Designation:D4454−85(Reapproved2009)StandardTestMethodforSimultaneousEnumerationofTotalandRespiringBacteriainAquaticSystemsbyMicroscopy1ThisstandardisissuedunderthefixeddesignationD4454;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(´)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1Thistestmethodcoversthedetectionandenumerationofaquaticbacteriabytheuseofanacridine-orangeepifluo-rescencedirect-microscopiccountingprocedure.Thistestmethodisapplicabletoenvironmentalwatersandpotablewaters.1.2Certaintypesofdebrisandothermicroorganismsmayfluoresceinacridine-orangestainedsmears.1.3Theproceduredescribedrequiresatrainedmicrobiolo-gistortechnicianwhoiscapableofdistinguishingbacteriafromotherfluorescingbodiesonthebasisofmorphologywhenviewedathighermagnifications.21.4Useofbrightlightpermitsdifferentiationofsinglebacteriawherereducedformazanisdepositedatthepolarends.1.5Approximately104cells/mLarerequiredfordetectionbythistestmethod.21.6Minimalcellsizewhichallowsthedetectionofforma-zandepositsisrepresentedbybacteriaof0.4µm.21.7ThevaluesstatedinSIunitsaretoberegardedasstandard.Nootherunitsofmeasurementareincludedinthisstandard.1.8Thisstandarddoesnotpurporttoaddressthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatorylimitationspriortouse.2.ReferencedDocuments2.1ASTMStandards:3D1129TerminologyRelatingtoWaterD1193SpecificationforReagentWater3.Terminology3.1Definitions—Fordefinitionsoftermsusedinthistestmethod,refertoTerminologyD1129.4.SummaryofTestMethod44.1AwatersampleistreatedwithanaqueoussolutionofINT-dye(2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazo-liumchloride)for20min.Thereactionthenisstoppedbyaddinga37%solutionofformaldehyde.Sampleisfilteredthrougha0.1-µmporesizepolycarbonateme...
