Designation:E1532–00StandardPracticeforDetectionofMycoplasmaContaminationofCellCulturesbyUseoftheBisbenzamideDNA-BindingFluorochrome1ThisstandardisissuedunderthefixeddesignationE1532;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(e)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1ThispracticecoverstheuseofcellculturesandDNA-bindingflurorochrometechniquestodetectmycoplasmacon-taminationofcellcultures.1.2Thispracticedoesnotcoveraxeniccultivationoridentificationofmycoplasmas.1.3Thisstandarddoesnotpurporttoaddressallofthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappro-priatesafetyandhealthpracticesanddeterminetheapplica-bilityofregulatorylimitationspriortouse.2.ReferencedDocuments2.1ASTMStandards:E1531PracticefortheDetectionofMycoplasmaContami-nationofCellCulturesbyGrowthonAgaroseMedium2E1533PracticeforIndirectDetectionofMycoplasmainCellCultureby48-6-Diamindino-2-2Phenylindole(DAPI)Staining2E1536PracticefortheDetectionofMycoplasmaContami-nationofBovineSerumbytheLargeVolumeMethod23.Terminology3.1Definitions:3.1.1axeniccultivation,n—cultivationfreefromotherlivingorganisms.3.1.2directmycoplasmadetection,n—demonstrationofcharacteristiccolonialgrowthonaxenicagarmedium.3.1.3mycoplasma(Mollicute),n—smallestprokaryotesca-pableofselfreplication.4.SignificanceandUse4.1Mycoplasmahyorhinis,cultivarastrains(1)3donotgrowonanyofthestandardmediausedformycoplasmacultivation.Thesestrains,whicharefoundascontaminantsincellcultures,aredetectedbyindirectmethods.4.2Aspecializedmediumhasbeendescribedbutitisnotyetinwideuse(2).4.3ThispracticeshouldbeusedinconjunctionwithPrac-ticeE1531.4.4Allcellculturestobeexaminedformycoplasmashouldundergoaminimumoftwopassagesinantibiototic-freetissueculturemediumbeforetesting.5.IndicatorCellCultures5.1BHK-21,3T6,andVeroarethemostwidel...
