1、Designation: E2967 15Standard Test Method forAssessing the Ability of Pre-wetted Towelettes to Removeand Transfer Bacterial Contamination on Hard, Non-PorousEnvironmental Surfaces Using the Wiperator1This standard is issued under the fixed designation E2967; the number immediately following the desi
2、gnation indicates the year oforiginal adoption or, in the case of revision, 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.INTRODUCTIONHigh-touch environmental surfa
3、ces (HITES) are increasingly being recognized as potentialvehicles for infectious agents (1-3).2Decontamination of such surfaces is almost always by either adisinfectant-spray-and-wipe procedure or, more commonly, by wiping with an applicator or toweletteprewetted with a disinfectant (4). In either
4、case, the microbicidal action of the disinfectant is combinedwith the physical action of wiping (mechanical removal). This standard, formulated after a criticalreview of available wipe test methods (5-8) and published standards, is based on the use of amechanical device (theWiperator; Appendix X1) d
5、esigned to wipe HITES under controlled conditionsand also to test the transfer of acquired microbial contamination to clean surfaces.1. Scope1.1 This standard is designed for use with a mechanizeddevice (the Wiperator; Appendix X1) to test pre-wetted tow-elettes.1.2 Two species of vegetative bacteri
6、a, one Gram-positivecoccus (Staphylococcus aureus) and one Gram-negative bacil-lus (Acinetobacter baumannii), representing important nosoco-mial pathogens, are used to separately contaminate disks ofmagnetized and brushed stainless steel in order to test thetowelettes for their relative ability to:1
7、.2.1 Decontaminate non-porous environmental surfacesexperimentally-contaminated with vegetative bacteria; and1.2.2 Transfer any acquired bacterial contamination on thetowelettes to clean surfaces.1.3 This test method is not meant for use with towelettes fordecontamination of skin.1.4 The values stat
8、ed in SI units are to be regarded as thestandard. The values given in parentheses, if any, are forinformation only.1.5 This test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at the appropriate biosafety level.1.
9、6 It is the responsibility of the investigator to determinewhether Good Laboratory Practice (GLP) regulations arerequired and to follow them where appropriate.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user
10、 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:3D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inac
11、tivators of Anti-microbial AgentsE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,and Sporicidal Activities of ChemicalsE2362 Practice for Evaluation of Pre-saturated or Impreg-nated Towelettes for Hard Surface DisinfectionE2756 Termi
12、nology Relating to Antimicrobial and AntiviralAgentsE2896 Test Method for Quantitative Petri Plate Method(QPM) for Determining the Effectiveness of Antimicro-bial Towelettes1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents
13、and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved May 1, 2015. Published February 2016. DOI: 10.1520/E2967-15.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit th
14、e ASTM website, www.astm.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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. U
15、nited States12.2 AOAC Standard:4AOAC Standards Method 961.02 Germicidal Spray Prod-ucts as Disinfectants. Official Methods of Analysis2.3 CFR Standards:540 CFR 160 Good Laboratory Practice Standards21 CFR 58 Good Laboratory Practice Regulations2.4 Other Reference:Organization for Economic Cooperatio
16、n and Development(OECD) Guideline No. 187; 2013 Guidance Document onQuantitative Methods for Evaluating the Activity of Mi-crobicides used on Hard, Non-Porous Surfaces63. Terminology3.1 For definitions of standard terms, please refer to Termi-nology E2756.3.2 Definitions of Terms Specific to This St
17、andard:3.2.1 boss, na Teflon cylinder designed to hold the testtowelette in place with the help of an O ring.3.2.2 eluate, nrecovered eluent that may contain the testorganism(s).3.2.3 eluent, nany liquid that is harmless to the testorganism and that is added to a carrier to recover the organ-ism(s)
18、in or on it.3.2.4 microbicidal action, nthe action of an agent leadingto killing of or irreversible damage to microorganisms.3.2.5 nosocomial infection, nan infection acquired duringstay in a healthcare setting.3.2.6 soil load, na mixture of one or more organic andinorganic substances added to the s
19、uspension of the testorganism to simulate the presence of bodily secretions,excretions, or other interfering substances. It presents the testsubstance with a challenge to overcome the chemical demandfrom the soil load and physical shielding of microbes that itmay provide (9).3.2.7 stock culture, nfr
20、ozen culture used to prepare testcultures.3.2.8 towelette, na pre-wetted piece of paper or cloth ofnatural or synthetic fibers for use in environmental or personalhygiene.4. Summary of Test Method4.1 The Wiperator is designed to simulate the orbital actionof wiping using prewetted towelettes. The pr
21、essure duringcontact, duration of wiping as well as the number of wipingstrokes in a given test can be preset, allowing for greaterprecision and reproducibility. The device can also test thetransfer of microbial contamination on a used towelette to aclean surface being wiped. Disks of brushed stainl
22、ess steel areused in this standard method as prototypical non-porousenvironmental surfaces. Each disk receives at its center 10 Lof the test organism, and the inoculum is dried. The disk is thenplaced in one of the two recessed areas on the Wiperatorscarrier platform.A4 cm2piece of control or test t
23、owelette is cutand mounted on a boss for placement in the Wiperatorsspindle (Appendix X1). The platform with the inoculated diskis raised to contact the towelette and activate wiping.To test fortransfer, a clean carrier in the second recessed area of theplatform is brought under the towelette and wi
24、ped. The carriersare separately transferred to a vial with an eluent containing avalidated neutralizer. The eluates are then assayed and log10CFU removed or transferred are calculated.5. Significance and Use5.1 Microbial decontamination of environmental surfacesby wiping is subject to many variables
25、 (4), and failure tostandardize them properly during testing of towelettes maygive inconsistent test data. (See Practice E2362 and TestMethod E2896.) In particular, precise control of the pressureapplied during wiping, the normally brief wiping times of afew seconds as well as the style and number o
26、f wiping strokesare difficult without a programmable mechanical device. TheWiperator has been designed and tested with these crucialfactors in mind. The method described here is to assess the roleof wiping in ridding non-porous environmental surfaces ofbacterial contamination using prewetted towelet
27、tes, and also todetermine if the used towelette can transfer viable contamina-tion to clean surfaces on contact.6. Equipment and Apparatus6.1 Bunsen Burner or an Incinerator, for sterilization ofinoculating loops.6.2 Centrifuge, for sedimentation of the cells of the testorganism(s).6.3 Colony Counte
28、r, for example, Quebec Colony Counter.6.4 Disks, 1 cm diameter and 0.7 mm thick made fromsheets of brushed and magnetized stainless steel (E2197;OECD 2013); further specifications on the disks and examplesfor their commercial sources are given in Appendix X2.6.5 Dissecting Microscope, 20 magnificati
29、on.6.6 Filter Sterilization System, a membrane filtration system(0.22-m pore diameter) for sterilization of heat-sensitivemedia and solutions.6.7 Incubator, to maintain an incubation temperature of3661C.6.8 Inoculating Loop, for transfer and spread-plating ofbacterial suspensions.6.9 Hot Air Oven, f
30、or drying of labware.6.10 Laminar Flow Biosafety Cabinet (Class II), for han-dling of microbial cultures at biosafety level 2 (BSL-2);procedures for the proper maintenance and use of such cabinetsare given in Ref (10, 11).6.11 Positive Displacement Pipette, a pipette and pipettetips that can accurat
31、ely dispense 10 L volumes.4Available from AOAC International, 481 North Frederick Ave., Suite 500,Gaithersburg, Maryland 20877-2417, http:/www.aoac.org.5Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.
32、access.gpo.gov.6Available from OECD Environment Directorate, Environment, Health access to an in-housevacuum line or a water faucet vacuum apparatus is required topull the samples through the membrane filters.7. Materials and Supplies87.1 Bovine Serum Albumin (BSA).7.2 Cryovials, 2-mL capacity.7.3 D
33、eionized Distilled Water (DDW), or water of equiva-lent quality, for making reagent solutions and culture media;can be produced in-house or obtained from a reputablecommercial source (D1129; D1193).7.4 Glass Beads (solid borosilicate), 3 mm diameter.7.5 Glycerol.7.6 J-Cloth Reusable Towels (unmedica
34、ted),9or equivalentmaterial for use as a control towelette.7.7 Membrane Filters (polyethylsulfone), 33 mm diameter(0.22 m pore diameter) for use with a syringe.7.8 Personal Protective Equipment, including gloves, lab-coat and safety glasses.7.9 Pipettors with Sterile Tips, including one 10-Lpositive
35、-displacement pipettor.7.10 Tryptic-soy Agar (TSA), in 100 mm diameter plasticPetri plates.7.11 Polypropylene Test Tubes, 15 mL capacity.7.12 Sodium Chloride (NaCl).7.13 Tryptic-soy Agar (TSA).7.14 Tryptic-soy Broth (TSB).7.15 Tryptone.7.16 Wide-mouth Nalgene Vials with Screw Caps, 30 mLcapacity.107
36、.17 Test OrganismsStaphylococcus aureus (ATCC 6538)and Acinetobacter baumannii (ATCC 19568).NOTE 1Ensure that S. aureus for subculture and any subsequent useproduces golden-colored colonies on TSA.8. Methods8.1 Preparation of Media and ReagentsAll autoclaving tobe in accordance with manufacturers in
37、structions or as speci-fied in the standard operating procedures of the laboratory:8.1.1 Recovery MediumTSA in 100 mm Petri platesprepared in-house or obtained from a reputable commercialsource; store the plates at 462C and use within three monthsfrom the date of preparation or within the shelf-life
38、 indicatedby the manufacturer.8.1.2 Culture BrothPrepare 400 mL of TSB according tothe manufacturers instructions and autoclave-sterilize; aliquot10 mL into each 15-mL test tube and store at 462C and usewithin three months from the date of preparation or within theshelf-life indicated by the manufac
39、turer.8.1.3 DiluentTryptone-sodium chloride (TSC) broth. Pre-pare by adding 1 g Tryptone and 8.5 g of NaCl to 1 L of DDWand autoclave sterilize.8.1.4 Eluent with NeutralizerPrepare in 1 L of TSC brothby adding:8.1.4.1 Saponin 30 g/L.8.1.4.2 L-histidine 1 g/L.8.1.4.3 Polysorbate-80 30 g/L.8.1.4.4 Aso
40、lectin from soybean 3 g/L.8.1.4.5 Sodium thiosulfate (Fisher) 5 g/L8.1.5 Store at 462C and use within three months from thedate of preparation.NOTE 2A different neutralizer may be used depending on the type(s)and level(s) of the active ingredient(s) in the towelette under test (E1054).However, the u
41、se of any such neutralizer must first be properly validated.8.1.6 Soil Load:8.1.6.1 Add 3.0 mg of BSA to 100 mL of TSC.8.1.6.2 Pass the solution through a 0.22-m pore diam.membrane filter mounted on a syringe, aliquot, and store at462C and use within three months from the date of prepara-tion; it ca
42、n be stored for up to one year at 2062C.NOTE 3A different soil load such as the one given in E2197 or theOECD guideline may be used instead.8.2 Preparation of Stock or Frozen Bacterial Stocks:8.2.1 Spread with an inoculating loop the bacterial cultureover a plate of TSA and incubate for 1862hat3661C
43、.7The sole source of supply of the Wiperator known to Subcommittee E35.15 atthis time is Filtaflex Ltd. (www.filtaflex.ca), Almonte, Ontario, Canada. If you areaware of alternative suppliers, please provide this information to ASTM Interna-tional Headquarters. Your comments will receive careful cons
44、ideration at a meetingof the responsible technical committee,1which you may attend. Alternatively, theuser may build the apparatus with assistance from Filtaflex.8If a specific source is not indicated, that item may be purchased from anyreputable scientific supplier.9The J-Cloth brand Reusable Towel
45、s, which can be autoclave sterilized, arewidely available in hardware stores in North America and elsewhere. They arecomposed of cellulosic fibers from wood pulp and biodegradable. Manufacturer:Associated Brands; Universal Product Code: 0 58354 51071 3. Address:Mississauga, ON, Canada; http:/ sole s
46、ource of supply of Nalgene vials (Catalog #2118-0001) known to thecommittee at this time is Nalge Nunc International, 75 Panorama Creek Dr.,Rochester, N.Y. 14625-2385. If you are aware of alternative suppliers, pleaseprovide this information to ASTM International Headquarters. Your comments willrece
47、ive careful consideration at a meeting of the responsible technical committee,1which you may attend.E2967 1538.2.2 Using an inoculating loop, pick up one discrete colonyfrom the plate and place it in 10 mLof TSB. Incubate the brothfor 1862hat3661C.8.2.3 Add 100 L of the broth culture to 100 mL of TS
48、Bcontaining glycerol at a final conc. of 10%. Incubate thesuspension at 3661C for 460.5 h.8.2.4 Aliquot 1.0 mL volumes into cryovials for storage at8062C.8.2.5 Use the frozen stocks within two years from the dateof their preparation.8.3 Preparation of Working Bacterial Cultures:8.3.1 Place 100 Lof a
49、 thawed suspension of S. aureus or A.baumannii inoculum into 10 mL of TSB. Incubate at 3661Cfor 1862 h for each days experimentation.8.3.2 Centrifuge the 10-mL suspension at 3,000xg for 20min and resuspend the pellet in 5 mL TSC.8.4 Stainless Steel Disk Carrier Preparation and Steriliza-tion:8.4.1 Soak disks in a non-ionic detergent solution11for atleast one hour to degrease prior to rinsing and sterilization;avoid extended soaking to minimize risk of corrosion.8.4.2 Rinse the disks in
