ASTM E2362-2004 Standard Practice for Evaluation of Pre-saturated or Impregnated Towelettes for Hard Surface Disinfection《硬表面消毒用预饱和或浸渍小毛巾的评估的标准实施规程》.pdf

1、Designation: E 2362 04Standard Practice forEvaluation of Pre-saturated or Impregnated Towelettes forHard Surface Disinfection1This standard is issued under the fixed designation E 2362; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio

2、n, the year 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 practice is designed to evaluate the antimicrobialactivity of pre-saturated or impregnated towele

3、ttes 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 This standar

4、d 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 health practices and to determine theapplicability of regulatory limitations prior to use.2. Referenced Documents2.1 AST

5、M Standards:2D 1193 Specification for Reagent WaterE 1054 Standard Test Methods for Evaluation of Inactiva-tors of Antimicrobial Agents Used in Disinfectant, Sani-tizer, Antiseptic or Preserved Products2.2 Federal Standard40 CFR, Part 160, Good Laboratory Practice Standards33. Terminology3.1 carrier

6、, 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 destroys pathogenic or potentially pathogen

7、ic 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 impregnated with testsubstance prior to the

8、time of its intended use.3.7 towelette, nA paper, cloth or non woven blendmaterial 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 carrier which has been inoculatedwith a test or

9、ganism 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-determined contact time. After the specifiedcon

10、tact 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 method may be used to determine if a pre-sat

11、uratedor 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 mycobacteria at 35 6 2 C and fungi at25 6 2 C).

12、6.2 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.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticidesand Alternat

13、ive Control Agents and is the direct responsibility of SubcommiteeE35.15 on Antimicrobial and Antiviral Agents. Current edition approved Oct. 1,2004. Published October 2004.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For A

14、nnual Book of ASTMStandards volume information, refer to the standards Document 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-Satura

15、ted or Impregnated Towelettes for Hard Surface Disinfection” StandardOperating Procedure for Testing of Towelette Disinfectants against Salmonellacholeraesuis , Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacte-rium bovis (BCG), EPA/OPP Microbiology Laboratory, Ft. Meade, MD. SOP#MB09-01,

16、 Revised 11/08/00.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.5 Spectrophotometercalibrated to 650 nm.6.6 Mixera vortex mixer is recommended.6.7 pH metera calibrated pH meter to determine the pH ofmedia.6.8 Nonporous Test Carri

17、ersborosilicate 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 size nontoxic clo-sures.6.11 Petri Dishes100 3 15 mm, glass and plastic, ster

18、ile.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 Bacteriological Culture Loop 4 mm inside diameterloop of platinum or platinum

19、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 Pipettesterile volumetric pipettes.6.20 Glass Jars100 mL or other appropriate vessel.6.21

20、 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.6.26 Glass Woolsterile grease free glass wool.6.27 Hot air ovenability to main

21、tain 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 AgarPseudomonas aeruginosa,7.1.3 Synthetic BrothSalmonella choleraesuis and Sta-phylococc

22、us 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 plates.7.3.2 Sabouraud Dextrose Agar slants.7.4 Neutralizing Subculture MediaA

23、 neutralizing growthmedium capable of supporting the growth of the test organismfollowing exposure to the test material in accordance withE 1054.7.5 Subculture Agar7.5.1 Tryptic Soy AgarBacteria.7.5.2 Middlebrook 7H11 AgarMycobacteria.7.5.3 Sabouraud Dextrose AgarFungi.7.6 Other subculture agars, br

24、oths 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 phosphate buffered water(PBDW), sterile saline or Butterfields Buffer.7.9 Carrier Preparation Solutions70-95 % isopropy

25、l al-cohol, deionized or distilled water.8. Test Organisms8.1 Bacteria Test Organisms:8.1.1 Staphylococcus aureus (ATCC 6538), Salmonellacholeraesuis (ATCC 10708), and Pseudomonas aeruginosa(ATCC 15442).8.1.2 Other bacterial organisms may be tested using appro-priate culture and subculture procedure

26、s.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 tested using appro-priate culture and subculture procedures.8.3 Fungi Test Organisms:8.3.1 Trichophyton mentagrophytes (ATCC 9533)8.3.2 Other fung

27、i strains may be tested using appropriateculture and subculture procedures.9. Preparation of Organism9.1 BacteriaMaintain stock cultures of S. aureus and S.choleraesuis on Nutrient Agar slants. Maintain stock culturesof P. aeruginosa on Cystine Trypticase Agar slants. Incubatefreshly subcultured sto

28、ck cultures for 48 6 4hat356 2 C,then refrigerate cultures at 2-8 C for up to 30 days. Stockcultures used for inoculation of broth cultures should notundergo more than 5 passages from the first subculture fromthe ATCC stock.9.1.1 Bacteria Inoculum PreparationFrom stock cultures,inoculate tubes conta

29、ining 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 of theculture into fresh culture broth. Make at least 3 but less than 15consecutive daily transfers prior to use as inoculum for testing.Incubate the f

30、inal 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.9.2 MycobacteriaMaintain a stock culture of Mycobacte-rium organisms on Middlebrook 7H11 or 7H9 agar slants bymonthly transfer and incubation for 15-3

31、0 days at 35 6 2Cfollowed by storage at 2-8 C.9.2.1 Mycobacteria Inoculum PreparationFrom stockculture, inoculate Modified Proskauer-Beck (MPB) Brothtubes and incubate 10-25 days at 35 6 2 C. Add 1.0 mL of0.1 % Tween 80 in saline to this 10-25-day culture, transfer toa sterile tissue grinder and gri

32、nd thoroughly. Finally transferthe appropriate volume of suspension from the grinder to avessel appropriate for spectrophotometric determination. Cali-brate a spectrophotometer to 650 nm using a MPB blank as the100 % transmittance point. Measure transmittance of the cul-ture suspension. Adjust cultu

33、re suspension with MPB to aE2362042target of 15-20 % transmittance to achieve a $ 1.0 3 104CFU/carrier concentration.9.3 FungiMaintain a stock culture of Trichophyton men-tagrophytes on Sabouraud Dextrose agar slants by transferringat less than or equal to 3 month intervals and incubate 10 daysat 25

34、62C, followed by storage at 2-8C.9.3.1 Conidial Suspension PreparationFrom stock cul-ture, inoculate Sabouraud Dextrose Agar plates and incubate at25 6 2 C for 10-15 days. Transfer growth, using a sterileswab, to a glass screw-top vessel containing 5 sterile glassbeads and 5 mL of 0.85-0.90 % steril

35、e saline per culture plate.Vortex mix the suspension for one min. Filter the conidialsuspension through sterile cotton gauze or glass wool toremove the hyphal fragments. Estimate the conidial concentra-tion by counting in a hemacytometer. Stock conidial suspen-sion can be stored at 2-8 C for one mon

36、th. On the day oftesting, standardize the conidial suspension using 0.85-0.9 %sterile saline to $ 1.0 3 106conidia/mL. A 0.2 mL aliquot ofTriton X-100 may be added to a 10 mL aliqout of conidialsuspension to facilitate spreading.9.4 Inocula used for Testing Pre-Cleaned SurfacesImmediately prior to u

37、se, 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, thoroughlyvortex mix the

38、prepared fungi suspension. Thoroughly vortexmix the prepared bacteria and mycobacteria 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 example, 19

39、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 with the test,

40、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 Petri dish andsterilize in a hot air oven for2hat180C.10.1.3

41、After sterilization, place each carrier into separateglass or plastic Petri dishes containing 2 pieces of sterile filterpaper. Transfer the required number of carriers for testingincluding a minimum of 2 carriers for the population control, 1carrier for each viability control, and 1 carrier for the

42、carriersterility 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 inside the above mentioned Petri dish.10.2.2 Spread the inoculum suspension evenly over thedesignated t

43、est 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 dry, but nolonger than 40 min.10.4

44、 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 performed withmore than one technicia

45、n. 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 potentialenvironmental contamination of th

46、e 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).10.4.3 The wiping procedure should be

47、 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.10.4.4 Following the treatment of the last carrie

48、r in the set,the towelette is aseptically placed in a separate sterile Petri dishand held for the prescribed exposure time starting whentreatment of the last carrier was begun.10.5 Carrier Subculture:10.5.1 Following the prescribed exposure time and usingseparate sterile forceps, transfer each carri

49、er into a vesselcontaining the appropriate amount of subculture/neutralizingmedia to completely cover the inoculated and treated area ofthe carrier and mix thoroughly.10.5.2 Following the same staggered interval used in 10.4.4,transfer the remaining carriers as described in 10.5.1.10.5.3 If necessary $30 min from original subculture,transfer carrier to a second vessel containing the appropriatesubculture/neutralizer media and mix thoroughly for furthertest substance neutralization by dilution (see Test Meth