1、Designation: E 2563 07An American National StandardStandard Test Method forEnumeration of Non-Tuberculosis Mycobacteria in AqueousMetalworking Fluids by Plate Count Method1This standard is issued under the fixed designation E 2563; the number immediately following the designation indicates the year
2、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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the detection and enumerationof
3、viable and culturable rapidly growing Mycobacteria (RGM),or non-tuberculosis Mycobacteria (NTM) in aqueous metal-working fluids (MWF) in the presence of high non-mycobacterial background population using standard micro-biological culture methods.1.2 The detection limit is one colony forming unit(CFU
4、)/mL metalworking fluid.1.3 This test method involves culture of organisms classi-fied as Level 2 pathogens, and should be undertaken by atrained microbiologist in an appropriately equipped facility.The microbiologist should also be capable of distinguishing thediverse colonies of Mycobacteria from
5、other microorganismcolonies 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 saf
6、ety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests
7、 Used for Enumerating 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 Test Method4.1 For recovery and enumer
8、ation of viable and 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 t
9、he enumeration of Mycobac-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 method allows for the recovery and enumeration ofviable and culturable, non-tuber
10、culosis, rapidly 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 ar
11、e commonmembers of 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 d
12、etection and enumeration 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
13、viable culturable (VC), viable-non culturable(VNC) and non viable (NV) counts) is usually the first step in1This test method is under the jurisdiction of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Meta
14、l Working Fluids.Current edition approved Oct. 1, 2007. Published October 2007.2For 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 o
15、nthe ASTM website.3Public Heatlth Microbiology: A Guide for the Level III Laboratory. Centers forDisease Control, U.S. Department of Health and Human Services, Atlanta, GA,1985.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.establis
16、hing any possible relationship between Mycobacteriaand occupational health concerns (for example, HP).5.3 The method can be employed in survey studies tocharacterize the viable-culturable mycobacterial populationdensities of metal working fluid field samples.5.4 This method is also applicable for es
17、tablishing themycobacterial resistance of metalworking fluid formulationsby determining mycobacterium survival by means of platecount technique.5.5 This method can also be used to evaluate the relativeefficacy of microbicides against Mycobacteria in metalworkingfluids.6. Interferences6.1 In some met
18、al 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 Sample dilution or smaller sample size can be used tominimize interference of non-targ
19、et 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, and other components)can interfere with the culturability of total viable Mycobacteriacou
20、nt 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 immersion lens, magnification10003.7.3 Staining tray or sink with running water and drying
21、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 Sterile 1mL Pipets, with 0.01mL divisions.8.6 Dilution Water Blanks, sterile, 9 mL.8.
22、7 Selective Mitchison Modified 7H11 Agar.8.8 Microscope Slides.8.9 Paraffnic Laboratory Film, 2.54 cm wide.8.10 Staining Reagents for Acid Fast Staining procedurefor staining Mycobacteria by the Kinyoun (cold) acid-fastprocedure.8.10.1 TB Quick Stain Reagents:8.10.1.1 Carbolfuchsin Reagent ABasic Fu
23、chsin 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 %) 30.0 mL, Alcohol (denatured95 % Ethanol or Methanol) 970 mL.8.10.1.3 Methylene Blue Reagent B:Methylene Blue 2.0 g, acid alcohol 1000 mL (acid/alcoho
24、l:3 mL 37 % HCl 97 mL in 9095 % 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 analyst must know and observe good laboratorypractices and safety procedures required in the
25、microbiologylaboratory in preparing, using and disposing of cultures,reagents and materials.10. Sampling and Test Specs and Units10.1 Use sterile screw-capped, non-breakable plastic screw-capped containers (100-200 mL) for metalworking fluid sam-pling for microbiological analysis. The sample should
26、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 possible, or refrigerate until analyzed. Maxi-mum sample storage time is 24 h at refrigeration temperat
27、ures.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, distribute 2 3 0.5mLaliquots (1 mL) directly from the liquid sample onto twoSelective Mitchison Modified 7H
28、11 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,(10-1dilution).11.4 Make decimal dilutions of the sample by diluting 1 mLof sample in 9mL sterile wat
29、er 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 from drying outduring the long term (minimum two weeks) incubation period.11.6 Incubate plates at
30、 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 as Acid Fast Bacilli using the Kinyuon AcidFast Staining method before reporting the results.11
31、.8 Confirm Mycobacteria colonies by means of Acid-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 spread it evenly with the loop.11.8.2 Heat-fix the slides over flame by gently passing theslide
32、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 at 75C for 10-20 minutes.11.8.3 Acid-Fast Stain the slides by using the modifiedKinyuon Acid Fa
33、st Staining Kit.E2563072NOTE 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 slides with Carbol Fuchsin TB quickstain Reagent A for 4-5 minutes.11.8.4.2 Rinse slide gently with wate
34、r.11.8.4.3 Flood slide with TB Acid Alcohol Decolorizer for15-30 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 Counter-stain slide with Methylene Blue/QuickStain Reagent B for 4-5 minutes. (Staining with Brilliant Gre
35、enfor 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 10003 magnification. Mycobacteria cells will retain thestain and will show characteristic morphology. Control slidewith stained Mycobacter
36、ia 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 viablemycobacterium count per ml of metalworking fluid sample:Mycobacterium per mL Metalworking Fluid 5Number of My
37、cobacteria Colonies multiplied by the sample dilution(1)13. Report13.1 Report results as number of viable and culturableMycobacteria per ml of metal working fluid sample.14. Precision and Bias14.1 Repeatability has been determined by a sample t-test.The results showed that the average difference in
38、measure-ments between two replicates made by the same analyst wasnot statistically significantly different than 0. The averagedifference between the two replicates was 2.739 (P = 0.76) andthe 95 % confidence interval for the average difference was-15,198 to 20,676.14.2 Reproducibility has not been d
39、etermined at this time.14.3 No information can be presented on the bias procedurein this test method for measuring Mycobacteria viable countsby means of plate count method because no material having anaccepted reference value is available.15. Keywords15.1 acid-fast bacteria; hypersensitivity pneumon
40、itis; 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 of this standard are expressly advised that determination of t
41、he 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 five years andif not revised, either reapproved or withdrawn. Yo
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43、s 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); or through the ASTM website(www.astm.org).E2563073
