1、Designation:D445585(Reapproved 2014)Standard Test Method forEnumeration of Aquatic Bacteria by EpifluorescenceMicroscopy Counting Procedure1This standard is issued under the fixed designation D4455;the number immediately following the designation indicates the year oforiginal adoption or,in the case
2、 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 describes a procedure for detection andenumeration of aquatic bacteria by the use
3、 of an acridine-orange epifluorescence direct-microscopic counting procedure.It is applicable to environmental waters.1.2 Certain types of debris and other microorganisms mayfluoresce in acridine orange-stained smears.1.3 The test method requires a trained microbiologist ortechnician who is capable
4、of distinguishing bacteria from otherfluorescing bodies on the basis of morphology when viewed athigher 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 t
5、his test method.21.6 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.7 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 establis
6、h 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 testmetho
7、d,refer to Terminology D1129.4.Summary of Test Method4.1 Enumeration of aquatic bacteria is obtained by passing awater sample through a 0.2-m polycarbonate membrane filter.4.2 The membrane filter is stained with acridine orangesolution.4.3 The stained filter is examined for fluorescing bacteriacells
8、 using a fluorescent microscope.4.4 The fluorescent bacteria are counted.Dilutions are takeninto consideration and bacterial concentrations established.5.Significance and Use5.1 Bacterial populations,as part of the microbial commu-nity in aquatic systems are actively involved in nutrientcycling.The
9、significance of these populations is often difficultto ascertain because of the presence of many physiologicaltypes.However,measurement of bacterial densities is usuallythe first step in trying to establish any relationship that mightexist between bacteria and other biochemical processes.45.2 Acridi
10、ne-orange epifluorescence direct-counting proce-dure cannot differentiate between viable and nonviable cells.5.3 This procedure cannot be used to convert directly thenumbers to total carbon biomass because of the naturalvariations in bacterial cell size.5.4 The acridine-orange epifluorescence direct
11、-microscopiccount is both quantitative and precise.5.5 This procedure is ideal for enumerating both pelagic andepibenthic bacteria in all fresh water and marine environ-ments.51This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D
12、19.24 on Water Microbiology.Current edition approved Jan.1,2014.Published March 2014.Originallyapproved in 1985.Last previous edition approved in 2009 as D4455 85(2009).DOI:10.1520/D4455-85R14.2The sole source of supply of the apparatus,Bacto Acridine Orange Stain,known to the committee at this time
13、 is Difco Laboratories,P.O.Box 1058,Detroit,MI 48201.If you are aware of alternative suppliers,please provide this informationto ASTM International Headquarters.Your comments will receive careful consid-eration at a meeting of the responsible technical committee,1which you may attend.3For referenced
14、 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.4Cherry,et al,“Temperature Influence on Bacterial Populations in AquaticSystems,”
15、Water Research,Vol 8,1974,pp.149155.5Daley,R.J.,“Direct Epifluorescence Enumeration of NativeAquatic Bacteria,”Native Aquatic Bacteria:Enumeration,Activity,and Ecology,ASTM STP 695,ASTM,1979,pp.2945.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United
16、 States1 5.6 The process can be employed in survey activities tocharacterize the bacteriological densities of environmentalwaters.5.7 The procedure can also be used to estimate bacterialdensities in cooling tower waters,process waters,and watersassociated with oil drilling wells.6.Apparatus6.1 Fluorescence Microscope,with oil-immersion objectivelens(100).6.2 Eye pieces,12.5,equipped with a net micrometer(10by 10 mm)(25 by 2-mm squares).6.3 Condenser,1.25,suitable for the microscope.6.4 High-Pres
