ASTM_E_2564_-_13.pdf

Designation:E256413An American National StandardStandard Practice forEnumeration of Mycobacteria in Metalworking Fluids byDirect Microscopic Counting(DMC)Method1This standard is issued under the fixed designation E2564;the number immediately following the designation indicates the year 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 describes a direct microscopic countingmethod(DMC)for the enumeration of the acid fast stainedmycobacteria population in metalworking fluids.It can be usedto detect levels of total mycobacteria population,includingculturable as well as non-culturable(possibly dead or mori-bund)bacterial cells.This practice is recommended for allwater-based metalworking fluids.1.2 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 determine the applica-bility of regulatory limitations prior to use.For additionalsafety information,see Laboratory Safety:Principle andPractices,4th Edition22.Referenced Documents2.1 ASTM Standards:3D2881 Classification for Metalworking Fluids and RelatedMaterials3.Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 acid-fast bacteria,na distinctive staining propertyof Mycobacteria due to their lipid-rich cell walls.3.1.1.1 DiscussionOnce stained,mycobacterium resist de-colorization when exposed to acidified organic solvents,andare therefore,informally designated acid-fast.3.1.2 non-tuberculousMycobacteria(NTM)environmental mycobacteria,not associated with tuberculosis.3.1.3 microscopic factor(MF),na calibrated conversionfactor for calculating the Mycobacterium count per mLsample.3.1.3.1 DiscussionThe average number of mycobacteriumcells per one microscopic field(or oil field,OIF)is multipliedby the MF to give the concentration of mycobacterium per mLof sample.3.1.4 oil immersion field(OIF),nthe circular area of amicroscopic field visible in the eye piece of the microscopeusing oil immersion objective.4.Summary of Practice4.1 The practice describes a semi quantitative test forenumerating acid fast stained environmental mycobacterium(AFB)from metal working fluids by direct microscopic count-ing(DMC)method4.It is used to determine total mycobacte-rium counts including culturable and possibly dead or mori-bund cells in the sample.This practice cannot be used todetermine the total viable mycobacterium population in thesample.A known sample volume(centrifuged or direct)isspread over a known area(1 cm2or similar)on a microscopeslide(marked by frosted or painted circles).Following differ-ential acid-fast staining5,the acid-fast cells are counted inseveral microscopic fields over the designated area.Thecalculation is based on using a calibrated microscope with aknown Microscopic Factor(MF).The MF is determined by themicroscopic area over which a known amount of sample wasspread,the number of microscopic fields in the marked circle,and the volume of sample examined.The number of acid faststained mycobacterium cells per microscopic field multipliedby the MF gives the mycobacterium number per mL of sample.5.Significance and Use5.1 During the past decade,it has become increasinglyapparent that non-tuberculous mycobacteria are common mem-bers of the indigenous MWF bacterial population.Measure-ment of mycobacterial cell count densities is an important step1This 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.Last previous edition approved in 2011 as E2564-11.DOI:10.1520/E2564-13.2Mary J.R.Gilchrist:Biosafety Precautions for Airborne Pathogens,inLaboratory Safety Principles and Practices,pp.67-76,1995,ASM Press3For 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 website.4Standard Methods for the Examination of Dairy Products,Chapter:10:DirectMicroscopic Methods for Bacteria or Somatic Cells,16th ed.America Public HealthAssociation,Inc.,Washington,DC,1978.5Ebersole L.L.:Acid-fast stain procedures,pp.3.5.1-3.5.11.In Clinical Micro-biology Procedures Handbook,Vol.1.American Society for Microbiology,1994,Washington,D.C.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 in establishing a possible relationship between mycobacteriaand occupational health related allergic responses,for example,Hypersensitivity Pneumonitis(HP)in persons exposed toaerosols of me