1、Designation: E2694 16 An American National StandardStandard Test Method forMeasurement of Adenosine Triphosphate in Water-MiscibleMetalworking Fluids1This standard is issued under the fixed designation E2694; the number immediately following the designation indicates the year oforiginal adoption or,
2、 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 quantifying t
3、he adenosine triphosphate (ATP)content associated with microorganisms found in water-miscible metalworking fluids (MWF).1.2 The ATP is measured using a bioluminescence enzymeassay, whereby light is generated in amounts proportional tothe concentration of ATP in the samples. The light is producedand
4、measured quantitatively as relative light units (RLU)which are converted by comparison with an ATP standard andcomputation to pg ATP/mL.1.3 This test method is equally suitable for use in thelaboratory or field.1.4 The test method detects ATP concentrations in the rangeof 4.0 pg ATP/mL to 400 000 pg
5、 ATP/mL.1.5 Providing interferences can be overcome, biolumines-cence is a reliable and proven method for qualifying andquantifying ATP. The method does not differentiate betweenATP from different sources, for example, from different typesof microorganisms, such as bacteria and fungi.1.6 The values
6、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, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety
7、 and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD4012 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in WaterD4840 Guide for Sample Chain-of-Custody Pro
8、ceduresD6161 Terminology Used for Microfiltration, Ultrafiltration,Nanofiltration and Reverse Osmosis Membrane ProcessesE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE1326 Guide
9、 for Evaluating Non-culture MicrobiologicalTestsE1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE2523 Terminology for Metalworking Fluids and Opera-tions2.2 Government Standards:329 CFR 1910.1000 Occupational Safety and Health Stan-dards; Air contamin
10、ants29 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories3. Terminology3.1 Definitions: For definition of terms used in this method,refer to Terminology standards D1129, D6161, and E2523.3.2 adenosine triphosphate (ATP), na molecule com-prised of a purine and three phosphate g
11、roups that serves as theprimary energy transport molecule in all biological cells.3.3 adenosine monophosphate (AMP), nthe moleculeformed by the removal of two molecules of phosphate (onepyrophosphate molecule) from ATP.3.4 aseptic, adjsterile, free from viable microbial con-tamination.3.5 biolumines
12、cence, nthe production and emission oflight by a living organism as the result of a chemical reactionduring which chemical energy is converted to light energy.3.6 biomass, nany matter which is or was a livingorganism or excreted from a microorganism (D6161).1This test method is under the jurisdictio
13、n of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Metal Working Fluids.Current edition approved Oct. 1, 2016. Published October 2016. Originallyapproved in 2009. Last previous edition approved in 2011 as
14、 E2694 - 11.DOI:10.1520/E2694-16.2For 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 Document Summary page onthe ASTM website.3Available from U.S. Governm
15、ent Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.*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.
16、United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barr
17、iers to Trade (TBT) Committee.13.7 culturable, adjmicroorganisms that proliferate as in-dicated by the formation of colonies on solid growth media orthe development of turbidity in liquid growth media underspecific growth conditions.3.8 Luciferase, na general term for a class of enzymes thatcatalyze
18、 bioluminescent reactions.3.9 Luciferin, na general term for a class of light-emittingbiological pigments found in organisms capable of biolumi-nescence.3.10 luminometer, nan instrument capable of measuringlight emitted as a result of non-thermal excitation.3.11 relative light unit (RLU), nan instru
19、ment-specificunit of measurement reflecting the number of photons emittedby the Luciferin-Luciferase driven hydrolysis of ATP to AMPplus pyrophosphate.3.11.1 DiscussionRLU is not an SI unit, however, RLUare proportional to ATP concentration.3.12 viable microbial biomass, nmetabolically active (liv-i
20、ng) microorganisms3.13 Acronyms:3.13.1 AMPadenosine monophosphate3.13.2 ATPadenosine triphosphate3.13.3 HDPEhigh density polyethylene3.13.4 MWFmetalworking fluid3.13.5 PPpolypropylene3.13.6 RLUrelative light unit4. Summary of Test Method4.1 A control assay is performed using 100 L of 1.0 ngATP/mL st
21、andard.4.2 A 5.0 mL sample of MWF is placed into a syringe andthen pressure- filtered through a 0.7 m, glass-fiber, in-linedepth filter.4.3 The retentate is then washed with a reagent to removeextra-cellularATP and other contaminants that might otherwiseinterfere with the ATP assay.4.4 The filter is
22、 air-dried.4.5 A lysing reagent is used to release ATP from microbialcells that have been captured on the glass-fiber filter, and thefiltrate is dispensed into an unused culture tube.4.6 The filtrate is diluted 1+9 with a buffer solution.4.7 A 100-L volume of diluted filtrate is transferred to anunu
23、sed culture tube into which 100 L of Luciferin-Luciferasereagent has previously been dispensed.4.8 The culture tube is placed into a luminometer and thelight intensity is read in RLU.4.9 RLU are converted to Log10pg ATP/mL of sample bycomputation.4.10 A procedure for differentiating between bacteria
24、l andfungal cATP-biomass is provided in Appendix X4.5. Significance and Use5.1 This method measures the concentration of ATP presentin the sample. ATP is a constituent of all living cells, includingbacteria and fungi. Consequently, the presence of ATP is anindicator of total microbial contamination
25、in metalworkingfluids. ATP is not associated with matter of non-biologicalorigin.5.2 Method D4012 validated ATP as a surrogate for cultur-able bacterial data (Guide E1326).5.3 This method differs from Method D4012 in that iteliminates interferences that have historically rendered ATPtesting unusable
26、 with complex organic fluids such as MWF.5.4 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 h to 48 h(or longer) required for culturable colonies to become visible,to approximat
27、ely five minutes.5.5 AlthoughATP data generally covary with culture data inMWF,4different factors affect ATP concentration than thosethat affect culturability.5.5.1 Culturability is affected primarily by the ability ofcaptured microbes to proliferate on the growth mediumprovided, under specific grow
28、th conditions. It have beenestimated that less than 1 % of the species present in anenvironmental sample will form colonies under any given setof growth conditions.55.5.2 ATP concentration is affected by: the microbial spe-cies present, the physiological states of those species, and thetotal bioburd
29、en (See Appendix X1).5.5.2.1 One example of the species effect is that the amountof ATP per cell is substantially greater for fungi than bacteria.5.5.2.2 Within a species, cells that are more metabolicallyactive will have more ATP per cell than dormant cells.5.5.2.3 The greater the total bioburden,
30、the greater the ATPconcentration in a sample.5.5.3 The possibility exists that the rinse step (11.15) maynot eliminate all chemical substances that can interfere with thebioluminescence reaction (11.39).5.5.3.1 The presence of any such interferences can beevaluated by performing a standard addition
31、test series asdescribed in Appendix X3.5.5.3.2 Any impact of interfering chemicals will be reflectedas bias relative to data obtained from fluid that does not containinterfering chemicals.6. Apparatus6.1 Culture tube, PP,12by55mm.6.2 Culture tube, PP, 17 by 100 mm with caps.6.3 Filter, 25 mm, steril
32、e, disposable, in-line, 0.7 m poresize, glass-fiber, depth-type with Luer-Lok inlet.4Passman et al. “Real-time Testing of Bioburdens in Metalworking Fluids usingAdenosine Triphosphate as a Biomass Indicator,” 2009 STLE Annual Meeting,Orlando, FL.5Sloan, W. T., C. Quince and Curtis, T. P., “The Uncou
33、ntables,” AccessingUncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.E2694 1626.4 Luminometer, using photomultiplier tube, capable ofdetecting light emission at 420 nm and with a cuvette chamberthat can hold a 12 by 55-mm culture tube.6.5 Macropipeter, adjustable, 1.0 to 5.0 mL.6.6
34、 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-mouth bottle,100 mL.NOTE 1ATP can adsorb onto glass surfaces. Consequently, PP orHDPE containers are s
35、trongly preferred.6.10 Syringe, Luer-Lok, 20 mL, PP, sterile, disposable.6.11 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.6.12 Test tube rack, 12 mm.6.13 Test tube rack, 17 mm.6.14 Waste receptacle, any container suitable for receivingand retaining filtrate fluid for ultimate disposal.7. Reagen
36、ts and Materials7.1 ATP standard, 1 ng ATP/mL7.1.1 Commercially available;6or7.1.2 Dilute 1 mg ATP into 1000 mL ATP dilution buffer toget a 1000-ng ATP/mL stock solution. Then, dilute 1.0 mL of1000 ng ATP/mL stock solution into 999.0 mL ATP dilutionbuffertogeta1ngATP/mL ATP standard.7.2 ATP extract
37、dilution buffer6(proprietary)7.3 ATP extraction reagent6(proprietary)7.4 Filter wash reagent6(proprietary)7.5 Luciferin-Luciferase reagent6(proprietary); store be-tween -20C and 4C; allow to equilibrate to ambient tempera-ture before using.8. Hazards8.1 The analyst must know and observe good laborat
38、orysafety practice in accordance with 29 CFR1910.1450.8.2 Inhalation or dermal exposure to MWF can pose healthproblems for personnel involved with MWF sampling. Provi-sion of personal protective equipment (PPE) in the form ofrespirators, protective clothing or both may be indicated (seePractice E149
39、7).8.3 Review material safety data sheets for materials in use atthe facility to identify potential hazards in order to determineappropriate PPE (see 29 CFR 1910.1000).9. Sampling and Test Specs and Units9.1 Sampling Site:9.1.1 Select sampling site that will yield a representativeMWF sample.9.1.2 Fo
40、r routine condition monitoring, select individualsump(s) or central systems that have actively circulating fluid.9.1.3 For diagnostic testing, select zones of pooled orstagnant MWF.9.2 Sampling:9.2.1 If practical, draw sample from the mid-point of thefluid reservoir, otherwise draw sample from below
41、 surface ofthe MWF at an accessible location.9.2.1.1 Microbial contamination will vary considerablywithin the fluid system and it is important to be consistent inselecting the sampling location; this should be appropriate forthe analysis objectives.9.2.2 Collect sample by removing lid from samplecon
42、tainer, immersing the open container (6.9), opening-down,below the fluid surface and inverting the container to allow itto fill with the sampled fluid.9.2.3 If the fluid depth is insufficient to permit 9.2.1, use asterile pipet to draw sample from the fluid and dispense it intothe sample container;
43、collecting at least 25 mL of sample.9.3 Sample Storage/Shipment:9.3.1 Label the sample container and follow accepted chain-of-custody procedures (Guide D4840).9.3.2 Optimally samples should be tested on-site as soon aspossible (50 mL.X2.1.3 Report actual volume filtered, and use this volume inEq 1 (
44、12.1).X2.2 Decreasing Test Sensitivity (Decreasing RLUobsbyDilution)X2.2.1 Many luminometers have an upper detection limit of100,000 RLU. If the luminescence is greater than the upperdetection limit, the RLU display defaults to an overload signal.X2.2.2 If the original test result yields a value gre
45、ater thanthe luminometers upper detection limit, run a 1+9 dilution ofthe diluted ATP extract (11.33).X2.2.2.1 To an unused 17 by 100-mm culture tube, add 9.0mL of ATP Extract Dilution Buffer (7.2).TABLE 1 Precision Data for Log10pg ATP/mLMaterialAverage RepeatabilityStandardDeviationReproducibility
46、StandardDeviationRepeatabilityLimitReproducibilityLimitxsrsRrREO0101 4.38 0.07 0.14 0.18 0.40EO0102 3.35 0.07 0.12 0.18 0.35E00103 1.85 0.30 0.32 0.83 0.91E00104 0.93 0.13 0.33 0.37 0.92SS0101 4.85 0.06 0.14 0.17 0.39SS0102 4.26 0.67 0.95 1.89 2.66SS0103 3.16 0.40 0.42 1.11 1.18SS0104 1.03 0.26 0.46
47、 0.74 1.28SO0101 4.59 0.07 0.10 0.21 0.28SO0102 3.90 0.06 0.21 0.17 0.58SO0103 1.66 0.09 0.29 0.25 0.82SO0104 0.89 0.25 0.53 0.70 1.48EO0201 4.21 0.06 0.67 0.16 1.87EO0202 2.46 0.15 0.56 0.42 1.58EO0203 1.06 0.25 0.39 0.70 1.09EO0204 0.80 0.26 0.55 0.74 1.53SS0201 1.74 0.44 0.49 1.22 1.36SS0204 0.74
48、 0.29 0.59 0.82 1.66SO0201 3.31 0.27 0.27 0.75 0.75SO0202 2.12 0.16 0.25 0.45 0.69SO0203 1.26 0.14 0.39 0.39 1.09E2694 166X2.2.2.2 Use the micropipeter to transfer 1.0 mL of thediluted ATP extract from step 11.33 to the 9.0 mL of ATPExtract Dilution Buffer prepared in X2.2.2.1.X2.2.2.3 Follow steps
49、11.33 through 11.39.X2.2.3 If the RLUobsis still greater than the luminometersupper detection limits, run a 1+99 dilution of the diluted ATPextract (11.33).X2.2.3.1 To an unused 17 by 100-mm culture tube, add 9.9mL of ATP Extract Dilution Buffer (7.2).X2.2.3.2 Use the micropipeter to transfer 0.1 mL of thediluted ATP extract from step 11.33 to the 9.9 mL of ATPExtract Dilution Buffer prepared in X2.2.3.1.X2.2.3.3 Follow steps 11.33 through 11.39.X2.2.4 Additional dilutions of the X2.2.3.2 dilution can bemade if necessary, until the RLUobsis below th
