1、Designation: D4412 15Standard Test Methods forSulfate-Reducing Bacteria in Water and Water-FormedDeposits1This standard is issued under the fixed designation D4412; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last r
2、evision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope*1.1 These test methods cover the procedure for the detectionand enumeration by the most probable number (MPN) tech-nique of sul
3、fate-reducing bacteria in water or water-formeddeposits.1.2 Two media preparations are provided. Medium A whichis prepared with reagent grade water, and Medium B which isprepared using the water to be sampled as the water source.Medium B is offered for those special conditions wheresulfate-reducing
4、bacterial strains have adapted to atypicalnon-fresh water environment.1.3 For the isolation and enumeration of thermophilicsulfate-reducing bacteria encountered in waters associated withoil and gas production, all broths, dilution blanks, and incuba-tions must be maintained at temperatures of at lea
5、st 45C andpreferably within 5C at the sample temperature.1.4 The sensitivity of these test methods can be increased bypurging the dilution blanks and tubes of media with nitrogenimmediately prior to use.1.5 The analyst should be aware that adequate collaborativedata for precision and bias statements
6、 as required by PracticeD2777 are not provided. See Section 11 for details.1.6 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 all of thesafety concerns, if any, associated with its
7、use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD
8、2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committee D19 on WaterD6503 Test Method for Enterococci in Water Using Enter-olert2.2 Other Standards:Standard Methods 9221 Multiple-Tube Fermentation Tech-nique for Members of the Coliform Group33. Terminology3.1 Def
9、initionsFor definitions of terms used in thisstandard, refer to Terminology D1129.3.1.1 most probable number, nstatistical method for de-termining bacterial density based on the Poisson distribution.D65033.2 Acronyms:3.2.1 SRB, nsulfate-reducing bacteria4. Summary of Test Methods4.1 Water and water
10、deposit samples and dilutions of thesesamples are dispensed into tubes of Starkeys medium (Aor B)following five tube MPN procedures. The tubes are sealed withliquid paraffin, and incubated at 20C for 21 days.4Positivereactions are indicated by the deposit of a black precipitate.5. Significance and U
11、se5.1 Sulfate-reducing bacteria are widely distributed in ma-rine and fresh water muds which, in consequence, frequently1These test methods are under the jurisdiction of ASTM Committee D19 onWater and are the direct responsibility of Subcommittee D19.24 on Water Micro-biology.Current edition approve
12、d July 15, 2015. Published August 2015. Originallyapproved in 1984. Last previous edition approved in 2009 as D4412 84 (2009).DOI: 10.1520/D4412-15.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandar
13、ds volume information, refer to the standards Document Summary page onthe ASTM website.3Available from Standard Methods, http:/standardmethods.org.4Starkey, R. L., “Characteristics and Cultivation of Sulfate-Reducing Bacteria,”Journal of the American Water Works Association, Vol 40, 1948, pp. 129112
14、98.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1are laden with the hydrogen sulfide produced by these organ-isms during dissimilatory sulfate reduction.5.2 It has bee
15、n reported that Desulfovibrio spp. can form asmuch as 10 g of sulfide per litre during active multiplication.Sulfate-reducing bacteria can cause the external or internalcorrosion of water or wastewater pipelines and pipelines forpetroleum and natural gas. The formation of galvanic cells bymassive gr
16、owth of sulfate-reducing bacteria under suitableconditions makes the corrosion much worse than just the effectof the hydrogen sulfide on the metal or concrete.6. Apparatus and Materials6.1 Anaerobic Incubator, 20C, if available, or conventional20C incubator.NOTE 1For thermophilic organisms use a 45C
17、 incubator.6.2 Pipets, sterile, 1 mL and 10 mL, “calibrated” to deliver.6.3 Test Tubes, with close fitting or airtight caps; 16 by 150mm and 20 by 150 mm.6.4 Test Tube Racks, of sufficient size to contain 16 and20-mm tubes.7. Reagents7.1 Purity of ReagentsReagent grade chemicals shall beused in all
18、tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society,5whensuch specifications are available.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mea
19、n Reagent Water Type IIconforming to Specification D1193. In addition, reagent waterused for these test methods must be sterile.7.3 Starkeys Medium A4(Modified):Sodium lactate (C3H5NaO3) 3.5 gAmmonium chloride (NH4Cl) 1.0 gDipotassium, hydrogen orthophosphate(K2HPO4)0.5 gMagnesium sulfate (MgSO47H2O
20、) 2.0 gSodium sulfate (Na2SO4) 0.5 gCalcium chloride (CaCl22H2O) 0.1 gThioglycollic acid 0.1 gAmmonium ferrous sulfate or ferrousammonium sulfate(NH4)2SO4FeSO46H2O)0.001 gWater (H2O) 1 L7.3.1 Double strength medium (2) is prepared as aboveexcept 500 mL of water are used instead of 1 L.7.3.2 Heat to
21、dissolve and dispense 9 mL of medium persingle strength tube, and 10 mL per double strength tube.7.3.3 Tubes should be of sufficient capacity to contain 1 mLof inoculum plus 9 mL of single strength medium or 10 mL ofinoculum plus 10 mL of 2 medium.7.3.4 pH of medium should be 7.2 after autoclavester
22、ilization, at 121C for 15 min.7.4 Starkeys Medium BThe medium is similar to thatdescribed in 7.3, 7.3.1, and 7.3.2 with the following modifica-tion:7.4.1 Water collected from the sample collection site is usedto prepare the medium outlined in 7.3. The water sample isfiltered to remove particulates (
23、1.2 m membrane filter) and thepH is recorded.7.4.1.1 After preparing the Medium B following 7.3.1,7.3.2, and 7.3.3, and prior to dispensing, check and adjust pH,if necessary to that of the original water used, then filtersterilize the medium by passage through 0.2-m filter andasceptically dispense i
24、nto presterilized tubes.7.5 Hydrogen Sulfide Test Reagent:7.5.1 Ferric Chloride Stock Solution (FeCl36H2O)Dissolve 13.5 g of ferric chloride in a mixture of 250 mL ofwater and 250 mL of HCl (sp gr 1.19). Store in an airtightamber container. Prepare fresh monthly.7.5.2 p-Aminodimethylaniline Dihydroc
25、hloride Stock Solu-tion:p-Aminodimethylaniline dihydrochloride(C8H12N22HCl)1.0 gHCl (6 N) 500 mLDissolve1gofp-aminodimethylaniline dihydrochloride in500 mL of 6 N HCl. Store for up to 1 month in an amberairtight container.7.6 Liquid ParaffnHeavy, sterile, or sterile mineral oil.7.7 Buffered Dilution
26、 Water, Stock Solution:7.7.1 Dissolve 34.0 g of KH2PO4in 500 mLof water, adjustpH to 7.2 with 1 N NaOH and dilute to 1 Lwith distilled water.This is called the stock phosphate solution.7.7.2 Dissolve 38 g of MgCL2in 1 L of distilled water.7.8 Buffered Dilution Water, Working SolutionAdd 1.25mL of st
27、ock buffered dilution water and 5 mL of MgCl2solution to 500 mL of water. Bring to 1 L with water. Mix welland dispense as 90 mL dilution blanks in screw-capped bottles.Sterilize by autoclaving at 121C for 15 min.8. Procedure8.1 Clean and disinfect the area with a cleaning solution thatleaves no res
28、idue.8.2 Set out and label five replicate tubes of 10-mL double-strength Starkeys medium, A or B, in the test tube rack.8.3 Set out and label five replicate tubes of 10-mL single-strength Starkeys medium, A or B, for each mL of sample ormL of sample dilution to be tested. Use two sets of fivereplica
29、te 10-mL tubes, each to contain 1 mL of sample or 1 mLof 1/10 dilution of sample.8.4 Prior to sample inoculation, heat tubes of media anddilution blanks in a water bath to 60C then cool rapidly to20C to ensure minimal oxygen levels.8.5 Shake sample thoroughly, at least 25 times; makedilutions starti
30、ng with 10 mL of sample into one 90-mLdilution blank.5Reagent Chemicals, 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.,
31、Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.D4412 152NOTE 2Organisms do not appear to be hypersensitive to smallamounts of oxygen.8.6 Pipet 10 mL of sample into each double-strength brothand 1 mL of sample or
32、 diluted sample into each set of fivesingle-strength broths.8.7 Maintain anaerobic conditions by layering 2 to 3 ml ofsterile liquid paraffin in each tube.8.8 Recap tubes and incubate at 20C for 21 days.8.9 Include sterile water samples with each test as negativecontrols.8.10 Positive reaction is in
33、dicated by the deposit of a blackprecipitate (sulfide).8.11 Confirm dubious results by the addition of 0.5 mL offerric chloride reagent followed by 0.5 mL ofp-aminodimethylaniline reagent to the MPN tube. Add reagentto the bottom of the tube using syringe or long Pasteur pipet.Apositive reaction, bl
34、ue color, occurs within 10 min if H2Sispresent.9. Calculation9.1 Compute the number of positive findings resulting frommultiple-portion decimal dilution planting as the combinationof positives and recorded as MPN (see Standard Methods9221).9.2 When more than three series of tubes are employed in ade
35、cimal series of dilutions, use the results from only three ofthese used in computing the MPN, for example:10mL1mL0.1mL0.01mL5/5 5/5 2/5 0/5 = 5-2-0 10 = 490/100 mL5/5 4/5 2/5 0/5 = 5-4-2 = 220/100 mL5/5 3/5 1/5 1/5 = 5-3-2 = 140/100 mL5/5 0/5 0/5 0/5 = 5-0-0 = 23/100 mL10. Report10.1 Perform a Log10
36、transformation and report the resultsas Log10MPN SRB/100 mL of sample.11. Precision and Bias11.1 Microbial populations are dynamic in culture. Conse-quently a full interlaboratory study is infeasible. However, anevaluation of method precision was performed and reported.611.2 Unless a large number of
37、 portions of sample areexamined, the precision of the MPN is rather low. For example,even when the sample contains one organism per millilitre,about 37 % of tubes inoculated with 1 mL of sample may beexpected to yield negative results because of irregular distri-bution of the bacteria in the sample
38、and the multiple attachmentof bacteria to particles. When five tubes, each with 1 mL ofsample, are employed under these conditions, a completelynegative result may be expected less than 1 % of the time.11.2.1 RepeatabilityThe difference between successivemeasured Log10MPN SRB/100 mL values obtained
39、by thesame operator from replicate subsamples of a given sample.The repeatability coefficient, r = 2.8 sr, where sris therepeatability standard deviation.11.2.2 ReproducibilityThe difference between two singleand independent Log10MPN SRB/100 mL values obtained bydifferent operators on replicate subs
40、amples of a given sampleunder nominally identical test conditions. The Reproducibilitycoefficient, R = 2.8 sR, where sRis the reproducibility standarddeviation.11.2.3 For both Starkeys Medium A (7.3) and StarkeysMedium B (7.4) Log10MPN/100 mL, results obtained on fivereplicate tests by two analysts
41、were statistically indistinguish-able at the 95 % confidence level, as determined by two-wayanalysis of variance (Fobs, 1,1= 0.26; Fcrit 1,1 =0.95= 4.49).Averages (X), standard deviations (srand sR), r and R aresummarized in Table 1.11.3 BiasSince there is no accepted reference materialsuitable for
42、determining the bias of the procedure, bias cannotbe determined.12. Keywords12.1 bacteria; enumeration; most probable number; MPN;SRB; sulfate reducing bacteria6Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D19-1116. ContactAST
43、M CustomerService at serviceastm.org.TABLE 1 Replicate Tests SummarizationMediumRepeatabilityReproducibilityAnalyst 1 Analyst 1Xsrr Xsrr XsrrStarkeys Medium A (7.3) 5.6 0.2 0.6 5.7 0.3 0.9 5.7 0.2 0.7Starkeys Medium B (7.4) 5.5 0.1 0.2 5.4 0.3 0.8 5.4 0.2 0.6D4412 153SUMMARY OF CHANGESCommittee D19
44、has identified the location of selected changes to this standard since the last issue (D4412 84(2009) that may impact the use of this standard. (Approved July 15, 2015.)(1) Revised Sections 2, 3, 9.1, 10.1, and 11. (2) Added Table 1 and Section 12.ASTM International takes no position respecting the
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47、quarters. 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 address shown below.This sta
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