1、Designation: D 6734 01Standard Test Method forLow Levels of Coliphages in Water1This standard is issued under the fixed designation D 6734; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in pare
2、ntheses 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 determination of coliphagesinfective for E. coli C in water. The test method is simple,inexpensive, and yields an indica
3、tion of water quality within6.5 h. This coliphage method can determine coliphages inwater down to 1 coliphage per volume of water sampled.1.2 The test method is applicable to natural fresh watersamples and to settled, filtered or finished water samples.1.3 This standard does not purport to address a
4、ll of thesafety concerns, if any, associated with its 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:2D 1129 Terminology
5、Relating to WaterD 1193 Specification for Reagent WaterD 3370 Practices for Sampling WaterD 4201 Test Method for Coliphages in Water3. Terminology3.1 For definitions of terms used in this test method, refer toTerminology D 1129.3.2 Description of Terms Specific to This Standard3.2.1 bacterial lawn,
6、nconfluent growth of bacteria cul-tured on an agar plate.3.2.2 coliphage, nbacterial virus capable of plaquing onthe wide-range E. coli host strain used in this assay.3.2.3 plaque, nthe circular zone of clearing (lysis) of thevisible growth of bacteria on a one or two layer agar plate,caused by the
7、action of one or more bacteriophage.3.2.4 plaque forming unit (PFU), nthe term used to reportthe number of plaques formed on an agar culture platepreviously seeded with a microorganism susceptible to abacteriophage. Although theoretically, each plaque developsfrom the action of a single bacteriophag
8、e, microbiologists usethe term, PFU, to acknowledge that a plaque may have beenformed from the action of two or more bacteriophage in closeproximity, which is indistinguishable from that formed by asingle phage.4. Summary of Test Method4.1 Ameasured water sample is adjusted to pH 6.0 with HClor NaOH
9、 and filtered through a positively-charged filter. Thecoliphages trapped in the filter are eluted with Trypticase SoyBroth (TSB) at pH 8.5. The total eluate is divided between fourTubes of melted modified nutrient agar (MNA) and E. coli Chost culture is added to each tube. The contents of each-tubea
10、re mixed and poured into a petri plate. The plates areincubated at 35C for 6 h. The coliphages present infect thehost bacteria and form plaques. The total number of plaques onthe four plates represents the number of coliphages in thevolume of water sample filtered.5. Significance and Use5.1 Coliphag
11、e organisms may serve as indicators of fecalcontamination. The presence of coliphages in water in theabsence of a disinfectant indicates the probable presence offecal contamination. The absolute relationship between thenumber of coliforms and coliphages in natural waters has notbeen conclusively dem
12、onstrated. Coliphages are generallymore resistant than coliforms to chlorination and may havesome advantage over coliforms as an indicator of treatmentefficiency in disinfected waters. The detection of coliphages ina water sample depends upon the use of a sensitive host strainin the coliphage assay.
13、 Coliphages may be detected by thisconcentration procedure in 6.5 h to provide important same-day information on the sanitary quality of water. The lowerdetection limit of this concentration procedure is 1 coliphageper volume of water sample tested.6. Interferences6.1 High salt concentrations, such
14、as these found in saline orbrackish water, interfere with this test method.6.2 Water sample turbidity in excess of 25 NTU (nepholo-metric turbidity units using Ratio Turbidimeter) results indecreased plaque formation because bacterial viruses are1This test method is under the jurisdiction of ASTM Co
15、mmittee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Nov. 10, 2001. Published January 2002.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book o
16、f ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.trapped with the particulate matter in the Zesa Plus filter andare not completely elu
17、ted by TSB at pH 8.5.6.3 Analysis for coliphage can be performed on settled andwastewaters filtered waters, disinfected waters or wastewaters;however, the relationship between coliphage and coliformbacteria will be different from that observed in natural freshwaters. Coliphage are less efficiently r
18、emoved by settling andfiltration than coliforms, and coliphage are generally moreresistant than coliforms to chlorination.7. Apparatus7.1 Water Bath,466 1C.7.2 Incubator,356 0.5C.7.3 Petri Plates, glass or plastic, sterile, 100 3 15 mm.7.4 Pipets, sterile T.D. bacteriological or Mohr, glass orplasti
19、c, 1 and 5 mL.7.5 Test Tubes, with airtight caps or screw caps, 16 3 125mm and 25 3 150 mm.7.6 Platinum Transfer Loop, 3 mm loop.7.7 Sterile Vials,123 75 mm with crimp or screw caps.7.8 Spectrophotometer, suitable for absorbance measure-ments at 520 nm.7.9 Freezer, with manual defrost.7.10 Filters,
20、Zeta Plus 60S positively charged 47 mm.37.11 Membrane Filtration Units, (filter base and funnel),reusable glass, plastic or stainless steel units wrapped withaluminum foil or kraft paper and sterilized, or disposable,sterile, plastic units.7.12 Vacuum Pump, capable of creating 15 psi pressure forfil
21、tration of wager.7.13 Vacuum Flasks, sterile 1 L.7.14 Turbidimeter, Hach ratio turbidimeter or equivalent.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications
22、of the commit-tee onAnalytical Reagents of theAmerican Chemical Society.4Other grades may be used, provided it is first ascertained thatthe reagent is of sufficiently high purity to permit its usewithout decreasing the accuracy of the determination.8.2 Purity of WaterUnless otherwise indicated, refe
23、rencesto water shall be understood to mean reagent water conformingto Specification D 1193, Type II.8.3 Host CultureEscherichia coli C, ATCC No. 13706.58.4 Trytpicase Soy Agar (TSA)68.4.1 Composition per Litre:Pancreatic Digest of Casein 15.0 gPapaic Digest of Soybean Meal 5.0 gSodium Chloride 5.0 g
24、Agar 15.0 gFinal pH 7.3 6 0.28.4.2 PreparationAdd 40 g or the dehydrated medium to1 L of water and mix well. Heat while stirring on a hot plate.Boil for 1 min or until completely dissolved. Dispense 8-10 mLquantities into screw-cap culture tubes.Autoclave for 15 min at121C (15 lbs pressure). Remove
25、from autoclave while stillmolten and incline tubes at appropriate angle for slants. Letcool to harden.8.5 Trypticase (Tryptic) Soy Broth7(TSB) and Glycerol8.5.1 Composition per Litre:Pancreatic Digest of Casein 17.0 gPapaic Digest of Soybean Meal 3.0 gSodium Chloride (NaCl) 5.0 gDipotassium Phosphat
26、e (K2PO4) 2.5 gDextrose 2.5 g8.5.2 PreparationAdd 30 g of the dehydrated mediumand 100 mL of glycerol to 900 mL of water. Mix well and heatgently to dissolve in a hot water bath. Dispense 5 mL volumesinto 16 mm screw-cap test tubes and 50 mL volumes into 125mL Erlenmyer flasks. Autoclave for 15 min
27、at 121C. Final pH7.3 6 0.2.8.6 pH Adjusted Tryptic Soy Broth8.6.1 PreparationAdd 30 g of dehydrated Tryptic SoyBroth to 1 L of water. Mix well and heat gently in a hot waterbath to dissolve.Add in NaOH drop-wise to raise the pH to 8.5.Dispense in 200 mL volumes in 250 mL screw-cap flasks andautoclav
28、e for 30 min at 121C.8.7 Modified Nutrient Agar (MNA):8.7.1 Composition per Litre:Nutrient Agar823.0 gNutrient broth 8.0 gStrontium Nitrate, Sr (NO3)0.23Ammonium Nitrate, NH4NO31.76 gSodium Chloride, NaCl 5.0 g8.7.2 PreparationAdd the ingredients to 1 L of water andmix well. Heat in boiling water ba
29、th until dissolved completely.Dispense 5.5 mLvolumes into 16 3 125 mm screw-cap culturetubes and autoclave for 15 min at 121C.9. Sampling9.1 Collect 1 L water samples in accordance with PracticeD 3370.10. Procedure10.1 Frozen Host Preparation:10.1.1 Inoculate 5 mL sterile TSB in a 16 3 125 mm cultur
30、etube with the E. coli C host culture from an agar slant or agarplate using a sterile loop. Incubate the inoculated TSB tube for18 h at 35C to allow the host to grow.10.1.2 Aseptically transfer the 5 mL of host culture from10.1.1 into 50 mL of sterile TSB + 10 % Glycerol in a 125 mLErlenmeyer flask.
31、 Incubate the culture at 35C until its absor-bance reaches 0.5 as measured at 520 nm with a spectropho-tometer previously calibrated with sterile TSB + Glycerol.3Zeta Plus 60S filters available from AMF Cuno, Meriden, CT 06450.4Reagent Chemicals, American Chemical Society Specifications , AmericanCh
32、emical Society, Washington, DC. For suggestions on the testing of reagents 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. Pharmaceutical Convention, Inc. (USPC), R
33、ockville,MD.5Available from the American Type Culture Collection, Rockville, MD 20854.6BBL 11043, Difco 0369, or equivalent.7BBL 11765, Difco 0370, or equivalent.8BBL 114472, Difco 0001, or equivalent.D673401210.1.3 Place the Erlenmeyer flask from 10.1.2 in an ice bathfor 15 min.10.1.4 Pipet 5 mL al
34、iquots of the E. coli C culture from10.1.3 into sterile vials.10.1.5 Seal the vials and store in a freezer at -20C for nomore than 9 weeks.NOTE 1Do not use a “frost-free” freezer because the freeze-thawcycles will kill the bacterial host.10.2 Assay Procedure:10.2.1 For each water sample, thaw a vial
35、 of frozen hostculture in a 46C water bath for 5 min then hold at roomtemperature until used.10.2.2 Place a measured volume of water sample (100 mL,200 mL, 500 mL; analyst selects volume) to be tested in asterile beaker and adjust the pH to 6.0 with 1 % HCL or 0.1NNaOH.10.2.3 Place 4 tubes containin
36、g 5.5 mL each of ModifiedNutrient Agar in boiling water to melt the agar. Transfer thetubes of melted agar to a 46C water bath and hold for 10 minto stabilize the temperature.10.2.4 Filter the measured water sample in 10.2.2 through aZeta Plus 60S positively charged filter contained in a vacuumfilte
37、r housing.10.2.5 Remove the filter housing containing the filter withadsorbed coliphages from the vacuum flask and place the filterhousing on a sterile vacuum flask.10.2.6 Elute adsorbed coliphages from the filter by applying10 mL pHAdjusted TSB and allowing a contact time of 10 minbefore vacuum is
38、applied. Add second 10 mL aliquot of pHAdjusted TSB to the filter and apply vacuous. Flush the filterwith three 1 mL washes of sterile water. (Eluate is thecombined filtrates from the pH Adjusted TSB elutions and thewater washes).10.2.7 Plate the total eluate volume from 10.2.6 (20-23 mL).Mix each a
39、liquot of the eluate (ca. 5 mL) with 5.5 mL moltenmodified Nutrient Agar from 10.2.3.10.2.8 Add 1.0 mL of thawed host culture from 10.2.1 toeach tube containing melted modified NutrientAgar and eluate.10.2.9 Gently mix the contents of each tube. Pour thecontents of each tube into a separate, labeled
40、 petri plate (4plates per water sample).10.2.10 Cover the 4 petri plates. Allow the agar to gel atroom temperature and incubate the plates at 35C.10.2.11 Count plaques after6h(6 0.5 h) of incubation.11. Calculation11.1 Count the plaques on each plate. Obtain the number ofplaques per total volume of
41、water sample filtered by adding theplaques counted on the four plates.Example:PlateNo. 1234No. Plaques 4255Total=16PFUIf original water sample filtered was 500 mL, report as 16PFU (plaque forming units) per 500 mL of water sample.12. Precision and Bias12.1 Single Laboratory Studies:12.1.1 Table 1 il
42、lustrates coliphage recovery from naturalwaters using the proposed coliphage method. Recovery ofcoliphages by the proposed method was compared to actualinput as determined by assay of 100 mL of water sample usingTest Method D 4201.12.1.2 Fig. 1 presents coliphage/total coliforms data fromnatural wat
43、er sources. Creeks, lakes, rivers, and reservoirs inthe Washington, D.C. area were. sampled an analyzed forcoliphages by the Test Method D 4201 and analyzed for totaland fecal coliforms by standard membrane filtration tech-niques. The best fit line was constructed by linear regressionanalysis of the
44、 data.12.1.3 Fig. 2 presents cotiphage-fecal coliform data fromnasural waters. Creeks, lakes, rivers and reservoirs in theWashington, D.C. area were sampled and analyzed for coliph-ages by the Test Method D 4201 and for total and fecalcoliforms by the standard membrane filtration technique. Thebest
45、fit line was constructed by linear regression analysis of thedata.12.1.4 Filtered-chlorinated water samples were collectedfrom water taps in a water treatment plant in the Washington,D.C. area. No chlorination occurred before settling or filtrationof the influent river water. Fig. 3 illustrates the
46、relationshipbetween coliphages and total coliforms found in filtered watersTABLE 1 Coliphage Recovery from Natural Waters by Proposed Coliphage Concentration MethodSamplemL WaterSample FilteredColiphages/100 mL asDetermined by ASTM Method No.Coliphages Recovered Using ThisMethod (20 mL eluate)% Reco
47、veredAPotomac River 250 52 122 93.8Potomac River 250 25 71 113Potomac River 250 23 55 95.7Potomac River 250 46 124 108Accotink Creek 250 10 22 88Potomac RiverB250 37 53 57Potomac River 250 524 1189 90.8Potomac River 250 52 116 89.2Cherokee Creek 500 1 5 100Potomac River 250 29 109 150Wilde Lake 250
48、1 2 80Cherokee Creek 500 7 32 91.4Potomac River 250 25 64 102.4ANumber of coliphages recovered by proposed method/coliphages in water sample filtered as determined by Test Method D 4201.BTurbidity of 35 NTU. All other sample turbidity readiness were #25 NTU.D6734013FIG. 1 Coliphage-Total Coliform Re
49、lationship in Natural Waters by Standard MethodFIG. 2 Coliphage-Fecal Coliform Relationship in Natural Waters by Standard MethodD6734014using the concentration technique. Statistical analysis of thedata by linear regression showed an r value (correlation) of0.82.12.1.5 Fig. 4 shows the relationship between coliphages andfecal coliforms found in filtered chlorinated waters using theconcentration technique. Statistical analysis of the data bylinear regression showed an r value (correlation) of 0.91.12.2 Interlaboratory Study:FIG. 3 Coliphage-Total Coliform Relat
