1、Designation: E2694 11E2694 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 adop
2、tion 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 The This test method provides a protocol for capturing, extracting and
3、quantifying the adenosine triphosphate (ATP) contentassociated with microorganisms found in water-miscible metalworking fluids (MWF).1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to theconcentration of ATP in the samples. The light i
4、s produced and measured quantitatively as relative light units (RLU) which areconverted by comparison with an ATP standard and computation to pg ATP/mL.1.3 This test method is equally suitable for use in the laboratory or field.1.4 The test method detects ATP concentrations in the range of 4.0 pg AT
5、P/mL to 400,000 400 000 pg ATP/mL.1.5 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifyingATP. The method does not differentiate betweenATP from different sources, for example, from different types of microorganisms,such as bacteria
6、and fungi.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to e
7、stablish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in WaterD4840 Guide for Sa
8、mple Chain-of-Custody ProceduresD6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration and Reverse Osmosis Membrane ProcessesE177 Practice for Use of the Terms Precision and Bias in ASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study to Determine the Precision
9、 of a Test MethodE1326 Guide for Evaluating Non-culture Microbiological TestsE1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE2523 Terminology for Metalworking Fluids and Operations2.2 Government Standards:329 CFR 1910.1000 Occupational Safety and Hea
10、lth Standards; Air contaminants29 CFR 1910.1450 Occupational Exposure to Hazardous Chemicals in Laboratories3. Terminology3.1 Definitions: For definition of terms used in this method, refer to Terminology standards D1129, D6161, and E2523.1 This test method is under the jurisdiction ofASTM Committee
11、 E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Healthand Safety Standards for Metal Working Fluids.Current edition approved Aug. 1, 2011Oct. 1, 2016. Published August 2011October 2016. Originally approved in 2009. Last previous edition approved in 2
12、0092011 asE2694 - 09.E2694 - 11. DOI:10.1520/E2694-11.DOI:10.1520/E2694-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the
13、 ASTM website.3 Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what
14、changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the o
15、fficial document.*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 States13.2 adenosine triphosphate (ATP), na molecule comprised of a purine and three phosphate groups that serv
16、es as the primaryenergy transport molecule in all biological cells.3.3 adenosine monophosphate (AMP), nthe molecule formed by the removal of two molecules of phosphate (onepyrophosphate molecule) from ATP.3.4 aseptic, adjsterile, free from viable microbial contamination.3.5 bioluminescence, nthe pro
17、duction and emission of light by a living organism as the result of a chemical reaction duringwhich chemical energy is converted to light energy.3.6 biomass, nany matter which is or was a living organism or excreted from a microorganism (D6161).3.7 culturable, adjmicroorganisms that proliferate as i
18、ndicated by the formation of colonies on solid growth media or thedevelopment of turbidity in liquid growth media under specific growth conditions.3.8 Luciferase, na general term for a class of enzymes that catalyze bioluminescent reactions.3.9 Luciferin, na general term for a class of light-emittin
19、g biological pigments found in organisms capable of biolumines-cence.3.10 luminometer, nan instrument capable of measuring light emitted as a result of non-thermal excitation.3.11 relative light unit (RLU), nan instrument-specific unit of measurement reflecting the number of photons emitted by theLu
20、ciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.3.11.1 DiscussionRLU is not an SI unit, however, RLU are proportional to ATP concentration.3.12 viable microbial biomass, nmetabolically active (living) microorganisms3.13 Acronyms:3.13.1 AMPadenosine monophosphate3.13.2 ATPadenos
21、ine 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 ng ATP/mL standard.4.2 A 5.0 mL sample of MWF is placed into a syringe and then pressure- fi
22、ltered through a 0.7 m, glass-fiber, in-line depthfilter.4.3 The retentate is then washed with a reagent to remove extra-cellular ATP 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 microbial cells t
23、hat have been captured on the glass-fiber filter, and the filtrateis 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 an unused culture tube into which 100 L of Luciferin-Luciferasereagent has previo
24、usly been dispensed.4.8 The culture tube is placed into a luminometer and the light intensity is read in RLU.4.9 RLU are converted to Log10 pg ATP/mL of sample by computation.4.10 A procedure for differentiating between bacterial and fungal cATP-biomass is provided in Appendix X4.5. Significance and
25、 Use5.1 This method measures the concentration of ATP present in the sample. ATP is a constituent of all living cells, includingbacteria and fungi. Consequently, the presence ofATPis an indicator of total microbial contamination in metalworking fluids.ATPis not associated with matter of non-biologic
26、al origin.5.2 Method D4012 validated ATP as a surrogate for culturable bacterial data (Guide E1326).E2694 1625.3 This method differs from Method D4012 in that it eliminates interferences that have historically rendered ATP testingunusable with complex organic fluids such as MWF.5.4 The ATP test prov
27、ides rapid test results that reflect the total bioburden in the sample. It thereby reduces the delay betweentest initiation and data capture, from the 36 h to 48 h (or longer) required for culturable colonies to become visible, toapproximately five minutes.5.5 Although ATP data generally covary with
28、 culture data in MWF4, different factors affect ATP concentration than those thataffect culturability.5.5.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, underspecific growth conditions. It have been estimated that less than 1 %
29、 of the species present in an environmental sample will formcolonies under any given set of growth conditions.55.5.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the totalbioburden (See Appendix X1).5.5.2.1 One example of the species
30、 effect is that the amount of ATP per cell is substantially greater for fungi than bacteria.5.5.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells.5.5.2.3 The greater the total bioburden, the greater the ATP concentration in a sample.5.5.3 T
31、he possibility exists that the rinse step (11.15) may not eliminate all chemical substances that can interfere with thebioluminescence reaction (11.39).5.5.3.1 The presence of any such interferences can be evaluated by performing a standard addition test series as described inAppendix X3.5.5.3.2 Any
32、 impact of interfering chemicals will be reflected as bias relative to data obtained from fluid that does not containinterfering chemicals.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 m pore-size, gla
33、ss-fiber, depth-type with Luer-Lok inlet.6.4 Luminometer, using photomultiplier tube, capable of detecting light emission at 420 nm and with a cuvette chamber that canhold 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 tip
34、s, 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 or HDPE containers are strongly preferred.6.10 Syringe, Luer-Lok , 20 mL, PP
35、, 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 receiving and retaining filtrate fluid for ultimate disposal.7. Reagents and Materials7.1 ATP standard, 1 ng ATP/mL7.1.1
36、 Commercially available6; or7.1.2 Dilute 1 mg ATP into 1000 mLATP dilution buffer to get a 1000-ng ATP/mL stock solution. Then, dilute 1.0 mL of 1000ng ATP/mL stock solution into 999.0 mL ATP dilution buffer to get a 1 ng ATP/mL ATP standard.7.2 ATP extract dilution buffer6(proprietary)4 Passman et
37、al. “Real-time Testing of Bioburdens in Metalworking Fluids using Adenosine Triphosphate as a Biomass Indicator,” 2009 STLE Annual Meeting, Orlando,FL.5 Sloan, W. T., C. Quince and Curtis, T. P., “The Uncountables,” Accessing Uncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.6 The
38、sole source of supply of the proprietaryATPdilution buffer,ATPextraction reagent, filter wash reagent, and Luciferin-Luciferase reagent, is LuminUltraTechnologiesLtd., Fredericton, New Brunswick, Canada, . If you are aware of alternative suppliers, please provide this information to ASTM Internation
39、alHeadquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.E2694 1637.3 ATP extraction reagent6(proprietary)7.4 Filter wash reagent6(proprietary)7.5 Luciferin-Luciferase reagent6(proprietary); store between -20C and 4C;
40、allow to equilibrate to ambient temperature beforeusing.8. Hazards8.1 The analyst must know and observe good laboratory safety practice in accordance with 29 CFR1910.1450.8.2 Inhalation or dermal exposure to MWF can pose health problems for personnel involved with MWF sampling. Provisionof personal
41、protective equipment (PPE) in the form of respirators, protective clothing or both may be indicated (see PracticeE1497).8.3 Review material safety data sheets for materials in use at the facility to identify potential hazards in order to determineappropriate PPE (see 29 CFR 1910.1000).9. Sampling an
42、d Test Specs and Units9.1 Sampling Site:9.1.1 Select sampling site that will yield a representative MWF sample.9.1.2 For routine condition monitoring, select individual sump(s) or central systems that have actively circulating fluid.9.1.3 For diagnostic testing, select zones of pooled or stagnant MW
43、F.9.2 Sampling:9.2.1 If practical, draw sample from the mid-point of the fluid reservoir, otherwise draw sample from below surface of the MWFat an accessible location.9.2.1.1 Microbial contamination will vary considerably within the fluid system and it is important to be consistent in selectingthe s
44、ampling location; this should be appropriate for the analysis objectives.9.2.2 Collect sample by removing lid from sample container, immersing the open container (6.9), opening-down, below the fluidsurface and inverting the container to allow it to fill with the sampled fluid.9.2.3 If the fluid dept
45、h is insufficient to permit 9.2.1, use a sterile pipet to draw sample from the fluid and dispense it into thesample 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 D4840).9.3.2 Optimall
46、y samples should be tested on-site as soon as possible (50 mL.X2.1.3 Report actual volume filtered, and use this volume in Eq 1 (12.1).X2.2 Decreasing Test Sensitivity (Decreasing RLUobs by Dilution)X2.2.1 Many luminometers have an upper detection limit of 100,000 RLU. If the luminescence is greater
47、 than the upperdetection limit, the RLU display defaults to an overload signal.E2694 167X2.2.2 If the original test result yields a value greater than the luminometers upper detection limit, run a 1+9 dilution of thediluted ATP extract (11.33).X2.2.2.1 To an unused 17 by 100-mm culture tube, add 9.0
48、 mL of ATP Extract Dilution Buffer (7.2).X2.2.2.2 Use the micropipeter to transfer 1.0 mL of the dilutedATP extract from step 11.33 to the 9.0 mL ofATP Extract DilutionBuffer prepared in X2.2.2.1.X2.2.2.3 Follow steps 11.33 through 11.39.X2.2.3 If the RLUobs is still greater than the luminometers up
49、per detection limits, run a 1+99 dilution of the diluted ATP extract(11.33).X2.2.3.1 To an unused 17 by 100-mm culture tube, add 9.9 mL of ATP Extract Dilution Buffer (7.2).X2.2.3.2 Use the micropipeter to transfer 0.1 mL of the dilutedATP extract from step 11.33 to the 9.9 mL ofATP Extract DilutionBuffer 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 be made if necessary, until the RLUobs is below the luminometers upperdetection limit.X2.2.5 Observe the R
