1、Designation: E 1482 04Standard Test Method forNeutralization of Virucidal Agents in Virucidal EfficacyEvaluations1This standard is issued under the fixed designation E 1482; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、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 use, in conjunction withevaluations of the virucidal efficacy, of disinfectant solutio
3、ns orpressurized disinfectant spray products intended for use oninanimate nonporous environmental surfaces or for otherspecial applications. The test method may be employed with allviruses and host systems.1.2 This test method should be performed only by personstrained in microbiology and virology.1
4、.3 This test method utilizes gel filtration technology. Theeffectiveness of the test method is dependent on the ratio of gelbed volume to sample size and uniformity in the preparation ofcolumns and centrifugation conditions. The effectiveness ofthis test method is maximized by investigator practice
5、andexperience with gel filtration techniques.1.4 This test method will reduce, but not necessarily elimi-nate, disinfectant toxicity while preserving the titer of inputvirus.1.5 The values stated in SI units are to be regarded as thestandard.1.6 This standard does not purport to address all of thesa
6、fety 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:2E 1052 Test Method for Efficac
7、y of Antimicrobial AgentsAgainst Viruses in SuspensionE 1053 Test Method for Efficacy of Virucidal Agents In-tended for Inanimate Environmental Surfaces3. Summary of Test Method3.1 After the exposure of a virus to a disinfectant, thevirus-disinfectant suspension is applied to a column of Sepha-dex3L
8、H60-120, or Sephacryl S-1000 Superfine. The column isplaced in a centrifuge and centrifuged to separate the virusfrom the disinfectant by gel filtration. The filtrate (the columnflow-through that contains the virus) is assayed in the appro-priate host system. The untreated virus control suspension i
9、ssimilarly gel filtered, and the virus titer of the filtrate isdetermined by assay of infectivity. The residual cytotoxicity ofthe disinfectant is determined by gel filtration of the disinfec-tant control under the same conditions. Results for the virusinactivation and disinfectant cytotoxicity of g
10、el filtrates arerecorded in the same manner as described in Test MethodsE 1052 and E 1053. The gel filtration procedures described inthis test method are a modification of the method of Blackwelland Chen.44. Significance and Use4.1 This test method is to be used for the removal ofvirucidal agents fr
11、om agent-virus mixtures, or from agent-neutralizer-virus mixtures, after the contact period and beforethe inoculation of these mixtures into host systems for assay ofinfectivity.4.2 The purpose of the test method is to reduce the concen-tration of agents and neutralizers in order to permit theevalua
12、tion of viral infectivity at dilutions that would otherwisebe toxic to the host.4.3 The test method is applicable to the testing of liquid andpressurized disinfectant products.4.4 This test method is compatible with organic soil loads,hard water, disinfectants containing organic solvents, andchemica
13、l neutralizers.5. Reagents and Materials5.1 Reagents:1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2004. Published October 2
14、004. Originallyapproved in 1992. Last previous edition approved in 2004 as E 1482 - 92 (2004).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document
15、 Summary page onthe ASTM website.3Sephadex is a registered trademark of Amersham Biosciences.4Blackwell, H. H., and Chen, J. H. S., “Effects of Various Germicidal Chemicalson H.EP.2 Cell Culture and Herpes simplex Virus,” Journal of the AOAC, Vol 53,1970, pp. 12291236.1Copyright ASTM International,
16、100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.1.1 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 of t
17、he American Chemical Society,where such specifications are available.5Other 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.5.1.2 Phosphate Buffered Saline (PBS) (see Dulbecco andV
18、ogt6).5.1.3 Sterile Distilled or Deionized Water.5.2 Sephadex Gel Filtration:5.2.1 Sephadex LH-60-120, compatible with organic sol-vents. (Sephacryl S-1000 Superfine may be substituted.)5.2.2 Syringe, 5 cc or 10 cc, disposable.5.2.3 Glass Wool, sterilized.5.2.4 Centrifuge Tube, 15 and 20 mL, conical
19、, sterile, anddisposable.5.2.5 Centrifuge, clinical, with rotor and shields capable ofholding 15 and 50-mL centrifuge tubes, and running at a r/minthat generates 600 3 g.5.2.6 Refrigerator, 4C.5.3 Labware:5.3.1 Pipettes, serological, 10, 5, and 1 mL, in 0.1-mLgraduations.5.3.2 Erlenmeyer Flask, ster
20、ile, 250 mL.5.3.3 Test Tube Rack or Holder, for 15 and 50-mL tubes.5.3.4 Test Tube, 18 by 150 mm.5.3.5 Laboratory Film,7or other sealing film.6. Procedure6.1 Suspend the Sephadex in a large excess of steriledistilled or deionized water in an Erlenmeyer flask. Use anamount of Sephadex sufficient for
21、the number of columns to beprepared (approximately 0.5 g of Sephadex per column). Closethe flask with a plastic film or closure and allow the Sephadexto swell overnight at 4C. Sterilize by autoclaving at 121C and15 lb of pressure for 15 min. Allow to cool to room tempera-ture.6.2 Select the syringe
22、size to be used depending on the sizeof the column desired. A 5-cc syringe is used for 3-cc columns(0.6 mL of sample to be added); a 10-cc syringe is used for 6-cccolumns (1.2 mL of sample to be added). Remove the cap fromthe syringe tip, remove the plunger from the syringe, and placethe syringe in
23、an 18 by 150-mm test tube or in a suitable tubeholder above a sink or liquid receptacle.6.3 Place a small wad of glass wool in the syringe to coverthe internal tip opening. The wad should have a diameterapproximately the same size as the internal syringe diameter,and it should be sufficiently thick
24、to hold the swollen Sephadexbeads while allowing water to pass readily.6.4 Swirl the Sephadex slurry and pipet Sephadex into thesyringe. Allow the excess water to drain, and repeat until thedesired bed size of Sephadex has formed. If the column is notused immediately, seal or plug the syringe tip, a
25、dd a layer ofdistilled water above the column, cover with parafilm, andstore at 4C.6.5 To use the column, allow the water to flow through, andthen equilibrate with PBS by passing 10 mL of PBS throughthe column.6.6 Cover the column with laboratory film or another film,and place the column in a steril
26、e 15 or 50-mL conicalcentrifuge tube. Place the tube with the column in the centri-fuge and centrifuge at approximately 600 3 g for 3 min toclear the void volume. Record the r/min used for this step.6.7 Remove the column, discard the void volume, and placethe column in a new tube.6.8 Gently pipet 0.
27、6 or 1.2 mL of the virus-disinfectantmixture (depending on the column size) onto the Sephadex,place the column in the centrifuge, and centrifuge again for 3min at exactly the same r/min as in the previous step.6.9 Remove the column from the centrifuge, collect thefiltrate (column flow-through), and
28、titrate for infectivity.6.10 The virus and disinfectant control samples are handledin the same manner.7. Spray Products7.1 Prior to applying the virus-product mixture to a Sepha-dex column, the volume of the mixture is adjusted to 2.0 mLwith an appropriate aqueous medium such as water, PBS,tissue cu
29、lture medium, or neutralizer solution.8. Chemical Neutralizers8.1 When utilized, the chemical neutralizer is added afterthe contact time, and the virus-disinfectant-neutralizer mixtureis applied to the Sephadex column.9. Precision and Bias9.1 A precision and bias statement cannot be made for thistes
30、t method at this time.10. Keywords10.1 cytotoxicity; disinfectant; gel filtration; neutralization;tissue culture; virucidal; virucidal neutralization method5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagen
31、ts notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.6Dulbecco and Vogt, Journal of Experimental Medicine, Vol 99,
32、 1954, p. 167.7The sole source of supply of the laboratory film known to the committee at thistime is American Can Company, Greenwich, CT 06830. If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful considerati
33、on at a meeting of theresponsible technical committee1, which you may attend.E1482042ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of th
34、e 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 five years andif not revised, either reapproved or withdrawn. You
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36、 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, PA 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).E1482043
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