1、Designation: E2563 13 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 year ofor
2、iginal 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 ofviable a
3、nd culturable rapidly growing Mycobacteria (RGM), ornon-tuberculosis Mycobacteria (NTM) in aqueous metalwork-ing fluids (MWF) in the presence of high non-mycobacterialbackground population using standard microbiological culturemethods.1.2 The detection limit is one colony forming unit(CFU)/mL metalw
4、orking fluid.1.3 This practice involves culture of organisms classified asLevel 2 pathogens, and should be undertaken by a trainedmicrobiologist in an appropriately equipped facility. The mi-crobiologist should also be capable of distinguishing thediverse colonies of Mycobacteria from other microorg
5、anismcolonies on a Petri dish and capable of confirming Mycobac-teria by acid fast staining method1.4 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
6、 practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumer
7、ating Bacteria2.2 Other Documents:3Kinyuon Acid-Fast Staining Procedure3. Terminology3.1 Definitions:3.1.1 rapidly growing mycobacteria (RGM)non-tuberculous Mycobacteria that grow and produce visible colo-nies in four to seven days.4. Summary of Practice4.1 For recovery and enumeration of viable and
8、 culturableMycobacteria population in metalworking fluid field samplesselective culture medium containing antimicrobial agents tosuppress bacterial and fungal contamination is recommended.(See Section 8). Standard microbiological spread and dropletplating techniques are used for the enumeration of M
9、ycobac-teria. After a minimum of 14 days incubation at 30C, theMycobacteria colonies are counted and confirmed by acid-faststaining technique specific for Mycobacteria.5. Significance and Use5.1 This practice allows for the recovery and enumeration ofviable and culturable, non-tuberculosis, rapidly
10、growing My-cobacteria (M.immunogenum, M.chelonae, M. absessus, M.fortuitum, and M.smegmatis) in the presence of high gramnegative background populations in metalworking fluid fieldsamples. During the past decade it has become increasinglyapparent that non-tuberculous Mycobacteria are commonmembers o
11、f the indigenous MWF bacterial population. Thispopulation is predominantly comprised of gram negativebacteria and fungi. Mycobacterial contamination of metal-working fluids has been putatively associated with hypersen-sitivity pneumonitis (HP) amongst metal grinding machinists.The detection and enum
12、eration of these organisms will aid inbetter understanding of occupational health related problemsand a better assessment of antimicrobial pesticide efficacy.5.2 The measurement of viable and culturable mycobacte-rial densities combined with the total mycobacterial counts(including viable culturable
13、 (VC), viable-non culturable(VNC) and non viable (NV) counts) is usually the first step inestablishing any possible relationship between Mycobacteriaand occupational health concerns (for example, HP).5.3 The practice can be employed in survey studies tocharacterize the viable-culturable mycobacteria
14、l populationdensities of metal working fluid field samples.1This practice is under the jurisdiction of ASTM Committee E34 on Occupa-tional Health and Safety and is the direct responsibility of Subcommittee E34.50 onHealth and Safety Standards for Metal Working Fluids.Current edition approved July 1,
15、 2013. Published July 2013. Originally approvedin 2007. Last previous edition approved in 2007 as E2563 - 07. DOI: 10.1520/E2563-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume inform
16、ation, refer to the standards Document Summary page onthe ASTM website.3Public Heatlth Microbiology:AGuide for the Level III Laboratory. Centers forDisease Control, U.S. Department of Health and Human Services, Atlanta, GA,1985.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West C
17、onshohocken, PA 19428-2959. United States15.4 This practice is also applicable for establishing themycobacterial resistance of metalworking fluid formulationsby determining mycobacterium survival by means of platecount technique.5.5 This practice can also be used to evaluate the relativeefficacy of
18、microbicides against Mycobacteria in metalworkingfluids.6. Interferences6.1 In some metal working fluid samples very high (106/mL) microbial background population levels; mainly gramnegative pseudomonads and fungi can interfere the enumera-tion of Mycobacteria by “overgrowth” on the agar surface.6.2
19、 Sample dilution or smaller sample size can be used tominimize interference of non-target bacterial and fungal den-sities. Replicates of sample dilutions could be also plated andthe results combined.6.3 In some metalworking fluid samples chemicals (antimi-crobial pesticides, functional additives, an
20、d other components)can interfere with the culturability of total viable Mycobacteriacount in the sample. If interference by chemicals is suspected,sample dilution may also overcome this interference but willreduce sensitivity.7. Apparatus7.1 Laboratory Incubator, 30 6 2C.7.2 Microscope with oil imme
21、rsion lens, magnification1000.7.3 Staining tray or sink with running water and drying rack.8. Reagents and Materials8.1 Test Tubes, with close fitting or airtight caps, 20 by 150mm, sterile.8.2 Test Tube Racks, sufficient size to hold 20 by 150mmtest tubes.8.3 Sterile Spreaders.8.4 Sterile Loops.8.5
22、 Sterile 1mL Pipets, with 0.01mL divisions.8.6 Dilution Water Blanks, sterile, 9 mL.8.7 Selective Mitchison Modified 7H11 Agar.8.8 Microscope Slides.8.9 Paraffnic Laboratory Film, 2.54 cm wide.8.10 Staining Reagents forAcid Fast Staining procedure forstaining Mycobacteria by the Kinyoun (cold) acid-
23、fast proce-dure.8.10.1 TB Quick Stain Reagents:8.10.1.1 Carbolfuchsin Reagent ABasic Fuchsin 17.0 g,Aqueous Phenol 1000 mL (aqueous solution of Phenol con-taining approximately 10 % water).8.10.1.2 TB-Decolorizer: Hydrochloric Acid (37 %) 30.0mL, Alcohol (denatured 95 % Ethanol or Methanol) 970 mL.8
24、.10.1.3 Methylene Blue Reagent B: Methylene Blue 2.0 g,acid alcohol 1000 mL (acid/alcohol: 3 mL 37 % HCl 97 mL in90 to 95 % Ethanol).NOTE 1Brilliant Green stain can be used instead of the MethyleneBlue stain (Brilliant Green 2.0 g, Sodium Hydroxide 0.02 g, Distilledwater 1000 mL).9. Hazards9.1 The a
25、nalyst must know and observe good laboratorypractices and safety procedures required in the microbiologylaboratory in preparing, using and disposing of cultures,reagents and materials.10. Sampling, Test Specimens, and Test Units10.1 Use sterile screw-capped, non-breakable plastic screw-capped contai
26、ners (100-200 mL) for metalworking fluid sam-pling for microbiological analysis. The sample should be arandom representative portion that is from the circulating tankas opposed to a pooled spillover or stagnant hose content.Collect approximately 100mL size samples. Analyze samplesimmediately if poss
27、ible, or refrigerate until analyzed. Maxi-mum sample storage time is 24 h at refrigeration temperatures.Follow sample documentation procedure in accordance withGood Laboratory Practices.11. Procedure11.1 Gently agitate sample to re-suspend any sediment.11.2 Using a sterile disposable pipet, distribu
28、te 2 0.5mLaliquots (1 mL) directly from the liquid sample onto twoSelective Mitchison Modified 7H11 Agar plate. Spread sampleevenly using a sterile spreader. The total count followingincubation for these two plates combined is the 100dilution.11.3 Spread plate 0.1mL sample over the agar surface,(10-
29、1dilution).11.4 Make decimal dilutions of the sample by diluting 1 mLof sample in 9mLsterile water dilution blank and plate 10-2to10-7dilutions using the Standard Droplet Plate dilution tech-nique.11.5 Allow plates to dry before inverting and sealing withparaffinic laboratory film to prevent media f
30、rom drying outduring the long term (minimum two weeks) incubation period.11.6 Incubate plates at 30C 6 2C for 14 days. Examinethe plates daily for growth. Plates with zero colonies after 14days may be evaluated as having 1 CFU.11.7 When colonies appear on plates verify at least tencolonies per plate
31、 as Acid Fast Bacilli using the Kinyuon AcidFast Staining method before reporting the results.11.8 Confirm Mycobacteria colonies by means ofAcid-FastStaining Method.11.8.1 Use a sterile loop to transfer a small amount of asuspicious Mycobacteria colony to a drop of water on themicroscope slide and s
32、pread it evenly with the loop.11.8.2 Heat-fix the slides over flame by gently passing theslide through the flame fast once or twice. The heat fixed slideshould be warm, not hot after flaming. In order to avoid overheating the slides the flaming can be substituted by a standardtemperature heat block
33、at 75C for 10 to 20 minutes.11.8.3 Acid-Fast Stain the slides by using the modifiedKinyuon Acid Fast Staining Kit.E2563 132NOTE 2Any other Acid-Fast Staining method can be used.11.8.4 If the Modified Kinyuon Acid-Fast Staining Methodis used follow the directions for staining:11.8.4.1 Flood the slide
34、s with Carbol Fuchsin TB quickstain Reagent A for 4 to 5 minutes.11.8.4.2 Rinse slide gently with water.11.8.4.3 Flood slide with TB Acid Alcohol Decolorizer for15 to 30 seconds.11.8.4.4 Rinse slide gently with water until the rinse wateris mostly clear.11.8.4.5 Gently remove excess water.11.8.4.6 C
35、ounter-stain slide with Methylene Blue/QuickStain Reagent B for 4 to 5 minutes. (Staining with BrilliantGreen for 30 seconds can replace Methylene Blue).11.8.4.7 Rinse slide under running water and dry slidecompletely before examining it under oil immersion objectiveat 1000 magnification. Mycobacter
36、ia cells will retain the stainand will show characteristic morphology. Control slide withstained Mycobacteria can also be used if the examiner isinexperienced in recognizing mycobacterial morphology.12. Calculation or Interpretation of Results12.1 Use the following equation to calculate the viablemy
37、cobacterium count per ml of metalworking fluid sample:Mycobacterium per mL Metalworking Fluid5 (1)Number of Mycobacteria Colonies multiplied by the sample dilution13. Report13.1 Report results as number of viable and culturableMycobacteria per ml of metal working fluid sample.14. Precision and Bias1
38、4.1 PrecisionSince precision will depend on the fluids,challenge microbes, and microbicide treatments used to per-form individual investigations, no statement on precision ismade.14.2 BiasSince there is no accepted reference materialsuitable for the bias in this practice, no statement on bias ismade
39、.15. Keywords15.1 acid-fast bacteria; hypersensitivity pneumonitis; met-alworking fluid; non-tuberculous Mycobacteria; rapidly grow-ing MycobacteriaASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users
40、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 and must be reviewed every fi
41、ve years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical
42、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, P
43、A 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); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E2563 133
