ASTM_D_4454_-_85_2009.pdf

Designation:D445485(Reapproved 2009)Standard Test Method forSimultaneous Enumeration of Total and Respiring Bacteriain Aquatic Systems by Microscopy1This standard is issued under the fixed designation D4454;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 test method covers the detection and enumerationof aquatic bacteria by the use of an acridine-orange epifluo-rescence direct-microscopic counting procedure.This testmethod is applicable to environmental waters and potablewaters.1.2 Certain types of debris and other microorganisms mayfluoresce in acridine-orange stained smears.1.3 The procedure described requires a trained microbiolo-gist or technician who is capable of distinguishing bacteriafrom other fluorescing bodies on the basis of morphology whenviewed at higher magnifications.21.4 Use of bright light permits differentiation of singlebacteria where reduced formazan is deposited at the polar ends.1.5 Approximately 104cells/mL are required for detectionby this test method.21.6 Minimal cell size which allows the detection of forma-zan deposits is represented by bacteria of 0.4 m.21.7 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.8 This standard does not purport to address the safetyconcerns,if any,associated with its use.It is the responsibilityof the user of this standard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2.Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD1193 Specification for Reagent Water3.Terminology3.1 DefinitionsFor definitions of terms used in this testmethod,refer to Terminology D1129.4.Summary of Test Method44.1 A water sample is treated with an aqueous solution ofINT-dye(2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazo-lium chloride)for 20 min.The reaction then is stopped byadding a 37%solution of formaldehyde.Sample is filteredthrough a 0.1-m pore size polycarbonate membrane filter(presoaked in sudan black solution or equivalent),and stainedwith acridine orange for 3 min.4.2 The filter is then air-dried and examined under oilimmersion for total bacteria under epifluorescence illuminationand for respiring bacteria under transmitted bright light illu-mination.5.Significance and Use5.1 Measurement of bacterial densities is generally the firststep in establishing a relationship between bacteria and otherbiochemical processes.5It is known that the classical platecount procedure underestimates bacterial densities while theepifluorescence direct microscopic procedure more accuratelydepicts the total numbers of nonviable or dormant and viablecells in a water sample.The acridine-orange INT-formazanreduction technique provides information on the total concen-trations of bacteria as well as that proportion which are activelyrespiring and thus involved in degradative processes.5.2 The acridine-orange INT-formazan reduction techniqueis both quantitative and precise.5.3 This procedure is ideal for enumerating both pelagic andepibenthic bacteria in all fresh water and marine environments.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved May 1,2009.Published June 2009.Originallyapproved in 1985.Last previous edition approved in 2002 as D4454 85(2002).DOI:10.1520/D4454-85R09.2DIFCO Technical InformationBactoAcridine Orange Stain,is available fromDifco Laboratories,P.O.Box 1058,Detroit,MI 48201.3For 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.4Zimmerman,et al,“Simultaneous Determination of Total Number of AquaticBacteria and the Number Thereof Involved in Respiration,”Applied and Environ-mental Microbiology,Vol 36,1978,pp.9269355Cherry,et al,“Temperature Influence on Bacterial Populations in AquaticSystems,”Water Res.,Vol 8,1974,pp.149155.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 5.4 The process can be employed in survey studies tocharacterize the bacteriological densities and activities ofenvironmental waters.6.Apparatus6.1 Fluorescence Microscope,with an oil immersion objec-tive lens(100).6.2 Eye Pieces,12.5,equipped with a net micrometer(10by 10 mm)(25 2-mm squares).6.3 Condenser,1.25,suitable for the microscope.6.4 High-Pressure Mercury Lamp,200-W,on a UV lightsource giving vertical illumination,and a filter unit H2(Leitz)6with BG12 and BG38 transmission filters or equivalents.6.5 Stage M