test Method D4012 in thatit utilizes filtration and wash steps designed to eliminateinterferences that have historically rendered ATP testing unus-able with complex organic fluids such as fuel, fuel/watermixtures and fuel-associated water.5.4 This test method differs from Te
34、st Method D7463 inseveral regards:D7687 1125.4.1 Test Method D7463 reports relative light units (RLU).Consistent with Test Method D4012 and E2694, this testmethod reports ATP concentration.5.4.2 This test method detects only cellular-ATP and it canbe used to detect cellular-ATP in fuels and fuel sto
35、cks fromwhich small quantities of water do not separate readily (forexample, ethanol blended gasoline containing $ 5% v/vethanol). Test Method D7463 cannot be used to recover ATPfrom fuels from which small quantities of water do not separatereadily (for example, ethanol blended gasoline containing $
36、5% v/v ethanol).5.4.3 This test method measures cellular-ATP in a singlemeasurement (as pg ATP/mL). Test Method D7463 detectstotal ATP (as RLU) and extra-cellular ATP (as RLU) using twoseparate analyses and permits computation of cellular-ATP (asRLU) as the difference between total and extracellular
37、 ATP.5.4.4 Test Method D7463 suggests a nominal 500 mL fuelsample volume. This test method suggests a nominal 20 mLfuel sample.5.5 This test method can be used with all fuels specified inSpecifications D396, D975, D1655, D2069, D2880, D3699,D6751, and D7467 and other fuels with nominal viscosities #
38、75 cSt at 20 6 2.5.6 The ATP test provides rapid test results that reflect thetotal bioburden in the sample. It thereby reduces the delaybetween test initiation and data capture, from the 36 to 48 h (orlonger) required for culturable colonies to become visible, toapproximately 5 min.5.7 AlthoughATP
39、data generally covary with culture data infuel, fuel/water mixtures, and fuel-associated water, differentfactors affect ATP concentration than those that affect cultur-ability.5.7.1 Culturability is affected primarily by the ability ofcaptured microbes to proliferate on the growth medium pro-vided,
40、under specific growth conditions. Consequently, a pro-portion of the active or inactive microbial population present ina sample may be viable but not detected by any one culturetest.45.7.2 ATP concentration is affected by: the microbial spe-cies present, the physiological states of those species, an
41、d thetotal bioburden (see Appendix X1).5.7.2.1 One example of the species effect is that the amountof ATP per cell is substantially greater for active fungal cellsthan bacteria.5.7.2.2 Within a species, cells that are more metabolicallyactive will have more ATP per cell than dormant cells, such asfu
42、ngal spores. Because fungal spores are more hydrophobicthan active fungal material (mycelium), spores may be the onlyindicator of fungal proliferation when fuel samples are takenfrom some fuel systems, but they will not be detected by a testfor ATP.5.7.2.3 The greater the total bioburden, the greate
43、r the ATPconcentration in a sample.5.7.3 The possibility exists that the rinse step (11.15) maynot eliminate all chemical substances that can interfere with thebioluminescence reaction (11.39).5.7.3.1 The presence of any such interferences can beevaluated by performing a standard addition test serie
44、s ordilution series as described in Appendix X4.6. Apparatus6.1 Culture Tube, sterile, disposable, PP, 12 by 55 mm.6.2 Culture Tube, sterile, disposable, PP, 17 by 100 mm withcaps.6.3 Filter, 25 mm, sterile, disposable, PP housing, in-line,0.7 m pore-size, glass-fiber depth-type with Luer-Lok inlet.
45、6.4 Luminometer, using photomultiplier tube, having aspectral range between 300 and 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 (6.1),providing a ratio of RLUbackground/RLUctrI(Refer to Section 10and App
46、endix X5) #0.01 and optimally having 5 decades oflinearity (refer to Appendix X2).NOTE 3The preliminary interlaboratory study and data presented inAppendix X6 and Table X4.2, respectively, were developed using aKikkoman Lumitester C-110, which provides nominally a 5000 RLUctrland 50 RLUbackground. A
47、lthough 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).NOTE 4It is the responsibility of the user to ensure that t
48、he luminom-eter selected for use meets the criteria listed in 6.4 and to consult with theluminometer manufacturer to ensure that use of the luminometer with theapparatus, reagents and materials described in Sections 6 and 7 does notresult in the inability of the instrument manufacturer to provide te
49、chnicalsupport or loss of instrument warranty.6.5 Macropipeter, adjustable, 1.0 to 5.0 mL.6.6 Micropipeter, adjustable, 100 to 1000 L.6.7 Pipet Tips, sterile, disposable, PP, 100 to 1000 L.6.8 Pipet Tips, sterile, disposable, PP, 1.0 to 5.0 mL.6.9 Sample Collection Container, sterile, wide-mouthbottle, PP or HDPE, 100 mL.NOTE 5ATP can adsorb onto glass surfaces. Consequently, PP orHDPE containers are strongly preferred.6.10 Syringe, Luer-Lok, 20 mL, PP, sterile, disposable.6.11 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.6.12 Test
