1、Designation: D 4455 85 (Reapproved 2002)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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method describes a procedure for detection andenumeration of aquatic bacte
3、ria 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 w
4、ho 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 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
6、 use.2. Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to Water3D 1193 Specification for Reagent Water3D 3370 Practices for Sampling Water from Closed Con-duits33. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D 1129.4. Summary o
7、f 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 using a fluorescent microscope.4.4 The
8、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 significance of these populations is
9、 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 Acridine-orange epifluorescence direct-c
10、ounting 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-microscopiccount is both quantita
11、tive 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 densities of environmentalwaters.5.7 The procedure can also be used to
12、 estimate bacterialdensities in cooling tower waters, process waters, and watersassociated 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).1This test metho
13、d is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Jan. 25, 1985. Published March 1985.2DIFCO Technical InformationBactoAcridine Orange Stain, is available fromDifco Laboratories, P.O. Box 1
14、058, Detroit, MI 48201.3Annual Book of ASTM Standards, Vol 11.01.4Cherry, et al, “Temperature Influence on Bacterial Populations in AquaticSystems,” Water Research, Vol 8, 1974, pp. 149155.5Daley, R. J., “Direct Epifluorescence Enumeration of Native Aquatic Bacteria,”Native Aquatic Bacteria: Enumera
15、tion, 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.6.3 Condenser, 1.253, suitable for the microscope.6.4 High-Pressure Mercury Lamp, 200 W, on a UV lightsource giving vertic
16、al 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 plain regular polyc
17、arbonate-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 chemicals shall beus
18、ed 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 water shall conform t
19、o 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 solution (40 g/L) anddi
20、lute 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 toCargille Type A).
21、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 Turn off the vacuu
22、m 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 and air dry for 1
23、5 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 range (410 nm).8.10 Co
24、unt 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 density per millilitr
25、e 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 are calculated fo
26、r 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 amagnification of
27、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 internal diameter o
28、f thefunnel.10. Report10.1 The results are reported as number of bacteria per 1 mLof the sample.11. 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.6Filter unit H2 with
29、BG12 and BG 38 transmission filters is available from LeitzInc., 24 Link Dr., Rockleigh, NJ 07647.7Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar
30、Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.8Supporting data for this test method have been filed at ASTM Headquarters.Request RR: D19-1118.D 4455 85 (2002)2ASTM I
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34、Standards, at the address 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
35、610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).TABLE 1 Precision and Bias of Acridine Orange Epifluorescence TechniqueNOTE 1Two separate predetermined samples (A and B) were prepared and dispatched to six laboratories for conducting a
36、n 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 cannot be estimated.Sample AABacteria/mLSample BABacteria/mLTotal (3104) Total (3106)Repeatability:BRepeatabilit
37、y: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 3.8mean 0.73 mean 9.7ST, Overall Precision 0.2 ST, Overall Precision 0.75SO, Single Operator Precision 0.37
38、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 (2002)3
