1、Designation: D 4454 85 (Reapproved 2002)Standard Test Method forSimultaneous Enumeration of Total and Respiring Bacteriain Aquatic Systems by Microscopy1This standard is issued under the fixed designation D 4454; the number immediately following the designation indicates the year oforiginal adoption
2、 or, in the 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 covers the detection and enumerationof aquatic bacteria by
3、 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 tr
4、ained 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 Appro
5、ximately 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 This standard does not purport to address the safetyconcerns, if any, associated with its use. It is the responsibilityo
6、f 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:D 1129 Terminology Relating to Water3D 1193 Specification for Reagent Water3D 3370 Practices for Sampling Wa
7、ter from Closed Con-duits33. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D 1129.4. 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 m
8、in. 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 oilimme
9、rsion 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.5
10、It 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 provide
11、s 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 a
12、ndepibenthic bacteria in all fresh water and marine environments.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 (1003).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 11.01.4Zimmerman, 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
15、on Bacterial Populations in AquaticSystems,” Water Res., Vol 8, 1974, pp. 149155.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.2 Eye Pieces, 12.53, equipped with a net micrometer (10by 10 mm) (25 3 2-mm squares).6.3 Condenser, 1.
16、253, 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 Micrometer, 2 by 200 parts.6.6 Membrane Filter Support, sterile, particle-free, frit
17、ted-glass, 25 mm.6.7 Funnel, 15-mL capacity or equivalent.6.8 Membrane Filter, sterile plain regular polycarbonate,25-mm (0.1-m pore size).6.9 Filter Apparatus, that should contain vacuum source,filtering flask, and a filtering flask as a water trap.6.10 Forceps (flat tip), Alcohol, Bunsen Burner, C
18、leanGlass Slides, and Cover Slips.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals 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 whensu
19、ch specifications are available.77.2 Purity of WaterUnless otherwise indicated, referencesto water shall conform to Specification D 1193, Type IAreagent water (Type I reagent water which has been filteredtwice through a 0.2-m filter to produce bacteria-free water).7.3 Phosphate Buffer SolutionDissol
20、ve 34.0 g of potas-sium dihydrogen phosphate (KH2PO4) in 500 mL of water.Adjust to pH 7.2 6 0.05 with the NaOH solution (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 solutio
21、n through a 0.2-m filter before use.7.5 Aqueous INT-Dye (0.2 %)Dissolve 200 mg of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloridein 100 mL of water.7.6 Sudan Dye SolutionDissolve 100 mg of Sudan BlackB or equivalent in 75 mL of absolute ethanol then add 75 mLof water and mix.7.7 Imm
22、ersion Oil, very low fluorescing (equivalent toCargille Type A).7.8 Formaldehyde, 37 % solution.8. Procedure8.1 Sample Processing:8.1.1 Place 10 mL of the sample into a clean, sterile testtube. Add 1 mL of 0.2 % aqueous INT-dye 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride.8.1.2 M
23、ix carefully and hold the sample in the dark at in situtemperature for approximately 20 min.8.1.3 Stop the reaction by adding 0.1 mL of 37 % formal-dehyde that also acts as preservative (at this stage the samplecan be stored at 4C up to one month).8.2 Membrane Filtration and Microscopic Examination:
24、8.2.1 Filter 1 mL of the (INT) treated/preserved samplethrough 0.1-m polycarbonate membrane which has beenpresoaked for 24 h in a solution of sudan black B (BDH) in50 % ethanol.8.2.2 Stain the filter with 3 mL of acridine orange for 3 min.8.2.3 Filter the acridine orange.8.2.4 Remove the filter, and
25、 air-dry for 15 s.8.2.5 Place the membrane on a clean slide on which hasbeen added .1 to 2 drops of very low fluorescing immersionoil.8.2.6 Place another drop of the immersion oil on top of themembrane and apply the cover slip.8.2.7 Count cells using incident fluorescent illumination in aviolet ligh
26、t wavelength range (410 nm) for total bacteria.8.2.8 Switch to bright field illumination and count cellsshowing only bright red spots (indication of respiring bacteria).8.2.9 Count 20 fields at random within the stained portion ofthe membrane.8.2.10 Count that portion of the field which lies within
27、themicrometer area.8.2.11 Calculate the average number of both total andrespiring bacteria per micrometer area.8.2.12 Use the procedure outlined below to determinebacterial densities per millilitre of water sample.8.2.13 Use Type IA water as a negative control and as acontrol against autofluorescing
28、 particle interference.9. Enumeration and Density Calculation9.1 Bacterial densities are calculated as follows:Bacterial density per mL 52.37 3 104n/d!where:n = average number of bacteria per net micrometer field,that is (total number of bacteria counted)/(number ofmicrometre fields counted), andd =
29、 dilution factor. 2.37 3 104is the membrane conversionfactor based on a magnification of 1562.5 (eyepiece12.53) 3 (objective 1003) 3 (Leitz Ploempak unit1.253).9.2 The conversion factor of 2.37 3 104for the magnifica-tion is obtained as follows:Wet Area of 252mm membrane/Area of micrometer!5204.3 mm
30、2/0.0086 mm2!52.37 3 104Wet area is determined by measuring internal diameter of thefunnel.10. Report10.1 Report results as total number of bacteria per millilitreof sample and as total number of active bacteria per millilitre.10.2 The results can also be expressed as the percentage ofmicrobial popu
31、lations that are actively respiring.6Filter unit H2 with BG12 and BG38 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
32、 notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.D 4454 85 (2002)211. Precision and Bias811.1 See Table 1 for th
33、e expression of precision for singleoperators as SO, and the overall precision as ST.11.2 See Table 1 for a statement on the bias of the testmethod.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users
34、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 responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every fi
35、ve 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 to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical
36、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 address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, P
37、A 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), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).8Supporting data for this test me
38、thod have been filed at ASTM Headquarters.Request RR: D19-1117.TABLE 1 Summary of Precision and BiasAcridine-Orange INT-Formazan Reduction Technique to Estimate Total andRespiring Aquatic BacteriaNOTE 1Two separate predetermined samples (A and B) were prepared and dispatched to three independent lab
39、oratories for conducting aninterlaboratory study to obtain a precision statement. The information from these laboratories is summarized in the table. The bias statement cannotbeincluded here because the persistent positive or negative deviation of the method value from the accepted true value cannot
40、 be estimated.Sample AABacteria/mLSample BABacteria/mLTotal (3106) Respiring (3104) Total (3106) Respiring (3104)Repeatability:BRepeatability:Bn 55 n 55mean 1.4 4.2 mean 9.6 2.9ST, Overall Precision 0.25 4.2 ST, Overall Precision 2.9 2.3 3 106SO, Single Operator Precision 0.14 3.1 SO, Single Operato
41、r Precision 1.8 2.3Reproducibility:CReproducibility:Cn 55 n 55mean 1.2 4.0 mean 6.96 0.4ST, Overall Precision 0.54 3.8 ST, Overall Precision 4.6 2.8 3 106SO, Single Operator Precision 0.11 1.4 SO, Single Operator Precision 0.4 0.1Awhere: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 4454 85 (2002)3
