1、Designation: D 4455 85 (Reapproved 2009)Standard Test Method forEnumeration of Aquatic Bacteria by EpifluorescenceMicroscopy Counting Procedure1This standard is issued under the fixed designation D 4455; the number immediately following the designation indicates the year oforiginal adoption or, in t
2、he 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 describes a procedure for detection andenumeration of aquatic bacter
3、ia by the use 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 wh
4、o is capable 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
5、detectionby this 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 sta
6、ndard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D 1129 Terminology Relating to WaterD 1193 Specification for Reagent Water3. Terminology3.1 DefinitionsFor definitions of terms u
7、sed in this testmethod, refer to Terminology D 1129.4. 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 f
8、luorescing bacteriacells 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 invol
9、ved in nutrientcycling. The 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 bio
10、chemical processes.45.2 Acridine-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
11、-orange epifluorescence direct-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.55.6 The process can be employed in survey activities tocharacterize the bacteriological densit
12、ies of environmentalwaters.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
13、2002 as D 4455 85 (2002).2The sole source of supply of the apparatus, Bacto Acridine Orange Stain,known to the committee at this time is Difco Laboratories, P.O. Box 1058, Detroit,MI 48201. If you are aware of alternative suppliers, please provide this informationto ASTM International Headquarters.
14、Your comments will receive careful consid-eration at a meeting of the responsible technical committee,1which you may attend.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, r
15、efer to the standards Document Summary page onthe ASTM website.4Cherry, et al, “Temperature Influence on Bacterial Populations in AquaticSystems,” Water Research, Vol 8, 1974, pp. 149155.5Daley, R. J., “Direct Epifluorescence Enumeration of NativeAquatic Bacteria,”Native Aquatic Bacteria: Enumeratio
16、n, Activity, and Ecology, ASTM STP 695,ASTM, 1979, pp. 2945.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.7 The procedure can also be used to estimate bacterialdensities in cooling tower waters, process waters, and watersassociat
17、ed with oil drilling wells.6. Apparatus6.1 Fluorescence Microscope, with oil-immersion objectivelens (1003).6.2 Eye pieces, 12.53, equipped with a net micrometer (10by 10 mm) (25 by 2-mm squares).6.3 Condenser, 1.253, suitable for the microscope.6.4 High-Pressure Mercury Lamp, 200 W, on a UV lightso
18、urce giving vertical illumination and a filter unit H2 (Leitz)6with BG12 and BG38 transmission filters or equivalent.6.5 Stage Micrometer, 2 by 200 parts.6.6 Membrane Filter Support (25 mm), sterile, particle-free, fritted-glass.6.7 Funnel, 15-mL capacity or equivalent.6.8 Membrane Filter, sterile p
19、lain regular polycarbonate-25mm, (0.2-m pore size).6.9 Filter Apparatus, containing vacuum source, filteringflask, and a filtering flask as a water trap.6.10 Forceps (flat tip), Alcohol, Bunsen Burner, CleanGlass Slides, and Cover Slips.7. Reagents and Materials7.1 Purity of ReagentsReagent grade ch
20、emicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society, whensuch specifications are available.77.2 Purity of WaterUnless otherwise indicated, referencesto wat
21、er shall conform to Specification D 1193 Type 1Areagent water (Type I reagent water filtered twice through a0.2-m filter to produce bacteria-free water).7.3 Phosphate Buffer SolutionDissolve 34.0 g of potas-sium dihydrogen phosphate (KH2PO4) in 500 mL of water.Adjust to pH 7.2 6 0.05 with NaOH solut
22、ion (40 g/L) anddilute to 1 L with water.7.4 Acridine Orange SolutionDissolve 10 mg of acridineorange in 100 mL of phosphate buffer. Filter small portions ofthe acridine orange solution through a 0.2-m filter before use.7.5 Isopropanol.7.6 Xylene.7.7 Immersion Oil, very low fluorescing (equivalent t
23、oCargille Type A).8. Procedure8.1 Place a 0.2-m membrane filter on the filter base andattach the funnel. Add 10 mL of buffered water to the funnelthen add 1 mL of the water sample or dilution (use 9-mLdilution blanks). Turn on the vacuum.8.2 Rinse the membrane with 5 mL of sterile reagent water.8.3
24、Turn off the vacuum and flood the membrane with theacridine orange solution. Allow to stand for 3 to 4 min, thenturn on the vacuum and filter through.8.4 Rinse the membrane with 0.5 mL of isopropanol. Do notexceed 10-s contact time.8.5 Rinse the membrane with 0.4 mL of xylene.8.6 Remove the membrane
25、 and air dry for 15 s.8.7 Place membrane on a clean microscope slide on whichhas been added 2 drops of fluorescence-free immersion oil.8.8 Place another drop of immersion oil on top of membraneand apply cover slip.8.9 Count cells using incident fluorescent illumination inviolet light wavelength rang
26、e (410 nm).8.10 Count 20 fields at random within the stained portion ofthe membrane.8.11 Count only that portion of the field which lies withinthe micrometer area.8.12 Calculate the average number of bacteria per microme-ter area.8.13 Use the procedure outlined in Section 9 to determinebacterial den
27、sity per millilitre of water sample.8.14 Type IA water is used as a negative control and as acontrol against autofluorescing particle interferences.8.15 Water sample may be preserved with 0.2 mL of 10 %formaldehyde per 10 mL of the sample.9. Enumeration and Density Calculation9.1 Bacterial densities
28、 are calculated for 25-mm filters asfollows:Bacterial Density/mL 5 2.37 3 104n/d!where:n = average number of bacteria per net micrometer field;that is (total number of bacteria counted)/(number ofmicrometer fields counted), andd = dilution factor.2.37 3 104is the membrane conversion factor based on
29、amagnification of 1562.5 (eyepiece 12.53) 3 (objective1003) 3 (condexer unit 1.253).9.2 The membrane conversion factor of 2.37 3 104for theabove magnification is obtained as follows:Wet area of 25 mm membrane/Area of micrometer!5 204.3 mm2/0.0086 mm2!5 2.37 3 104Wet area is determined by measuring i
30、nternal diameter of thefunnel.10. Report10.1 The results are reported as number of bacteria per 1 mLof the sample.6The sole source of supply of the apparatus, filter unit H2 with BG12 and BG38 transmission, known to the committee at this time is Leitz Inc., 24 Link Dr.,Rockleigh, NJ 07647.7Reagent C
31、hemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand Nati
32、onal Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.D 4455 85 (2009)211. Precision and Bias811.1 See Table 1 for the expression of single operatorprecision as SOand overall precision as ST.11.2 See Table 1 for a statement on the bias of the testmethod.ASTM International takes no
33、 position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own respons
34、ibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed t
35、o ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the add
36、ress shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone),
37、 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).8Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR: D19-1118.TABLE 1 Precision and Bias of Acridine Orange Epifluorescence TechniqueNOTE 1T
38、wo separate predetermined samples (A and B) were prepared and dispatched to six laboratories for conducting an interlaboratory study toobtain a precision statement. A bias statement cannot be included here because the positive or negative deviation of the method value from the acceptedtrue value can
39、not be estimated.Sample AABacteria/mLSample BABacteria/mLTotal (3104) Total (3106)Repeatability:BRepeatability:Bn 5 n 5mean 0.62 mean 8.6ST, Overall PrecisionB0.28 ST, Overall Precision 1.5SO, Single OperatorBPrecision 0.14 SO, Single Operator Precision 0.52Reproducibility:CReproducibility:Cn 3.25 n
40、 3.8mean 0.73 mean 9.7ST, Overall Precision 0.2 ST, Overall Precision 0.75SO, Single Operator Precision 0.37 SO, Single Operator Precision 0.89Awhere:ST=the average standard deviation calculated by pooling the sum of the squares, andSO=the square root of the quotient extracted from the sum of the individual analyst variances divided by the number of analysts.BReading of five (5) slides from a sample.CReading of one (1) slide five times from a sample.D 4455 85 (2009)3
