1、Designation: E2362 09Standard 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 revision,
2、 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 antimicrobialactivity of pre-saturated or impregnated towelette
3、s when usedas a hard surface disinfectant.1.2 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLPs) are required andto follow them when appropriate.1.3 This practice should be performed only by those trainedin microbiological techniques.1.4 The values stat
4、ed 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 and
5、 health practices and to determine theapplicability of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inactivators of An-timicrobial Agents2.2 Federal Standard40 CFR, Part 160 Good Laboratory Pr
6、actice Standards33. Terminology3.1 carrier, na transportable surface onto which a testorganism will be inoculated and dried. The carrier will betreated with the test substance and subcultured for survivors.3.2 CFU, ncolony forming units3.3 disinfectant, na physical or chemical agent or processthat d
7、estroys pathogenic or potentially pathogenic microorgan-isms in/on surfaces or objects.3.4 impregnated, adjsaturated with test substance.3.5 neutralizer, na component used to render an activeagent incapable of destroying organisms by chemical orphysical means.3.6 pre-saturated, adjto be filled or im
8、pregnated with testsubstance prior to the time of its intended use.3.7 towelette, nA paper, cloth or non-woven blend mate-rial used as a transporter for a cleaning and/or disinfectionagent.4. Summary of Practice44.1 A towelette impregnated or pre-saturated with a testsubstance is used to treat a car
9、rier which has been inoculatedwith a test organism after an aliquot of a test organism has beeninoculated, evenly distributed, and dried onto the carrier. Thecarrier is wiped using the pre-saturated or impregnated tow-elette simulating the application of the test substance and thenheld for a pre-det
10、ermined contact time. After the specifiedcontact time, the test substance remaining on the carrier isneutralized and the carrier is subcultured to recover survivingtest organism. The used towelette, after the contact time, is alsocultured for surviving test organism.5. Significance and UseThis test
11、method may be used to determine if a pre-saturatedor impregnated towelette demonstrates antimicrobial effective-ness as a disinfectant on hard surfaces.6. Apparatus6.1 Incubatorany calibrated incubator that maintains atemperature specific for propagation of organisms. (for ex-ample, bacteria and myc
12、obacteria at 35 6 2 C and fungi at25 6 2 C).1This practice is under the jurisdiction of ASTM Committee E35 on Pesticidesand Alternative Control Agents and is the direct responsibility of SubcommiteeE35.15 on Antimicrobial Agents.Current edition approved Dec. 1, 2009 Published December 2009. Original
13、lyapproved in 2004. Last previous edition approved in 2004 as E2362 04. DOI:10.1520/E2362-09.2For 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
14、Summary page onthe ASTM website.3Available from the Superintendent of Documents, U.S. Government PrintingOffice, Washington D.C. 204024United States Environmental Protection Agency, Efficacy Data Requirements,“Pre-Saturated or Impregnated Towelettes for Hard Surface Disinfection” StandardOperating P
15、rocedure for Testing of Towelette Disinfectants against Staphylococcusaureus and Pseudomonas aeruginosa, EPA/OPP Microbiology Laboratory, Ft.Meade, MD. SOP# MB09-02, Revised 12/31/06.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.
16、2 Sterilizerany suitable steam sterilizer that producesthe conditions of sterilization is acceptable.6.3 Test Toweletteswith instructions for use.6.4 Timer (Stop-clock)a calibrated timer that displays minand s.6.5 Spectrophotometercalibrated to 650 nm.6.6 Mixera vortex mixer is recommended.6.7 pH me
17、tera calibrated pH meter to determine the pH ofmedia.6.8 Nonporous Test Carriersborosilicate glass slides, 253 75 3 2 mm slides, pre-cleaned (or other hard surfaces andsizes as appropriate).6.9 Glass Culture Tubes20 3 150 or 25 3 150 mmwithout lip or equivalent.6.10 Culture Tube Closuresappropriate
18、size nontoxic clo-sures.6.11 Petri Dishes100 3 15 mm, glass and plastic, sterile.6.12 Balancea calibrated balance sensitive to 0.1 g.6.13 Micropipettorcalibrated for dispensing 10 L.6.14 Forcepssterilizable forceps.6.15 Sterilizer Apparatusa bunsen burner or other appro-priate heat sterilizer.6.16 B
19、acteriological Culture Loop 4 mm inside diameterloop of platinum or platinum alloy wire or sterile disposableplastic loops of appropriate size.6.17 Colony Counterany one of several types may beused, for example Quebec.6.18 Glovessterile gloves not possessing antimicrobialproperties.6.19 Pipettesteri
20、le volumetric pipettes.6.20 Glass Jars100 mL or other appropriate vessel.6.21 Filter Paper9 cm (Whatman No. 2, or equivalent)sterilized 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
21、.26 Glass Woolsterile grease free glass wool.6.27 Hot air ovenability to maintain 180C.6.28 Tissue grindersterile disposable or sterilizable glass.6.29 Orbital Shakercalibrated shaker.7. Reagents7.1 Culture MediaBacteria7.1.1 Nutrient BrothPseudomonas aeruginosa,7.1.2 Cystine Trypticase AgarPseudomo
22、nas aeruginosa,7.1.3 Synthetic BrothSalmonella enterica and Staphylo-coccus aureus.7.1.4 Nutrient Agar.7.1.5 Fluid Thioglycollate Broth.7.2 Culture MediaMycobacteria7.2.1 Middlebrook 7H11 or 7H9 Agar Slants.7.2.2 Modified Proskauer-Beck Broth.7.3 Culture MediaFungi7.3.1 Sabouraud Dextrose Agar plate
23、s/Potato DextroseAgar.7.3.2 Sabouraud Dextrose Agar slants.7.4 Neutralizing Subculture MediaA neutralizing growthmedium capable of supporting the growth of the test organismfollowing exposure to the test material in accordance withE1054.7.5 Subculture Agar7.5.1 Tryptic Soy Agar with or without sheep
24、 bloodBacteria.7.5.2 Middlebrook 7H11 AgarMycobacteria.7.5.3 Sabouraud Dextrose AgarFungi.7.6 Other subculture agars, broths and neutralizers may beused where appropriate.7.7 SoilBlood Serum, such as heat inactivated fetal bo-vine serum or other appropriate alternative soil.7.8 Dilution Fluidsterile
25、 phosphate buffered water(PBDW), sterile saline or Butterfields Buffer. (See Specifica-tion D1193.)7.9 Carrier Preparation Solutions70 to 95 % isopropylalcohol, deionized or distilled water.8. Test Organisms8.1 Bacteria Test Organisms:8.1.1 Staphylococcus aureus (ATCC 6538), Salmonella en-terica (AT
26、CC 10708), and Pseudomonas aeruginosa (ATCC15442).8.1.2 Other bacterial organisms may be tested using appro-priate culture and subculture procedures.8.2 Mycobacteria Test Organisms:8.2.1 Mycobacterium chelonae (ATCC 35752).8.2.2 Mycobacterium bovis (ATCC 35743)8.2.3 Other mycobacteria strains may be
27、 tested using appro-priate culture and subculture procedures.8.3 Fungi Test Organisms:8.3.1 Trichophyton mentagrophytes (ATCC 9533)8.3.2 Other fungi strains may be tested using appropriateculture and subculture procedures.9. Preparation of Organism9.1 BacteriaMaintain stock cultures of S. aureus and
28、 S.enterica on Nutrient Agar slants. Maintain stock cultures of P.aeruginosa on Cystine Trypticase Agar. Incubate freshly sub-cultured stock cultures for 48 6 4hat356 2 C, thenrefrigerate cultures at 2 to 8 C for up to one month. Stockcultures used for inoculation of broth cultures should notundergo
29、 more than 5 passages from the first subculture fromthe ATCC frozen stock.9.1.1 Bacteria Inoculum PreparationFrom stock cultures,inoculate tubes containing 10 mL of the appropriate freshculture broth and incubate for 24 6 4hat356 2 C. Using a4 mm inside diameter transfer loop, transfer one loopful o
30、f theculture into fresh culture broth. Make at least 3 but less than 30consecutive daily transfers prior to use as inoculum for testing.Incubate the final transfer for 48 6 4 h, and use these culturesin the test. Aseptically remove the pellicle from the P. aerugi-nosa culture before use in the test.
31、9.2 MycobacteriaMaintain a stock culture of Mycobacte-rium organisms on Middlebrook 7H11 or 7H9 agar slants bymonthly transfer and incubation for 15 to 30 days at 35 6 2Cfollowed by storage at 2 to 8C.E2362 0929.2.1 Mycobacteria Inoculum PreparationFrom stockculture, inoculate Modified Proskauer-Bec
32、k (MPB) Brothtubes and incubate 10 to 25 days at 35 6 2 C. Add 1.0 mL of0.1 % Tween 80 in saline to this 10 to 25-day culture, transferto a sterile tissue grinder and grind thoroughly. Finally transferthe appropriate volume of suspension from the grinder to avessel appropriate for spectrophotometric
33、 determination. Cali-brate a spectrophotometer to 650 nm using a MPB blank as the100 % transmittance point. Measure transmittance of the cul-ture suspension. Adjust culture suspension with MPB to atarget of 8 to 12 % transmittance to achieve a $ 1.0 3 104CFU/carrier concentration.9.3 FungiMaintain a
34、 stock culture of Trichophyton men-tagrophytes on Sabouraud Dextrose or Potato Dextrose agarslants by transferring at less than or equal to 3 month intervalsand incubate 10 days at 2 56 2C, followed by storage at 2 to8C. Alternatively, a water stock may be prepared and held atambient temperature.9.3
35、.1 Conidial Suspension PreparationFrom stock cul-ture, inoculate Sabouraud Dextrose or Potato Dextrose agarplates and incubate at 25 6 2 C for 10 to 15 days. Transfergrowth, using a sterile swab, to a glass screw-top vesselcontaining 5 sterile glass beads and 5 mLof 0.85-0.90 % sterilesaline or othe
36、r appropriate diluent per culture plate. Vortex mixthe suspension for one min. Filter the conidial suspensionthrough sterile cotton gauze or glass wool to remove the hyphalfragments. Estimate the conidial concentration by counting ina hemacytometer. Stock conidial suspension can be stored at2-8 C fo
37、r one month. On the day of testing, standardize theconidial suspension using 0.85 to 0.9 % sterile saline to $ 1.03 106conidia/mL. A 0.2 mL aliquot of Triton X-100 may beadded to a 10 mL aliqout of conidial suspension to facilitatespreading.9.4 Inocula used for Testing Pre-Cleaned SurfacesImmediatel
38、y prior to use, thoroughly vortex mix the pre-pared fungi suspension. Thoroughly vortex mix the preparedbacteria and mycobacteria suspension and allow to settle for$10 min prior to use.9.5 Inocula used for Testing Formulations as Disinfectantson Soiled SurfacesImmediately prior to use, thoroughlyvor
39、tex mix the prepared fungi and mycobacterial suspension.Thoroughly vortex mix the prepared bacteria suspension andallow to settle for $10 min prior to use. Transfer an aliquot ofthe suspension into a sterile tube and add an appropriatevolume of blood serum (soil) to yield a 5 % organic soil load(for
40、 example, 19 mL of the test organism suspension plus 1 mLfetal bovine serum). Perform a sterility control of the bloodserum by adding 1.0 mL of serum to 9.0 mL of appropriaterecovery broth and incubate with the test.9.6 Organism PuritySubculture each test organism to theappropriate agar, incubate wi
41、th the test, and examine for purity.10. Procedure10.1 Preparation of Carriers:10.1.1 Test carriers should be submerged in 70 to 95 % ethylor isopropyl alcohol, rinsed with deionized or distilled water.10.1.2 Place the test carriers into a large glass dish andsterilize in a hot air oven for $2hat$180
42、C.10.1.3 After sterilization, place each carrier horizontallyinto separate glass or plastic Petri dishes containing 2 pieces ofsterile filter paper. Transfer the required number of carriers fortesting including a minimum of 2 carriers for the populationcontrol, 1 carrier for each viability control,
43、and 1 carrier for thecarrier sterility control.10.2 Inoculation of Carriers:10.2.1 Using a pipette or 4.0 mm inside diameter (id) loop,transfer 0.01 mL of the test organism to the nonporous carrierwhich has been placed horizontally inside the above mentionedPetri dish.10.2.2 Spread the inoculum susp
44、ension evenly over thedesignated test area and within 3 mm of the edge using thesterile pipette tip or 4.0 mm id loop used for inoculation andrecover with the Petri dish lid.10.3 Carrier Drying:10.3.1 Place all Petri dishes containing inoculated carriersinto an incubator equilibrated at 3562C until
45、dry, but nolonger than 40 min.10.4 Carrier Treatment/Application of Product:10.4.1 Wipe the inoculated test area according to labelinstructions or the procedure under test. The wiping procedureshould closely simulate the direction for intended use.NOTE 1The carrier treatment phase is most easily per
46、formed withmore than one technician. The technician performing the wiping (treat-ment) procedure must wear sterile gloves prior to handling the toweletteunder test and must not touch anything except the towelette under test andinoculated carriers. Aseptic technique is critical in minimizing potentia
47、lenvironmental contamination of the carrier subcultures.10.4.2 The area of the towelette used for wiping should berotated so as to expose a new surface of the towelette surfacein the course of each carrier treatment. One towelette may beused to treat multiple carriers (typically 10 carrier sets/wipe
48、).10.4.3 The wiping procedure should be performed at stag-gered intervals so as to allow for the prescribed exposure timebefore subculture.NOTE 2The technician should allow enough time between eachcarrier wiping (for example, 30 s) to allow for the exact exposure timeprior to subculturing. The expos
49、ure time starts immediately following thecompletion of treatment.10.4.4 OptionalFollowing the treatment of the last carrierin the set, the towelette or liquid expressed from the toweletteis aseptically placed in a separate sterile Petri dish and held forthe prescribed exposure time starting when treatment of the lastcarrier ended.10.5 Carrier Subculture:10.5.1 Following the prescribed exposure time and usingseparate sterile forceps, transfer each carrier into a vesselcontaining the appropriate amount of
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