1、Designation: D 4994 89 (Reapproved 2002)Standard Practice forRecovery of Viruses from Wastewater Sludges1This standard is issued under the fixed designation D 4994; 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice is used for the recovery of viruses fromwastewater sludges and favors the enteroviruses.1.2 Both procedures a
3、re applicable to raw, digested, anddewatered sludges.SectionsProcedure AAdsorption 6 to 10Procedure BSonication 11 to 151.3 This practice was tested on standardized sludges asdescribed in 10.1 and 17.1. It is the users responsibility toensure the validity of this practice for untested matrices.1.4 T
4、his standard does not purport to address all 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.1.5 Only adequately
5、trained personnel should be allowed toperform these procedures and should use safety precautionsrecommended by the U.S. Public Health Service, Center forDisease Control,2for work with potentially hazardous biologi-cal organisms.2. Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to
6、 Water3D 1193 Specification for Reagent Water33. Terminology3.1 DefinitionsFor definitions of terms used in this prac-tice, refer to Terminology D 1129.4. Significance and Use4.1 Although many laboratories are presently isolating vi-ruses from sludge, a valid comparison of data generated has notbeen
7、 possible because of the lack of a standard test method(s).5. Apparatus5.1 Centrifuge(s), refrigerated, capable of attaining10 000 3 g, screw-capped 100-mL centrifuge bottles that canwithstand 10 000 3 g, and 250-mL screw-capped centrifugebottles capable of withstanding 2 500 3 g.5.2 pH Meter, measu
8、ring to an accuracy of at least 0.1 pHunit, equipped with a combination-type electrode. Calibratewith standard buffers.5.3 Filter Apparatus, for membrane sterilization,4with47-mm diameter filter holder and 50-mL slip-tip syringe (see7.7 for type of filter material).6. Purity of Reagents6.1 Purity of
9、 ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where such specifications are available.5Other grades may beused, prov
10、ided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.6.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D 1193, Type II.1Thi
11、s practice is under the jurisdiction of ASTM Committee D19 on Water andis the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Oct. 27, 1989. Published March 1990.2Richardson, J. H., and Barkley, W. E., Biological Safety in Microbiological andBiomedical Lab
12、oratories, 2nd. edition, U.S. Dept. of Health and Human Services,Public Health Service, Center for Disease Control, and National Institutes of Healthand Human Services, 1988.3Annual Book of ASTM Standards, Vol 11.01.4The Swinnex filter (No. SX0047000, available from Millipore Corp., 80AshbyRd., Bedf
13、ord, MA 01730, or equivalent, has been found suitable for this purpose.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChe
14、micals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.PROCEDURE AADSORPTION7. Reagen
15、ts and Materials7.1 Aluminum Chloride Solution (12.07 g/L)Dissolve12.07 g of aluminum chloride (AlCl36H2O) in 500 mL ofwater and dilute to 1000 mL. Autoclave AlCl3solution at121C for 15 min.7.2 Buffered Beef Extract SolutionDissolve 10 g of beefextract powder,61.34gofNa2HPO47H2O, and 0.12 g of citri
16、cacid in 100 mL of water in a screw-cap flask by stirring forabout2honamagnetic stirrer.Autoclave at 121C for 15 min.7.3 Disodium Hydrogen Phosphate Solution (4 g/100 mL)Dissolve4gofdisodium hydrogen phosphate(Na2HPO47H2O) in 100 mL of water and autoclave at 121Cfor 15 min.7.4 Hydrochloric Acid (1 +
17、 1)Add 1 volume of concen-trated HCl (sp gr 1.19) to 1 volume of water.7.5 Hydrochloric Acid (1 + 9)Add 1 volume of concen-trated HCl (sp gr 1.19) to 9 volumes of water.7.6 Sodium Hydroxide Solution (4 g/100 mL)Dissolve 4.0g of dry sodium hydroxide (NaOH) in water and dilute to 100mL.7.7 Filters, Di
18、sc, Membrane, 47-mm3.0-, 0.45-, and0.25-m pore size which must be cut to proper size from sheetfilters.7Disassemble filter holder. Place filter with 0.25-mpore size on support screen of filter holder and stack theremaining filters on top in order of increasing pore size.Reassemble and tighten filter
19、 holder. Filters stacked in-tandemas described tend to clog more slowly when turbid material isfiltered through them. Prepare several filter stacks.8. Summary of Procedure8.1 The adsorption procedure relies upon adsorption ofviruses from the liquid phase to the sludge solids, which areconcentrated b
20、y centrifugation. The supernatant is discarded.Viruses are desorbed from the solids by physicochemicalmeans and further concentrated by organic flocculation. De-contamination is accomplished by filtration.9. Procedure9.1 Conditioning of SludgeIn the absence of experiencethat dictates otherwise, use
21、100-mL volumes for liquid sludgesand 100-g quantities for digested, dewatered sludges.9.1.1 Measure 100 mL of well-mixed sludge in a graduated100-mL cylinder. Mix sludge vigorously immediately before itis poured into cylinder because sludge solids, which containmost of the viruses, begin to settle o
22、ut immediately aftermixing stops.9.1.2 Place stir bar into a 250-mL beaker.9.1.3 Pour the 100-mL of measured sludge from the cylin-der into the 250-mL beaker. If necessary, pour sludge severaltimes from beaker to cylinder and back to remove all sludgesolids to beaker. Take care to avoid formation of
23、 aerosols.9.1.4 Place beaker on magnetic stirrer, and stir at speedsufficient to develop vortex.9.1.5 Add 1 mL of AlCl3solution to sludge. Final concen-tration of AlCl3in sludge is approximately 0.0005 M.9.1.6 Place combination-type pH electrode into sludge andadjust pH of sludge to 3.5 6 0.1 with H
24、Cl (1 + 1). If pH fallsbelow 3.5, readjust with NaOH solution (4 g/100 mL). Ifsludge adheres to electrodes, clean electrodes by moving themup and down gently in mixing sludge. pH meter must bestandardized at pH 4.9.1.7 Continue mixing for 30 min. Check pH of the sludgeat frequent intervals. If the p
25、H drifts up, readjust to 3.5 6 0.1with HCl (1 + 9). If the pH drifts down, readjust with NaOHsolution (4 g/100 mL).9.1.8 Turn stirrer off and remove pH electrode from sludge.9.1.9 Remove cap from a screw-capped centrifuge bottleand pour conditioned sludge into centrifuge bottle. To preventtransfer o
26、f stir bar into centrifuge bottle when decantingsludge, hold another stir bar or magnet against bottom ofbeaker. Remove sludge that adheres to stir bar in the beaker bymanipulation with a stirring rod. If necessary, pour sludgeseveral times from centrifuge bottle to beaker and back toremove all slud
27、ge solids to bottle. Take care to avoid formationof aerosols.9.1.10 Replace and tighten cap on centrifuge bottle.9.1.11 Centrifuge conditioned sludge at 2 500 3 g for 15min at 4C. Discard supernatant.9.2 Elution of Viruses from Sludge Solids:9.2.1 Add stir bar to the centrifuge bottle that containss
28、edimented, conditioned sludge.9.2.2 Add 100 mL of buffered beef extract solution to thesedimented, conditioned sludge. The volume of buffered beefextract solution used to elute viruses from the conditionedsludge is equal to the original volume of the sample volume(see 9.1).9.2.3 Replace and tighten
29、cap on centrifuge bottle.9.2.4 Place centrifuge bottle on magnetic stirrer and stir atspeed sufficient to develop vortex. To minimize foaming(which may inactivate viruses), do not mix faster than neces-sary to develop vortex. Care must be taken to prevent bottlefrom toppling. Stabilize bottle as nec
30、essary.9.2.5 Continue mixing for 30 min.9.2.6 Turn stirrer off and remove stir bar from centrifugebottle.9.2.7 Replace and tighten cap on centrifuge bottle andcentrifuge conditioned sludge-eluate mixture at 10 000 3 g for30 min at 4C.9.2.8 Remove cap from centrifuge bottle. Decant superna-tant fluid
31、 (eluate) into beaker and discard sediment.9.2.9 Place a filter holder that contains a filter stack asdescribed in 7.7 on a 250-mL Erlenmeyer receiving flask.9.2.10 Load 50-mL syringe with eluate.9.2.11 Place tip of syringe into filter holder.9.2.12 Force eluate through filter stack into 250-mL rece
32、iv-ing flask. Take care not to break off tip of syringe and tominimize pressure on receiving flask, because such pressuremay splinter or topple the flask. If filter stack begins to clogbadly, empty loaded syringe into beaker containing unfiltered6Extract available from Grand Island Biological Corp.,
33、 3175 Staley Rd, GrandIsland, NY 14072, or equivalent, has been found suitable for this purpose.7Duo-Fine series sheet filters, available from Filterlite Corp., 2033 Green SpringDr., Timonium, MD 21093, or equivalent, have been found suitable for this purpose.D 4994 89 (2002)2eluate, fill syringe wi
34、th air, and inject air into filter stack toforce residual eluate from filters. Continue filtration procedurewith another filter holder and filter stack. Discard contaminatedfilter holders and filter stacks. Repeat 9.2.9 through 9.2.12 asoften as necessary to filter entire volume of eluate. Disas-sem
35、ble each filter holder and examine bottom filters to becertain they have not ruptured. If a bottom filter has ruptured,repeat 9.2.10 through 9.2.12 with new filter holders and filterstacks.9.2.13 Refrigerate eluate immediately at 4C, and maintainat that temperature until it is assayed for viruses (s
36、ee 9.3). Thenumber of cell cultures necessary for the viral assay may bereduced by concentrating the viruses in the beef extract by theorganic flocculation procedure. Some loss of virus may occurwith this procedure. If viruses in eluates are to be concentrated,proceed immediately to 9.4. If further
37、concentration is notrequired and if assay for viruses cannot be undertaken within 8h, distribute eluate into sterile sample bottles, cap tightly, andstore immediately at 70C.9.3 Viral Assay:9.3.1 At time of viral assay, rapidly thaw the frozen con-centrate at 37C and proceed with usual viral assay.
38、At least10 % of the isolates should be confirmed by second passage.9.4 Procedure for Concentrating Viruses from Sludge Elu-ates (Organic Flocculation Concentration)It is preferable toassay eluted viruses in the beef extract eluate without concen-trating them because some loss of viruses may occur in
39、concentration. However, the numbers of cell cultures neededfor assays may be reduced by concentrating the viruses in theeluate. Significant further loss of viruses may occur with thecurrently available beef extract which may not produce suffi-cient floc to adsorb all of the suspended virions.9.4.1 P
40、our eluate from 9.2.13 into a graduated cylinder andrecord the volume.9.4.2 Pour eluate into 600-mL beaker.9.4.3 For every 3 mL of beef extract eluate, add 7 mL ofsterile water to the 600-mL beaker. The concentration of beefextract is now 3 %. This dilution is necessary because 10 %beef extract ofte
41、n does not process well by the organicflocculation concentration procedure.9.4.4 Pour the diluted, filtered beef extract into a graduatedcylinder and record the total volume.9.4.5 Decant diluted filtered beef extract into 600-mL bea-ker and add a stir bar.9.4.6 Place beaker on magnetic stirrer and s
42、tir at a speedsufficient to develop vortex. To minimize foaming (which mayinactivate viruses), do not mix faster than necessary to developvortex.9.4.7 Insert combination-type pH electrode into diluted,filtered beef extract and add HCl (1 + 9) slowly until pH ofbeef extract reaches 3.56 0.1. A floccu
43、late or precipitate willform. If pH drops below 3.4, add NaOH solution (4 g/100 mL)until pH is 3.56 0.1. Avoid reducing pH below 3.4 becausesome inactivation of viruses may occur. Continue to stir for 30min.9.4.8 Turn stirrer off, remove electrode from beaker, anddistribute contents of beaker evenly
44、 among centrifuge bottles.To prevent transfer of stir bar into a centrifuge bottle, holdanother stir bar or magnet against bottom of beaker whendecanting contents.9.4.9 Replace and tighten caps on centrifuge bottles andcentrifuge the flocculated beef extract suspension at 2 500 3 gfor 15 min at 4C.
45、Pour off and discard supernatants.9.4.10 Place a small stir bar into each centrifuge bottle thatcontains flocculate and replace covers loosely.9.4.11 Measure a volume of Na2HPO47H2O solution equalto120 of the volume recorded in 9.4.4. Divide this volumeequally among the flocculates in the centrifuge
46、 bottles.9.4.12 Replace and tighten-down caps on centrifuge bottles,and place each on a magnetic stirrer. Stir flocculates slowlyuntil dissolved completely. Support bottles as necessary toprevent toppling. Avoid foaming which may inactivate oraerosolize viruses. Flocculates may be partially dissipat
47、ed withspatula before or during stirring procedure.9.4.13 Remove caps from centrifuge bottles and combinethe dissolved flocculates in a small beaker. To prevent transferof stir bars into beaker, hold another stir bar or magnet againstthe bottom of centrifuge bottle when decanting dissolvedflocculate
48、s.9.4.14 Measure pH of dissolve flocculate. If pH is above orbelow 7.0 to 7.5, adjust to within this range with either HCl(1 + 9) or NaOH solution (4 g/100 mL).9.4.15 Refrigerate final concentrate immediately at 4C, andmaintain at that temperature until assay for viruses is under-taken. If assay for
49、 viruses cannot be undertaken within 8 h,transfer dissolved precipitates to sterile sample bottles, captightly, and store immediately at 70C.9.4.16 At the time of viral assay, rapidly thaw the frozenconcentrate at 37C and proceed with usual viral assay.At least10 % of the isolates should be confirmed by second passage.10. Precision and Bias10.1 Eight independent laboratories participated in theevaluation of this recovery procedure for viruses in sludges.Five standardized sludges were utilized in the study: (1)Anaerobic, high rate, digested (mesophil
