1、Designation: E2896 12Standard Test Method forQuantitative Petri Plate Method (QPM) for Determining theEffectiveness of Antimicrobial Towelettes1This standard is issued under the fixed designation E2896; the number immediately following the designation indicates the year oforiginal adoption or, in th
2、e 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.INTRODUCTIONThis test method provides a standardized approach to quantitatively determine the effe
3、ctiveness ofantimicrobial towelettes (wipes) in treating hard non-porous surfaces contaminated with Staphylo-coccus aureus, Pseudomonas aeruginosa, and Salmonella enterica. This test method addresses theneed for a user-friendly, relevant, and reproducible procedure.21. Scope1.1 This test method prov
4、ides detailed instructions forperforming a quantitative evaluation of antimicrobial efficacyof a towelette when challenged against Staphylococcus aureus,Pseudomonas aeruginosa and Salmonella enterica. Themethod may be used with other microbial strains, thoughmodification may be necessary to accommod
5、ate recovery.1.1.1 Antimicrobial towelettes, designed to decontaminatehard, non-porous surfaces, are diverse in size, matrixcomposition, and packaging.1.1.2 Antimicrobial towelettes also vary in label claims anduse directions.1.2 This quantitative method does not differentiate betweenmechanical remo
6、val of inoculum from a surface and chemicalinactivation of the test microbe; rather, product efficacy isconsidered a combination of both attributes of a towelette-based formulation.1.3 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLPStandards40 CFR,Part
7、 160 of FIFRA) are required and to follow them whenappropriate.1.4 This standard may involve the use of hazardousmaterials, chemicals and infectious microorganisms and shouldbe performed only by persons with formal training in micro-biology.1.5 Strict adherence to the protocol is necessary for theva
8、lidity of the test results.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard does not address specific product perfor-mance standards established by regulatory authorities; seeSection 10, Note 2 for details.1
9、.8 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 health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Do
10、cuments2.1 Other Documents:AOAC Official Method 961.02, Germicidal Spray Productsas Disinfectants. Revised 20123AOAC Official Method 955.15, Use-Dilution Method forTesting Disinfectants against Staphylococcus aureus. Re-vised 20123AOAC Official Method 964.02, Use-Dilution Method forTesting Disinfect
11、ants against Pseudomonas aeruginosa.Revised 20123AOAC Official Methods 955.14, Use-Dilution Method forTesting Disinfectants against Salmonella enterica. Re-vised 2012340 CFR, Part 160 Federal Insecticide, Fungicide and Roden-ticideAct (FIFRA); Good Laboratory Practice Standards41This test method is
12、under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 15, 2012. Published December 2012. DOI:10.1520/E2896-12.2Samalot-Freire, L., Tomasino
13、, S. F., and Hasan, J. A., The Quantitative PetriPlate Method (QPM): A New Method for Assessing the Efficacy of AntimicrobialTowelettes, Presented at the 125thAnnual Meeting of theAOAC International, NewOrleans, LA, 2011.3Available from AOAC International, 481 North Frederick Ave., Suite 500,Gaither
14、sburg, Maryland 20877-2417, http:/www.aoac.org.4Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19
15、428-2959. United States13. Terminology3.1 Definitions:3.1.1 antimicrobial towelette (wipe), na piece of porousmaterial soaked in an antimicrobial liquid that is meant fordecontamination of hard non-porous environmental surfaces bywiping.3.1.2 dilution blank, ntubes of phosphate buffered saline(PBS)
16、or phosphate buffered dilution water (PBDW) or similarinert phosphate buffer solution.3.1.3 carriers, nglass petri plates (150 by 20 mm).3.1.4 colony forming units (CFU), nnumber of microor-ganisms that form colonies (clusters of microorganisms visiblygrowing on the surface of a membrane filter) as
17、a means ofenumerating the total number of viable microorganisms in asample.3.1.5 quality control (QC), nthe procedures, products orservices that meet a laboratorys specified standards of quality.4. Summary of Test Method4.1 This test method provides detailed instructions forperforming a quantitative
18、 evaluation of antimicrobial efficacyof a towelette when challenged against Staphylococcus aureus,Pseudomonas aeruginosa and Salmonella enterica. Themethod may be used with other microbial strains, thoughmodification may be necessary to accommodate recovery.4.2 Antimicrobial towelettes, for which di
19、sinfecting andsanitizing label claims are made, are suitable products to beevaluated with this test method.4.3 Petri plates, inoculated with a suspension of vegetativebacteria, are used as the test carriers.4.3.1 Each petri plate is inoculated with five spots (10 Leach) of the test organism and allo
20、wed to dry.4.4 The bacteria are exposed to the test chemical byapplying a towelette to the inner bottom surface of a plate usinga prescribed pattern of wiping, followed by exposure to the testchemical for a specific contact time. Bacterial population afterexposure is determined by neutralizing the r
21、esidual test sub-stance (liquid), scraping the petri plate surface, and collectingthe inoculum-neutralizer suspension. The pooled suspension isserially diluted, filtered and plated onto recovery media (Tryp-tic Soy Agar).4.5 Viable CFUs for control and treated carriers are enu-merated using membrane
22、 filtration.4.6 The mean log10density (LD) recovered from treatedcarriers is compared to the mean log10density recovered fromthe control carriers. This calculation is used to determine theefficacy of the product based on the mean log10reduction (LR)value.5. Significance and Use5.1 The glass petri pl
23、ate provides a closed system forenumeration and easy application of a pre-saturated or impreg-nated antimicrobial towelette by an analyst.5.2 Inoculation of carriers (five 10 L spots of microbialsuspension) is conducted using a template and a positivedisplacement pipette, thereby ensuring a precise
24、inoculumlevel and uniform distribution of inoculum.5.3 A single towelette is tested per carrier, thereby ensuringcomparable treatment among carriers and eliminating thelikelihood of cross-contamination between carriers.5.4 The circular motion of the product application (wipeoutside to inside, lift t
25、owelette to invert and wipe inside tooutside) is a relevant motion that ensures uniform coverage andcontact of disinfectant with the inoculated surface.5.5 The addition of neutralizer to the treated plates ensuresthorough neutralization at the end of the products contact time.This test method provid
26、es a procedure for performing neutral-ization verification to confirm that the microbicidal and/ormicrobistatic activity of a test substance has been brought to anundetectable level at the end of the contact time.5.6 The design of the test method minimizes any loss ofviable organisms through carrier
27、 wash-off.5.7 This test method provides for optional use of an organicsoil load as dictated by a products label claim.5.8 It is optional to adjust (either dilute or concentrate) theinoculum level to achieve desired control carrier counts and toaccommodate different product performance standards.6. A
28、pparatus6.1 Biosafety cabinet (BSC, Type B2, Class II)Certified,recommended for maintaining an aseptic work environment.6.2 Petri platesGlass petri plates used as test carriers (150by 20 mm).6.3 MicropipetteCalibrated.6.4 Positive displacement pipetteCalibrated, used to dis-pense 10 L aliquots (5 to
29、tal spots) of a challenge suspensiononto the test carriers.6.5 TimerAny certified timer that can display time inseconds.6.6 Sterile test tubesReusable or disposable 20 by 150mm for dilution blanks and cultures/subcultures or otherappropriate size.6.7 Test tube racksAny convenient size.6.8 Sterile ce
30、ll scraperTo scrape carriers for removal ofbacteria during harvesting (for example, scraper blade dimen-sions = 1.8 to 3.0 cm).6.9 Sterile plate spreaderMay be used to spread inoculumon plate surface.6.10 CryovialTo store frozen stock cultures (for example,1.5 mL capacity).6.11 Conical tubesSterile,
31、 50 mL. To collect neutralizer/product/bacterial suspensions from treated carriers andneutralizer/bacterial suspensions from control carriers afterinoculum has been dislodged by scraping and neutralized.6.12 Vortex mixer.6.13 Serological pipettesSterile single-use pipettes (forexample, 25.0, 10.0, 5
32、.0, 2.2, 1.0 mL capacity).E2896 1226.14 Sterile membrane filtersTo filter serial dilutions forcell enumeration (0.22 m pore size, polyethersulfone). Filtra-tion units (reusable or disposable) may be used.6.15 Sterile surgical glovesTo handle antimicrobial tow-elette when folding and wiping inoculate
33、d petri plate carrier.6.16 Autoclave (steam sterilizer)To sterilize media andreagents.7. Media and Reagents7.1 Culture Media:7.1.1 Trypticase Soy Broth (TSB)For use in rehydratinglyophilized/frozen vegetative culture of test microorganism.Prepare TSB according to manufacturers instructions.7.1.2 AOA
34、C Nutrient Broth (AOAC NB)For use in culturemaintenance of Salmonella enterica. Prepare media in accor-dance with the instructions from AOAC methods.37.1.3 Synthetic Broth (AOAC SB)For use in culture andsubculture preparation of Staphylococcus aureus, Pseudomo-nas aeruginosa and Salmonella enterica.
35、 Prepare media inaccordance with the instructions from AOAC methods.37.1.4 Trypticase Soy Agar (TSA)For use as a recoverymedium for bacterial enumeration and purity checks. PrepareTSA according to manufacturers instructions. Equivalentcommercially prepared agar culture medium may be pur-chased. TSA
36、with 5 % sheep blood may be substituted.7.1.5 Nutrient Agar (NA)For use in propagation. Dissolve1.5 % Bacto Agar (Difco) in AOAC NB and adjust pH to 7.2to 7.4 (blue-green with bromothymol blue), autoclave (at121C for 20 min).7.1.6 Mannitol Salt Agar (MSA)Selective solid mediumfor S. aureus. Prepare
37、MSA according to manufacturersinstructions.7.1.7 Cetrimide Agar (CA)Selective solid medium for P.aeruginosa. Prepare CA according to manufacturers instruc-tions.7.1.8 Neutralizer MediumTo use as chemical neutralizer(for example, letheen broth, letheen broth with 0.1 % sodiumthiosulfate).NOTE 1Commer
38、cially dehydrated media that conform to the recipesprovided in AOAC Method 961.02 may be substituted.7.2 Reagents:7.2.1 Cryoprotectant solutionTSB with 15 % v/v glyc-erol. Sterile solution is used in the preparation of frozen stockcultures.7.2.2 Organic soilOrganic burden added to inoculum,consisten
39、t with product label claims (for example, fetal bovineserum, horse serum, heat-inactivated fetal bovine serum).7.2.3 De-ionized waterPurified water with mineral ionsremoved through pre-treatment, deionization and filters.Alternatively, reagent grade water (ultrapure water) may beused.7.2.4 Phosphate
40、-buffered saline stock solution (PBS-SS)Prepare 10 stock solution of PBS by dissolving 492 g PBSpowder in 5 L of deionized water.7.2.5 Phosphate-buffered saline (PBS) 1 SolutionDilute1:10 1 part (PBS-SS) 10 solution) plus 9 parts deionizedwater to obtain 1 solution, distribute into bottles and auto-
41、clave for 20 min at 121C.8. Test Organisms8.1 Staphylococcus aureusATCC 6538. The organism is agram-positive, coccus-shaped bacterium, that when plated ontogeneral growth media (for example, Trypticase Soy Agar)produces small, circular, yellow, glistening colonies within 24hat366 1C.8.2 Pseudomonas
42、aeruginosaATCC 15442. The organismis a gram-negative, rod-shaped bacterium, that when platedonto general growth media (for example, Trypticase SoyAgar)produces flat, opaque to off-white, round, spreading colonieswithin 24 h at 36 6 1C.8.3 Salmonella enterica (subsp. enterica serovarcholeraesuis)ATCC
43、 10708. The organism is a gram-negative,rod-shaped bacterium, that when plated onto general growthmedia (for example, Trypticase Soy Agar) produces entire,glistening, circular, smooth, translucent, low, convex colonieswithin 24 h at 36 6 1C.9. Generation of Frozen Stock Cultures9.1 All cultures are
44、reconstituted per the manufacturersinstructions as presented in sections 9.2-9.4.9.2 Using tubes containing 5 to 6 mL of TSB for S. aureusand P. aeruginosa and NB for S. enterica, aseptically withdraw0.5 to 1.0 mL and rehydrate the lyophilized culture.9.3 Aseptically transfer the entire rehydrated p
45、ellet backinto the source tube of TSB or NB. Mix well.9.4 Incubate for 24 6 2hat366 1C.9.5 Using a sterile plate spreader, inoculate a sufficientnumber of TSA plates for S. aureus and P. aeruginosa or NAfor S. enterica (for example, 5 to 10 plates per organism) with100 L each of the culture.9.6 To v
46、erify purity, conduct streak-isolation for S. aureusand P. aeruginosa using a general growth medium such asTSA. Use of selective media such as MSA for S. aureus andCA for P. aeruginosa may be employed as an option forpresumptive identification testing.9.7 Incubate all plates for 24 6 2hat366 1C.9.8
47、Following incubation, add 5 mL cryoprotectant solutionto the surface of each agar plate.9.9 Resuspend the cells in this solution using a sterile platespreader and aspirate the cell suspension from the surface ofthe agar.9.10 Transfer suspension into a sterile vessel.9.11 Mix the pooled contents of t
48、he vessel thoroughly.9.12 Immediately after mixing, pipette approximately 0.5 to1.0 mL of the collected suspension into a cryovial.9.13 Place cryovials in a 70C (or lower) freezer for longterm storage; these are the frozen stock suspensions that maybe used for testing for up to 18 months after prepa
49、ration.AnewATCC culture must be purchased after 18 months to generatenew frozen stock cultures.E2896 12310. Test Organism Preparation10.1 Defrost a single cryovial at room temperature andbriefly vortex to mix. Defrosting should be rapid to avoid lossin the viability of the preserved cells (for example, expose torunning water to thaw). Each cryovial is for single use only.10.2 Add 100 L of the thawed stock suspension to a tubecontaining 10 mL of SB (that is, primary) and incubate at 36 61C for 18 to 24 h.10.3 For S.
