1、Designation: E 2694 09An American National StandardStandard Test Method forMeasurement of Adenosine Triphosphate in Water-MiscibleMetalworking Fluids1This standard is issued under the fixed designation E 2694; 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. Scope1.1 The method provides a protocol for capturing, extract-ing and quantifying the a
3、denosine triphosphate (ATP) contentassociated with microorganisms found in water-miscible met-alworking 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 mea
4、sured quantitatively as relative light units (RLU)which are converted by comparison with an ATP standard andcomputation to pg ATP/mL.1.3 This method is equally suitable for use in the laboratoryor field.1.4 The method detects ATP concentrations in the range of4.0 pg ATP/mL to 400,000 pg ATP/mL.1.5 P
5、roviding 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 stated in SI
6、are to be regarded as standard.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 applica-bility of regulatory limitatio
7、ns prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 4012 Test Method for Adenosine Triphosphate (ATP)Content of Microorganisms in WaterD 4840 Guide for Sample Chain-of-Custody ProceduresD 6161 Terminology Used for Microfiltration, Ultrafiltra-tion, Nanofi
8、ltration and Reverse Osmosis Membrane Pro-cessesE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for Enumerating BacteriaE 1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE 2523 Terminology for Metalworking Fluids and Opera-tions
9、2.2 Government Standards:329 CFR 1910.1000 Occupational Safety and Health Stan-dards; Air contaminants29 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories3. Terminology3.1 Definitions:For definition of terms used in this method, refer to Termi-nology standards D 1129, D 6161,
10、 and E 2523.3.2 adenosine triphosphate (ATP), na molecule com-prised of a purine and three phosphate groups 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 (onepyrophosphat
11、e molecule) from ATP.3.4 aseptic, adjsterile, free from viable microbial con-tamination.3.5 bioluminescence, 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 wa
12、s a livingorganism or excreted from a microorganism (D 6161).3.7 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.1This test method is under the jurisd
13、iction 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 May 1, 2009. Published June 2009.2For referenced ASTM standards, visit the ASTM website, www.astm.org,
14、 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. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC
15、 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.8 Luciferase, na general term for a class of enzymesthat catalyze bioluminescent reactions.3.9 Luciferin, na general term for a class of light-emittin
16、gbiological 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 instrument-specificunit of measurement reflecting the number of photons emittedby the Lucif
17、erin-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-ing) microorganisms3.13 Acronyms:3.13.1 AMPadenosine monophosphate3.13.2 ATPadenosine
18、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 standard.4.2 A 5.0 mL sample of MWF is placed into a syringe andthen pressure- filtered
19、 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 air-dried.4.5 A lysing reagent is used to release ATP from microbialcells that have
20、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 anunused culture tube into which 100 L of Luciferin-Luciferasereagent has previously been
21、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.5. Significance and Use5.1 This method measures the concentration of ATP presentin the sample. ATP is a constituent of all living cells, i
22、ncludingbacteria and fungi. Consequently, the presence of ATP is anindicator of total microbial contamination in metalworkingfluids. ATP is not associated with matter of non-biologicalorigin.5.2 Method D 4012 validated ATP as a surrogate for cultur-able bacterial data (Guide E 1326).5.3 This method
23、differs from Method D 4012 in that iteliminates interferences that have historically rendered ATPtesting unusable 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 an
24、d data capture, from the 36 h to 48 h(or longer) required for culturable colonies to become visible,to approximately five minutes.5.5 Although ATP data covary strongly with culture data inMWF4, different factors affect ATP concentration than thosethat affect culturability.5.5.1 Culturability is affe
25、cted primarily by the ability ofcaptured microbes to proliferate on the growth medium pro-vided, under specific growth conditions. It have been estimatedthat less than 1 % of the species present in an environmentalsample will form colonies under any given set of growthconditions.55.5.2 ATP concentra
26、tion is affected by: the microbial spe-cies present, the physiological states of those species, and thetotal bioburden (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 a
27、re more metabolicallyactive will have more ATP per cell than dormant cells.5.5.2.3 The greater the total bioburden, 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 thebioluminescenc
28、e reaction (11.39).5.5.3.1 The presence of any such interferences can beevaluated by performing a standard addition 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 chemic
29、als.6. Apparatus6.1 Culture tube, PP, 12 by 55 mm.6.2 Culture tube, PP, 17 by 100 mm with caps.6.3 Filter, 25 mm, sterile, disposable, in- line, 0.7 mpore-size, glass-fiber, depth-type with Luer-Lok inlet.6.4 Luminometer, using photomultiplier tube, capable ofdetecting light emission at 420 nm and w
30、ith a cuvette chamberthat can hold a 12 by 55-mm culture tube.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
31、, wide-mouth bottle,100 mL.NOTE 1ATP 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 tube rack,12mm.6.13 Test tube rack,17mm.6.14 Waste re
32、ceptacle, any container suitable for receivingand retaining filtrate fluid for ultimate disposal.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
33、., “The Uncountables,” AccessingUncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.E26940927. Reagents and Materials7.1 ATP standard, 1 ng ATP/mL7.1.1 Commercially available6;or7.1.2 Dilute 1 mg ATP into 1000 mL ATP dilution buffer toget a 1000-ng ATP/mL stock solution. Then, dilute
34、 1.0 mL of1000 ng ATP/mL stock solution into 999.0 mL ATP dilutionbuffertogeta1ngATP/mL ATP standard.7.2 ATP extract 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; a
35、llow to equilibrate to ambient tempera-ture before using.8. Hazards8.1 The analyst must know and observe good laboratorysafety practice in accordance with 29 CFR 1910.1450.8.2 Inhalation or dermal exposure to MWF can pose healthproblems for personnel involved with MWF sampling. Provi-sion of persona
36、l protective equipment (PPE) in the form ofrespirators, protective clothing or both may be indicated (seePractice E 1497).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 a
37、nd Test Specs and Units9.1 Sampling Site:9.1.1 Select sampling site that will yield a representativeMWF sample.9.1.2 For 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.
38、9.2 Sampling:9.2.1 If practical, draw sample from the mid-point of thefluid reservoir, otherwise draw sample from below 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 sampl
39、ing location; this should be appropriate forthe analysis objectives.9.2.2 Collect sample by removing lid from sample con-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
40、insufficient to permit 9.2.1, use asterile pipet to draw sample from the fluid and dispense it intothe sample container; 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 D 4840).9.3.2 Optimally sam
41、ples should be tested on-site as soon aspossible (50 mL.X2.1.3 Report actual volume filtered, and use this volume inEq1(12.1).TABLE 1 Precision Data for Log10pg ATP/mLMaterial Average RepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibilityLimitxSrSRrR1 3.530 0
42、.028 0.065 0.079 0.1832 0.227 0.116 0.135 0.326 0.3773 1.350 0.022 0.022 0.063 0.0634 0.079 0.094 0.099 0.262 0.2785 0.658 0.016 0.061 0.045 0.1716 0.178 0.093 0.093 0.261 0.2617 3.140 0.058 0.135 0.163 0.3788 3.364 0.012 0.099 0.033 0.277Average 1.566 0.154E2694095X2.2 Decreasing Test Sensitivity (
43、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 greater thanthe luminometers upper detectio
44、n 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).X2.2.2.2 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 prepare
45、d in X2.2.2.1.X2.2.2.3 Follow steps 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
46、 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 the luminom
47、etersupper detection limit.X2.2.5 Observe the RLU value from the luminometer andmultiply it by the appropriate dilution factor (that is, 10 for a1+9 dilution; 100 for a 1+99 dilution). Record the result asRLUobsand use this value in Eq 1.X3. EVALUATING THE RELATIONSHIP BETWEEN LOG10RLU AND LOG10PG A
48、TP/ML IN EMULSIFIED OIL, SEMI-SYNTHETIC AND SYNTHETIC MWFX3.1 Neat stocks of emulsifiable oil (EO), semi-syntheticMWF (SS) and synthetic MWF (S) were used to prepare ATPextracts by way of steps 11.1 through 11.29.X3.2 Prepared ATP extracts were diluted (11.31 through11.33) and doped with a 100,000 p
49、g ATP/mL stock solution togive 10 000, 1000, 100, and 10 pg ATP/mL.X3.3 These doped Diluted ATP extracts were analyzed forATP concentration by way of steps 11.34 through 11.39. Testresults are shown in Table X3.1 and Fig. X3.1.X3.4 Two-way Analysis of Variance (ANOVA) was com-puted to determine whether fluid-type affected the relationshipbetween RLU and ATP concentration. The ANOVA summary(Table X3.2) demonstrates that the only significant source ofvariation was the ATP concentration. There was no significanteffect due to fluid type.TABLE X3.1 ATP Sta
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