1、Designation: E2362 09E2362 15Standard Practice forEvaluation of Pre-saturated or Impregnated Towelettes forHard Surface Disinfection1This standard is issued under the fixed designation E2362; the number immediately following the designation indicates the year oforiginal adoption or, in the case of r
2、evision, the year of last revision. 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 designed to evaluate the antimicrobial activity of pre-saturated or impregnated
3、towelettes when used as ahard surface disinfectant.1.2 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLPs) are required and tofollow them when appropriate.1.3 This practice should be performed only by those trained in microbiological techniques.1.4 The
4、values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 Appropriate modifications to the practice may be required when testing organisms not specified herein.1.6 This standard does not purport to address all of the safety concerns, if
5、 any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1054 Test
6、 Methods for Evaluation of Inactivators of Antimicrobial Agents2.2 Federal Standard40 CFR, Part 160 Good Laboratory Practice Standards33. Terminology3.1 carrier, na transportable surface onto which a test organism will be inoculated and dried. The carrier will be treated withthe test substance and s
7、ubcultured for survivors.3.2 CFU, ncolony forming units3.3 disinfectant, na physical or chemical agent or process that destroys pathogenic or potentially pathogenic microorganismsin/on surfaces or objects.3.4 impregnated, adjsaturated with test substance.3.5 neutralizer, na component used to render
8、an active agent incapable of destroying organisms by chemical or physicalmeans.3.6 pre-saturated, adjto be filled or impregnated with test substance prior to the time of its intended use.3.7 towelette, nA paper, cloth or non-woven blend material used as a transporter for a cleaning and/or disinfecti
9、on agent.1 This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommiteeSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Dec. 1, 2009Oct. 1, 2015 Published December 2009Oc
10、tober 2015. Originally approved in 2004. Last previous edition approved in 20042009as E2362 04.E2362 09. DOI: 10.1520/E2362-09.10.1520/E2362-15.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvo
11、lume information, refer to the standards Document Summary page on the ASTM website.3 Available from the Superintendent of Documents, U.S. Government Printing Office, Washington D.C. 20402This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication
12、of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be consider
13、ed the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14. Summary of Practice44.1 A towelette impregnated or pre-saturated with a test substance is used to treat a carrier which has been inoculated with atest organis
14、m after an aliquot of a test organism has been inoculated, evenly distributed, distributed to an inoculation area ofapproximately one square inch (approximately 625 mm), and dried onto the carrier. The carrier is wiped using the pre-saturatedor impregnated towelette simulating the application of the
15、 test substance and then held for a pre-determined contact time. Afterthe specified contact time, the test substance remaining on the carrier is neutralized and the carrier is subcultured to recoversurviving test organism. The used towelette, after the contact time, is also cultured for surviving te
16、st organism.5. Significance and Use5.1 This test method practice may be used to determine if a pre-saturated or impregnated towelette demonstrates antimicrobialeffectiveness as a disinfectant on hard surfaces. This practice provides survivor results in the form of a qualitative endpoint (growthposit
17、ive versus growth negative). The results generated by following this practice do not provide for specific quantitativereductions.6. Apparatus6.1 Incubatorany calibrated incubator that maintains a temperature specific for propagation of organisms. (for example,bacteria and mycobacteria at 35 6 2 C36
18、6 1 C and fungi at 25 6 2 C).27.5 6 2.5 C).6.2 Sterilizerany suitable suitable, calibrated steam sterilizer that produces the conditions of sterilization is acceptable.6.3 Test Toweletteswith instructions for use.6.4 Timer (Stop-clock)a calibrated timer that displays min and s.6.5 Spectrophotometerc
19、alibrated to 650 nm.6.6 Mixera vortex mixer is recommended.6.7 pH metera calibrated pH meter to determine the pH of media.6.8 Nonporous Test Carriersborosilicate glass slides, 25 mm 75 2 mm slides, pre-cleaned (or other hard surfaces and sizesas appropriate).6.9 Glass Culture Tubes20 mm 150 ormm, 25
20、 mm 150 mm without lip or equivalent.mm, and 38 mm 100 mm or 38mm 200 mm without lip, or equivalent, sterile.6.10 Culture Tube Closuresappropriate size nontoxic closures.6.11 Petri Dishes100 mm 15 mm, glass and plastic, sterile.6.12 Balancea calibrated balance sensitive to 0.1 g.6.13 Micropipettorca
21、librated for dispensing 10 L.6.14 Forcepssterilizable forceps.or pre-sterilized.6.15 Sterilizer Apparatusa bunsen burner or other appropriate heat sterilizer.6.16 Bacteriological Culture Loop 4 mm inside diameter loop of platinum or platinum alloy wire or sterile disposable plasticloops of appropria
22、te size.6.17 Colony Counterany one of several types may be used, for example Quebec.6.18 Glovessterile gloves not possessing antimicrobial properties.6.19 Pipettesterile volumetric pipettes.6.20 Glass Jars100 mL or other appropriate vessel.6.21 Filter Paper9 cm (Whatman No. 2, or equivalent) sterili
23、zed prior to use.6.22 Thermometercalibrated thermometer.6.23 Glass Beads3 5 mm sterile beads.6.24 Gauzesterile cotton gauze.6.25 Hemacytometercalibrated hemacytometer.6.26 Glass Woolsterile grease free glass wool.4 United States Environmental Protection Agency, Efficacy Data Requirements, “Pre-Satur
24、ated or Impregnated Towelettes for Hard Surface Disinfection” StandardOperating Procedure for Testing of Towelette Disinfectants againstDisinfectant Towelette Test Against Staphylococcus aureus and aureus, Pseudomonas aeruginosa, andSalmonella enterica, EPA/OPP Microbiology Laboratory, Ft. Meade, MD
25、. SOP# MB09-02,MB09-05, Revised 12/31/06.1/30/13.E2362 1526.27 Hot air ovenability to maintain 180C.180C.6.28 Refrigeratorcalibrated to maintain 5 6 3C.6.29 Ultra-Cold Freezer, Calibrated to maintain -70C6.30 Tissue grinderGlass Tissue Grinder or Macerator, sterile disposable or sterilizable glass.s
26、terile.6.31 Orbital ShakerSterile cryovials, calibrated shaker. (for example, 1.5 mL with screw cap)6.32 Centrifuge, calibrated.7. Reagents7.1 Culture MediaBacteria7.1.1 Nutrient Broth or Synthetic BrothPseudomonas aeruginosa,7.1.2 Cystine Trypticase AgarPseudomonas aeruginosa,7.1.3 Synthetic BrothS
27、almonella enterica and Staphylococcus aureus.7.1.4 Nutrient Agar.7.1.4 Fluid Thioglycollate Broth.7.1.5 Tryptic Soy Broth (TSB)7.1.6 Tryptic Soy Broth with 15% v/v glycerol (Cyroprotectant solution)7.2 Culture MediaMycobacteria7.2.1 Middlebrook 7H11 or 7H9 Agar Slants.7.2.2 Modified Proskauer-Beck B
28、roth.7.3 Culture MediaFungi7.3.1 Sabouraud Dextrose Agar plates/Potato Dextrose Agar.plates/Glucose Agar plates.7.3.2 Sabouraud Dextrose Agar slants/Glucose Agar slants.7.4 Neutralizing Subculture MediaA neutralizing growth medium capable of supporting the growth of the test organismfollowing exposu
29、re to the test material in accordance with E1054. For Mycobacterium, horse serum (which may be supplementedwith additional neutralizers) is recommended.7.5 Subculture Agar7.5.1 Tryptic Soy Agar with or without sheep bloodBacteria.7.5.2 Middlebrook 7H11 AgarMycobacteria.7.5.3 Sabouraud Dextrose Agar
30、or Glucose AgarFungi.7.6 Subculture MediaMycobacteria7.6.1 Modified Proskauer-Beck Broth57.6.2 Kirchners Medium57.6.3 Middlebrook 7H9 Broth or TB broth7.7 Other subculture agars, broths and neutralizers may be used where appropriate.7.8 SoilBlood Serum, such as heat inactivated fetal bovine serum or
31、 other appropriate alternative soil.7.9 Dilution Fluidsterile phosphate buffered water (PBDW), sterile saline or Butterfields Buffer. (See Specification D1193.)7.10 Sterile saline + 0.05% v/v Triton X-1007.11 Sterile 0.1% v/v Polysorbate (Tween) 807.12 Carrier Preparation Solutions70 to 95 % isoprop
32、yl alcohol, deionized or distilled water.8. Test Organisms8.1 BacteriaBacterial Test Organisms:8.1.1 Staphylococcus aureus (ATCC 6538), Salmonella enterica (ATCC 10708), and Pseudomonas aeruginosa (ATCC15442).15442)-received lyophilized.8.1.2 Other bacterial organisms may be tested using appropriate
33、 culture and subculture procedures.8.2 MycobacteriaMycobacterial Test Organisms:Organism:8.2.1 Mycobacterium bovisMycobacterium chelonae(BCG) (Organon (ATCC 35752). teknika or ATCC 35743)8.2.2 Mycobacterium bovis (ATCC 35743)8.2.2 Other mycobacteriamycobacterial strains may be tested using appropria
34、te culture and subculture procedures.8.3 FungiFungal Test Organisms:5 AOAC Official Method 965.12 Tubcerculocidal Activity of Disinfectants. AOAC International, Chapter 6.E2362 1538.3.1 Trichophyton mentagrophytes (ATCC 9533)8.3.2 Other fungi strains may be tested using appropriate culture and subcu
35、lture procedures.9. Preparation of Organism9.1 BacteriaBacteria6Preparation of frozen stock cultures for S. enterica, S. aureus, and P. aeruginosa.Maintain stockcultures ofUsing a tube containing S. aureus56 mL TSB, aseptically withdraw 0.5 to 1.0 mL and S. entericarehydrate the onNutrient Agar slan
36、ts. Maintain stock cultures lyophilized culture. Aseptically transfer the entire rehydrated pellet back into theoriginal tube of P. aeruginosabroth. Mix on Cystine Trypticase Agar. Incubate freshly subcultured stock cultures for 48 6 4 h at35 6 2 C, then refrigerate cultures at 2 to 8 C for up to on
37、e month. Stock cultures used for inoculation of broth cultures shouldnot undergo more than 5 passages from the first subculture from the ATCC frozen stock.well. Incubate for 24 6 2 h at 36 6 1C.Using a sterile spreader, inoculate a sufficient number of TSA plates (for example, 5 to 10 plates per org
38、anism) with 100 L eachof the culture. Incubate plates at 36 6 1C for 24 6 2 h. Following incubation, add 5 mL cryoprotectant solution (TSB with 15%v/v glycerol) to the surface of each agar plate. Resuspend the cells in this solution using a sterile spreader or a sterile swab andaspirate the cell sus
39、pension from the surface of the agar. Transfer suspension into a sterile vessel. Repeat by adding another 5 mLcryoprotectant to the agar plates, resuspend the cells, aspirate suspension and pool with the initial cell suspension. Alternately, 10mL cryoprotectant solution may be added per plate for re
40、suspending with subsequent aspiration. Mix the pooled contents of thevessel thoroughly. Immediately after mixing, pipet approximately 1.0 mL quantities of the diluted suspension into cryovials. Placeand store cryovials in 70C or below freezer; these are the frozen stock cultures. Each cryovial is co
41、nsidered as single use only.Store stock cultures up to 18 months. Reinitiate stocks using a new lyophilized culture.9.1.1 Bacteria Inoculum PreparationFromFor S. aureus and S. enterica,stock cultures, inoculate defrost a single cryovial atroom temperature and briefly vortex to mix.Add 10 L of the th
42、awed frozen stock to a tube containing 10 mL synthetic broth andthen vortex to mix. Incubate at 36 6 1C for 24 6 2 h. Briefly vortex the 24 h culture prior to transfer. For this final subculturestep, inoculate a sufficient number of 20 150 mm tubes containing 10 mL of the appropriate fresh culture b
43、roth and incubate for24 6 4 h at 35 6 2 C. Using a 4 mm inside diameter transfer loop, transfer one loopful of the culture into fresh culture broth.Make at least 3 but less than 30 consecutive daily transfers prior to use as inoculum for testing. Incubate the final transfer for48 6 4 h, and use thes
44、e synthetic broth with 10 L per tube of the 24 h synthetic broth culture; incubate 48 to 54 h at 36 6 1C.Using a Vortex-style mixer, mix synthetic broth test cultures 3 to 4 s and let stand 10 min at room temperature before continuing.Remove the upper portion of each culture, leaving behind any debr
45、is or clumps, and transfer to a sterile flask or tube; pool culturesin the test. Aseptically remove the pellicle from theflask and swirl to mix. Aliquot a P. aeruginosasufficient volume culture beforeuse in the test.of culture into a sterile test tube.9.1.1.1 For each bacterium, one daily transfer i
46、s required prior to the inoculation of a final test culture. Daily cultures may besubcultured for up to 5 d; each daily transfer may be used to generate a test culture. For the purpose of achieving the carrier countrange, final cultures may be adjusted by dilution in growth medium or by concentratio
47、n using centrifugation (for example, 5000g for 20 min) resuspending the pellet in the appropriate volume of sterile test culture medium.9.1.2 For P. aeruginosa, defrost a single cryovial at room temperature and briefly vortex to mix. Each cryovial should be singleuse only. Add 10 L of the thawed fro
48、zen stock to a tube containing 10 mL broth (synthetic or nutrient broth) and then vortex tomix. Incubate at 36 6 1C for 24 6 2 h. Do not vortex the 24 h culture prior to transfer. For this final subculture step, inoculatea sufficient number of 20 150 mm tubes containing 10 mL broth (synthetic or nut
49、rient) with 10 L per tube of the 24 h brothculture; incubate 48 to 54 h at 36 6 1C. Do not shake 48 to 54 h test culture. The pellicle from the 48 to 54 h cultures must beremoved from the broth either by decanting the liquid aseptically into a sterile tube, by gently aspirating the broth away from thepellicle using a pipet, or by removal with a vacuum. Avoid harvesting pellicle from the bottom of the tube.9.1.2.1 Any disruption of the pellicle resulting in dropping, or br
