1、Designation: E2563 13E2563 18 An American National StandardStandard Practice forEnumeration of Non-Tuberculosis Mycobacteria in AqueousMetalworking Fluids by Plate Count Method1This standard is issued under the fixed designation E2563; the number immediately following the designation indicates the y
2、ear 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 practice covers the detection and enumeration of
3、 viable and culturable rapidly growing Mycobacteria (RGM), ornon-tuberculosis Mycobacteria (NTM) in aqueous metalworking fluids (MWF) in the presence of high non-mycobacterialbackground population using standard microbiological culture methods.1.2 The detection limit is one colony forming unit (CFU)
4、/mL metalworking fluid.1.3 This practice involves culture of organisms classified as Level 2 pathogens, and should be undertaken by a trainedmicrobiologist in an appropriately equipped facility. The microbiologist should also be capable of distinguishing the diversecolonies of Mycobacteria from othe
5、r microorganism colonies on a Petri dish and capable of confirming Mycobacteria by acid fastacid-fast staining methodmethod.1.4 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 establish app
6、ropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles f
7、or the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating MethodsE1326
8、Guide for Evaluating Non-culture Microbiological TestsE2523 Terminology for Metalworking Fluids and Operations2.2 Other Documents:Document:3KinyuonKinyoun Acid-Fast Staining Procedure3. Terminology3.1 For definitions of terms used in this standard, refer to Terminology E2523.3.2 Definitions:3.2.1 ra
9、pidly growing mycobacteria (RGM)non-tuberculous Mycobacteria that grow and produce visible colonies in four toseven days.4. Summary of Practice4.1 For recovery and enumeration of viable and culturable Mycobacteria population in metalworking fluid field samples,selective culture medium containing ant
10、imicrobial agents to suppress bacterial and fungal contamination is recommended. (See1 This practice is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Healthand Safety Standards for Metal Working Fluids.Curren
11、t edition approved July 1, 2013Oct. 1, 2018. Published July 2013October 2018. Originally approved in 2007. Last previous edition approved in 20072013 as E2563- 07. 13. DOI: 10.1520/E2563-13.10.1520/E2563-18.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer
12、Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Public Heatlth MicrobiologyHealth Microbiology: A Guide for the Level III Laboratory.Laboratory, Centers for Disease Control, U.S. Department of Health
13、and HumanServices, Atlanta, GA, 1985.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM re
14、commends 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 official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Section 8)
15、Standard microbiological spread and droplet plating techniques are used for the enumeration of Mycobacteria.My-cobacteria (see Practices D5465). After a minimum of 14 days incubation at 30C,30 C, the Mycobacteria colonies are countedand confirmed by acid-fast staining technique specific for Mycobact
16、eria.5. Significance and Use5.1 This practice allows for the recovery and enumeration of viable and culturable, non-tuberculosis, rapidly growingMycobacteria (M.immunogenum,M. immunogenum,M.chelonae,M. chelonae,M. absessus,M. fortuitum, and M.smegmatisM.smegmatis) in the presence of high gram negati
17、ve Gram-negative background populations in metalworking fluid field samples.During the past decade, it has become increasingly apparent that non-tuberculous Mycobacteria are common members of theindigenous MWF bacterial population. This population is predominantly comprised of gram negative Gram-neg
18、ative bacteria andfungi. Mycobacterial contamination of metalworking fluids has been putatively associated with hypersensitivity pneumonitis (HP)amongst metal grinding metalgrinding machinists. The detection and enumeration of these organisms will aid in betterunderstanding of occupational health re
19、lated health-related problems and a better assessment of antimicrobial pesticide efficacy.5.2 The measurement of viable and culturable mycobacterial densities (Guide E1326), combined with the total mycobacterialcounts (including viable culturable (VC), viable-non culturableviable nonculturable (VNC)
20、 and non viable nonviable (NV)counts)counts), is usually the first step in establishing any possible relationship between Mycobacteria and occupational healthconcerns (for example, HP).5.3 The practice can be employed in survey studies to characterize the viable-culturable mycobacterial population d
21、ensities ofmetal working metalworking fluid field samples.5.4 This practice is also applicable for establishing the mycobacterial resistance of metalworking fluid formulations bydetermining mycobacterium survival by means of plate count technique.5.5 This practice can also be used to evaluate the re
22、lative efficacy of microbicides against Mycobacteria in metalworking fluids.6. Interferences6.1 In some metal working metalworking fluid samples, very high (106/mL) microbial background population levels;levels,mainly gram negative Gram-negative pseudomonads and fungi, can interfere with the enumera
23、tion of Mycobacteria by“overgrowth” on the agar surface.6.2 Sample dilution or smaller sample size can be used to minimize interference of non-target bacterial and fungal densities.Replicates of sample dilutions could be also plated and the results combined.6.3 In some metalworking fluid samples, ch
24、emicals (antimicrobial pesticides, functional additives, and other components) caninterfere with the culturability of total viable Mycobacteria count in the sample. If interference by chemicals is suspected, sampledilution may also overcome this interference but will reduce sensitivity.7. Apparatus7
25、.1 Laboratory Incubator, Laboratory Incubator, 30 6 2C.2 C.7.2 Microscope, Microscope with oil immersion lens, magnification 1000.7.3 Staining Tray or Sink, Staining tray or sink with running water and drying rack.8. Reagents and Materials8.1 Test Tubes, with close fitting close-fitting or airtight
26、caps, 20 by 150 mm, 150 mm, sterile.8.2 Test Tube Racks, sufficient size to hold 20 by 150mm150 mm test tubes.8.3 Sterile Spreaders.8.4 Sterile Loops.8.5 Sterile 1mL 1 mL Pipets, with 0.01mL0.01 mL divisions.8.6 Dilution Water Blanks, sterile, 9 mL.8.7 Selective Mitchison Modified 7H11 Agar.8.8 Micr
27、oscope Slides.8.9 Paraffnic Laboratory Film, 2.54 cm wide.8.10 Staining Reagents, for Acid Fast Staining acid-fast staining procedure for staining Mycobacteria by the Kinyoun (cold)acid-fast procedure.8.10.1 TB Quick Stain Reagents:8.10.1.1 Carbolfuchsin Reagent ABasic Fuchsinfuchsin 17.0 g, Aqueous
28、 Phenolaqueous phenol 1000 mL (aqueous solutionof Phenolphenol containing approximately 10 % water).E2563 1828.10.1.2 TB-Decolorizer: TB-DecolorizerHydrochloric Acid (37 %) 30.0 mL, Alcohol acid (37 %) 30.0 mL, alcohol(denatured 95 % Ethanol or Methanol) 970 mL.ethanol or methanol) 970 mL.8.10.1.3 M
29、ethylene Blue Reagent B: BMethylene Blue 2.0 g, acid alcohol 1000 mL (acid/alcohol: 3 mL 37 % HCl 97 mL in90 to 95 % Ethanol).ethanol).NOTE 1Brilliant Green stain can be used instead of the Methylene Blue stain (Brilliant Green 2.0 g, Sodium Hydroxidesodium hydroxide 0.02 g,Distilleddistilled water
30、1000 mL).9. Hazards9.1 The analyst must know and observe good laboratory practices and safety procedures required in the microbiology laboratoryin preparing, using, and disposing of cultures, reagents, and materials.10. Sampling, Test Specimens, and Test Units10.1 Use sterile screw-capped, non-break
31、able sterile, non-breakable, plastic screw-capped containers (100-200 (100 to 200 mL)for metalworking fluid sampling for microbiological analysis. The sample should be a random representative portion that is fromthe circulating tank, as opposed to a pooled spillover or stagnant hose content. Collect
32、 approximately 100mL100 mLsize samples.Analyze samples immediately if possible, or refrigerate until analyzed. Maximum sample storage time is 24 h at refrigerationtemperatures. Follow sample documentation procedure in accordance with Good Laboratory Practices.good laboratory practices.11. Procedure1
33、1.1 Gently agitate sample to re-suspend any sediment.11.2 Using a sterile disposable pipet, distribute 2 0.5mL0.5 mL aliquots (1 mL) directly from the liquid sample onto twoSelectiveselective Mitchison Modifiedmodified 7H11 Agar plate.agar plates. Spread sample evenly using a sterile spreader. Theto
34、tal count following incubation for these two plates combined is the 100 dilution.11.3 Spread plate 0.1mL0.1 mL sample over the agar surface, (10-11 dilution).11.4 Make decimal dilutions of the sample by diluting 1 mLof sample in 9mL9 mLsterile water dilution blank and plate 10-22to 10-77 dilutions u
35、sing the Standard Droplet Platestandard droplet plate dilution technique.11.5 Allow plates to dry before inverting and sealing with paraffinic laboratory film to prevent media from drying out duringthe long term (minimum two weeks) incubation period.11.6 Incubate plates at 30C30 C 6 2C2 C for 14 day
36、s. Examine the plates daily for growth. Plates with zero colonies after14 days may be evaluated as having 1 CFU.11.7 When colonies appear on plates, verify at least ten colonies per plate asAcid Fast Bacilli acid-fast bacilli using the KinyuonAcid Fast Staining Kinyoun acid-fast staining method befo
37、re reporting the results.11.8 Confirm Mycobacteria colonies by means of Acid-Fast Staining Method.acid-fast staining method.11.8.1 Use a sterile loop to transfer a small amount of a suspicious Mycobacteria colony to a drop of water on the microscopeslide and spread it evenly with the loop.11.8.2 Hea
38、t-fix the slides over flame by gently passing the slide through the flame fast once or twice. The heat fixed heat-fixedslide should be warm, not hot after flaming. In order to avoid over heating overheating the slides, the flaming can be substitutedby a standard temperature heat block at 75C75 C for
39、 10 to 20 minutes.min.11.8.3 Acid-Fast StainAcid-fast stain the slides by using the modified Kinyuon Acid Fast Staining Kit.Kinyoun acid-faststaining kit.NOTE 2Any other Acid-Fast Stainingacid-fast staining method can be used.11.8.4 If the Modified Kinyuon Acid-Fast Staining Method is usedmodified K
40、inyoun acid-fast staining method is used, followthe directions for staining:11.8.4.1 Flood the slides with Carbol Fuchsin carbolfuchsin TB quick stain Reagentreagent A for 4 to 5 minutes.min.11.8.4.2 Rinse slide gently with water.11.8.4.3 Flood slide with TB Acid Alcohol Decolorizeracid alcohol deco
41、lorizer for 15 to 30 seconds.s.11.8.4.4 Rinse slide gently with water until the rinse water is mostly clear.11.8.4.5 Gently remove excess water.11.8.4.6 Counter-stain slide with Methylene Blue/Quick Stain ReagentBlue/quick stain reagent B for 4 to 5 minutes.min.(Staining with Brilliant Green for 30
42、secondss can replace Methylene Blue).Blue.)11.8.4.7 Rinse slide under running water and dry slide completely before examining it under oil immersion objective at 1000magnification. Mycobacteria cells will retain the stain and will show characteristic morphology. Control slide with stainedMycobacteri
43、a can also be used if the examiner is inexperienced in recognizing mycobacterial morphology.12. Calculation or Interpretation of Results12.1 Use the following equation to calculate the viable mycobacterium count per mlmL of metalworking fluid sample:E2563 183Mycobacterium per mL Metalworking Fluid5
44、(1)Number of Mycobacteria Colonies multiplied by the sample dilutionMycobacterium per mL metalworking fluid5 (1)Number of Mycobacteria colonies multiplied by the sample dilution13. Report13.1 Report results as number of viable and culturable Mycobacteria per mlmL of metal working metalworking fluid
45、sample.14. Precision and Bias14.1 PrecisionSince precision will depend on the fluids, challenge microbes, and microbicide treatments used to performindividual investigations, no statement on precision is made.14.2 BiasSince there is no accepted reference material suitable for the bias in this practi
46、ce, no statement on bias is made.15. Keywords15.1 acid-fast bacteria; hypersensitivity pneumonitis; metalworking fluid; non-tuberculous Mycobacteria; rapidly growingMycobacteriaASTM International takes no position respecting the validity of any patent rights asserted in connection with any item ment
47、ionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee
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49、of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail
