1、Designation:E256313An 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 oforigin
2、al 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 and culturab
3、le 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 metalworking fluid.1.3
4、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 microorganismcolonies on a
5、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 practices and determi
6、ne 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 Enumerating Bacteria2.2 Other
7、 Documents:3Kinyuon Acid-Fast Staining Procedure3.Terminology3.1 Definitions:3.1.1 rapidlygrowingmycobacteria(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 culturableMycobacteria popu
8、lation 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 Mycobac-teria.After a minimum
9、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 growing My-cobacteria(M.immunogenu
10、m,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 of the indigenous MWF bacterial population.
11、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 enumeration of these organisms will aid inbetter u
12、nderstanding 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(VC),viable-non culturable(VNC)and non viable(
13、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 mycobacterial populationdensities of metal working fluid field sam
14、ples.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 1,2013.Published July 2013.Originally approvedin 2007.La
15、st 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 information,refer to the standards Document Summary page onthe ASTM webs
16、ite.3Public Heatlth Microbiology:A Guide 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 Conshohocken,PA 19428-2959.United States1 5.4 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