1、Designation: D4994 89 (Reapproved 2014)Standard Practice forRecovery of Viruses from Wastewater Sludges1This standard is issued under the fixed designation D4994; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last rev
2、ision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () 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 are
3、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. It is the users responsibility to ensure thevalidity of this practice for untested matrices.1.4 The values st
4、ated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This 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 a
5、nd health practices and determine the applica-bility of regulatory limitations prior to use.1.6 Only adequately 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 pote
6、ntially hazardous biologi-cal organisms.2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD1193 Specification for Reagent Water3. Terminology3.1 DefinitionsFor definitions of terms used in thispractice, refer to Terminology D1129.4. Significance and Use4.1 Although many l
7、aboratories are presently isolating vi-ruses from sludge, a valid comparison of data generated has notbeen possible because of the lack of a standard test method(s).5. Apparatus5.1 Centrifuge(s), refrigerated, capable of attaining10 000 g, screw-capped 100-mL centrifuge bottles that canwithstand 10
8、000 g, and 250-mL screw-capped centrifugebottles capable of withstanding 2 500 g.5.2 pH Meter, measuring 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,4,5with47-mm diameter filter hol
9、der and 50-mL slip-tip syringe (see7.7 for type of filter material).6. Purity of Reagents6.1 Purity of 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 o
10、f the American Chemical Society,where such specifications are available.6Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.1This practice is under the jurisdiction of ASTM Comm
11、ittee D19 on Water andis the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Jan. 1, 2014. Published March 2014. Originallyapproved in 1989. Last previous edition approved in 2009 as D4494 89 (2009).DOI: 10.1520/D4994-89R14.2Richardson, J. H., and Barkley,
12、 W. E., Biological Safety in Microbiological andBiomedical Laboratories, 2nd edition, U.S. Dept. of Health and Human Services,Public Health Service, Center for Disease Control, and National Institutes of Healthand Human Services, 1988.3For referenced ASTM standards, visit the ASTM website, www.astm.
13、org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4The sole source of supply of the apparatus, Swinnex filter (No. SX0047000),known to the committee at this time is Millipore C
14、orp., 80Ashby Rd., Bedford, MA01730.5If you are aware of alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive careful consider-ation at a meeting of the responsible technical committee,1which you may attend.6Reagent Chemicals, American
15、Chemical Society Specifications, AmericanChemical 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
16、. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specificat
17、ion D1193, Type II.PROCEDURE AADSORPTION7. Reagents 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 beefextrac
18、t powder,5,71.34gofNa2HPO47H2O, and 0.12 g ofcitric acid in 100 mL of water in a screw-cap flask by stirringfor about2honamagnetic stirrer. Autoclave at 121C for 15min.7.3 Disodium Hydrogen Phosphate Solution (4 g/100 mL)Dissolve4gofdisodium hydrogen phosphate(Na2HPO47H2O) in 100 mL of water and aut
19、oclave at 121Cfor 15 min.7.4 Hydrochloric Acid (1+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 (NaO
20、H) in water and dilute to 100mL.7.7 Filters, Disc, Membrane, 47-mm3.0-, 0.45-, and0.25-m pore size which must be cut to proper size from sheetfilters.5,8Disassemble filter holder. Place filter with 0.25-mpore size on support screen of filter holder and stack theremaining filters on top in order of i
21、ncreasing pore size.Reassemble and tighten filter 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 ph
22、ase to the sludge solids, which areconcentrated by 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 a
23、bsence of experiencethat dictates otherwise, use 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, wh
24、ich containmost of the viruses, begin to settle out 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 sludge
25、solids to beaker. Take care to avoid formation of 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
26、sludge andadjust pH of sludge to 3.5 6 0.1 with HCl (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. Chec
27、k pH of the sludgeat frequent intervals. If the pH 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 sl
28、udge into centrifuge bottle. To preventtransfer of 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 centr
29、ifuge bottle to beaker and back toremove all sludge 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 2500 g for 15 minat 4C. Discard supernatant.9.2 Elution of Viruses from Sludge Solids:9.2.1 Add st
30、ir bar to the centrifuge bottle that containssedimented, 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 sam
31、ple volume(see 9.1).9.2.3 Replace and tighten 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 preven
32、t bottlefrom toppling. Stabilize bottle as necessary.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 g for30 min at 4C.9.2.8 Remove cap from
33、centrifuge bottle. Decant superna-tant fluid (eluate) into beaker and discard sediment.7The sole source of supply of the apparatus, extract, known to the committee atthis time is Grand Island Biological Corp., 3175 Staley Rd, Grand Island, NY14072.8The sole source of supply of the apparatus, Duo-Fin
34、e series sheet filters, knownto the committee at this time is Filterlite Corp., 2033 Green Spring Dr., Timonium,MD 21093.D4994 89 (2014)29.2.9 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 Pla
35、ce tip of syringe into filter holder.9.2.12 Force eluate through filter stack into 250-mL receiv-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 s
36、yringe into beaker containing unfilteredeluate, fill syringe with 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 as
37、often as necessary to filter entire volume of eluate. Disas-semble 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
38、 maintainat that temperature until it is assayed for viruses (see 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
39、 are to be concentrated,proceed immediately to 9.4. If further 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
40、frozen con-centrate at 37C and proceed with usual viral assay. 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 withou
41、t concen-trating them because some loss of viruses may occur inconcentration. 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
42、suffi-cient floc to adsorb all of the suspended virions.9.4.1 Pour 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 no
43、w 3 %. This dilution is necessary because 10 %beef extract often 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
44、and add a stir bar.9.4.6 Place beaker on magnetic stirrer and stir 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
45、 + 9) slowly until pH ofbeef extract reaches 3.5 6 0.1. A flocculate or precipitate willform. If pH drops below 3.4, add NaOH solution (4 g/100 mL)until pH is 3.5 6 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, remo
46、ve electrode from beaker, anddistribute contents of beaker evenly 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 f
47、locculated beef extract suspension at 2500 gfor 15 min at 4C. 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. Div
48、ide this volumeequally among the flocculates in the centrifuge 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 inactiva
49、te oraerosolize viruses. Flocculates may be partially dissipated 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 dissolvedflocculates.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 fin
