1、Designation: E2274 16Standard Test Method forEvaluation of Laundry Sanitizers and Disinfectants1This standard is issued under the fixed designation E2274; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A
2、 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 test method is designed to evaluate sanitizing/disinfectant laundry detergents/additives for use in traditionaltop-loading automa
3、tic clothes washing operations. This testmethod is designed predominantly to provide testing withrepresentative vegetative bacteria but can also be designed toaccommodate the testing of fungi and viruses.NOTE 1This test method does not evaluate sanitizing/disinfectantlaundry detergent/additives for
4、use in front-loading or top-loading, lowwater high efficiency automatic clothes washing operations.1.2 This test method is intended to compliment Test MethodE2406 and is to be used in the cases where this test method isdetermined to be the worse case scenario for product usage.1.3 Knowledge of micro
5、biological techniques is requiredfor these procedures.1.4 It is the responsibility of the investigator to determinewhether Good Laboratory Practices (GLP) are required and tofollow them where appropriate (see section 40 CFR, 160 or asrevised.)1.5 The values stated in SI units are to be regarded asst
6、andard. No other units of measurement are included in thisstandard.NOTE 2In this test method, metric units are used for all applications,except for distance in which case inches are used.1.6 Appropriate modifications to the test method may berequired when the testing organisms are not specified here
7、in.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 of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Reference
8、d Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE1054 Test Methods for Evaluation of Inactivators of Anti-micro
9、bial AgentsE2406 Test Method for Evaluation of Laundry Sanitizersand Disinfectants for Use in High Efficiency WashingOperationsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Other Standards:AATCC Test Method 70 Water Repellency; Tumble JarDynamic Absorption Test3OCSPP 810.2400 :
10、Disinfectants and Sanitizers for Use onFabrics and Textiles Efficacy Data Recommendations440 CFR, Part 160 , Good Laboratory Practice Standards53. Terminology3.1 For definitions of terms used in this test method refer toTerminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 activ
11、e antimicrobial ingredienta substance added toa formulation intended specifically for the inhibition or inac-tivation of microorganisms.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Su
12、bcommittee E35.15 on Antimicrobial Agents.Current edition approved April 15, 2016. Published May 2016. Originallyapproved in 2003. Last previous edition approved in 2009 as E2247 09. DOI:10.1520/E2274-16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Se
13、rvice at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.4Availab
14、le from United States Environmental ProtectionAgency (EPA),WilliamJefferson Clinton Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460,http:/www.epa.gov.5Available from U.S. Government Publishing Office Bookstore 710 NorthCapitol Street N.W. Washington, DC, http:/www.gpo.gov/about/bookstore.htm
15、Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.2 antimicrobial agent(s)an active ingredient designedto suppress the growth or action of microorganisms.3.2.3 carrier count controlprocedure used to determinethe initial number of mi
16、croorganisms on a fabric carrierfollowing the inoculation and drying procedure.3.2.4 diluentsterile deionized water, sterile distilled wateror sterile synthetic AOAC hard water that may be used toprepare the active test formulation, vehicle control or productcontrol for use in the test procedure.3.2
17、.5 diluted product solutiontest formulation, vehiclecontrol, or product control diluted to use concentration.3.2.6 numbers controlin assessing sanitizer levelperformance, procedure used to determine the number ofmicroorganisms remaining on the fabric carriers and in thewash water following the test
18、procedure in the presence of thediluent. This may also be performed using diluent or phosphatebuffer dilution water with surfactant.3.2.7 product controla formulation with or without anactive ingredient(s) used for comparison to the test formula-tion.3.2.8 test formulationa formulation containing an
19、 antimi-crobial agent(s).3.2.9 vehicle controlthe test formulation without the ac-tive ingredient(s) used for comparison to the test formulation.3.2.10 wash waterthe liquid contained in the exposurechamber previously exposed to either uninoculated fabric orfabric inoculated with the challenge microo
20、rganism.4. Summary of Test Method4.1 Under simulated laundry conditions, sets of inoculatedfabric swatches are placed into diluted product solution andagitated.After a specified contact time, the wash water and thetest fabric are individually cultured either quantitatively (sani-tizer efficacy) or q
21、ualitatively (disinfectant efficacy).NOTE 3See appropriate regulatory guidance document for the mini-mum number of replicates required to meet a specific claim.5. Significance and Use5.1 The procedure in this test method is used to evaluate theactivity of a test reagent (antimicrobial agent/active i
22、ngredient)or formulation in the reduction or complete kill of the bacterialpopulation in fabric and wash water following a single wash.The water to fabric ratio in common top loading machines isdynamic and varies by region. The proper water to fabric ratioand temperature for the worse-case scenario
23、should be deter-mined prior to testing. This test method may need to modifiedif the worse-case scenario is determined to be in top loadinghigh efficiancy washing machine6. Apparatus6.1 Colony Counter, any of several types may be used, forexample, Quebec.6.2 Incubator, any incubator that can maintain
24、 the optimumtemperature, 62C, for growth of the challenge microorgan-ism(s).6.3 Sterilizer, any suitable steam sterilizer producing theconditions of sterility is acceptable.6.4 Timer (Stop-clock), any calibrated device that can beread for minutes and seconds.6.5 Exposure Chamber, container with clos
25、ure that canwithstand sterilization. Should be large enough to hold a singlestainless steel spindle yet allow diluted product solution tocompletely contact the entire fabric spindle during the tum-bling period.NOTE 4Standard lids may form a vacuum seal when steam sterilized.To avoid, prior to steril
26、ization place a piece of paper between lid and jar.6.6 Stainless Steel spindles, Spindles are fabricated from asingle continuous piece of stainless steel wire, (116 in. diameterand bent to contain 3 horizontal extensions, 2 in. longconnected by 2 vertical sections approximately 2 in. long.)They are
27、shaped so that vertical sections form 150 angles, freeends of 2 outer horizontal extensions are sharpened to a point.Use as carrier for wrapping fabric ballast. See Fig. 1.6.7 Agitator, tumbling device to rotate Exposure Chamberthrough 360 vertical orbit of 4 to 8 in. diameter at 45 to 60rpm or comp
28、arable tumbling devices such as, launderometer ortumble jar described in AATCC Test Method 70.6.8 Micropipettor (and Pipet Tips), suitable to deliver 0.01to 0.03 mL volume.6.9 Forceps, large and small, sterile.6.10 Safety Pins, sterile.6.11 Stapler and Staples.6.12 Balance, with a platform to accomm
29、odate 15 6 0.1 gof test fabric.6.13 Sterile Glass Beads, Average size 3 to 4 mm.6.14 Filter Sterilization System for Media and Reagents, amembrane or cartridge filtration system (0.22 m pore diam-eter). Required for sterilizing heat-sensitive solutions.6.15 Membrane Filtration System for Capture of
30、the TestOrganism(s), sterile 47 mm diameter Polyethersulfone (PES)membrane filters (0.45 m pore diameter) and holders for suchfilters.FIG. 1 Stainless Steel Spindel Schematic (top view and sideview).E2274 1627. Reagents and Materials7.1 Petri Dishes, sterile 100 15 mm glass and plastic.Required for
31、performing standard plate counts and used inpreparation of contaminated fabric carriers.7.2 Bacteriological Pipets, sterile, various sizes.7.3 Test Fabric, approximately 80 by 80 threads/in.bleached, desized, plain-weave cotton print cloth and withoutbluing or optical brighteners.NOTE 5Other test fa
32、brics/blends may be used at the discretion of theinvestigator.7.4 Dilution Fluid, AOAC Phosphate buffer dilution water6or other suitable diluent containing appropriate neutralizers forserial dilution of test samples.7.5 Water for Dilution of Formulations under Test:7.5.1 Water, sterile, deionized or
33、 distilled, equivalent to orbetter than Type 3, see Specification D1193.7.5.2 AOAC Synthetic Hard Water.67.5.3 All water used for preparation of test solutions shall besterile.7.6 Purity of Reagentsreagent grade chemicals shall beused in all tests.7.6.1 Sodium carbonate.7.6.2 Alkaline nonionic wetti
34、ng agent with HLB(hydrophilic-lipophilic balance) value of approximately 13.Prepare solution containing 0.5% nonoxynol-10 class ofethoxylated alkyl phenols, for example Tergitol NP-10 orTriton X-100 an 0.5% Na2CO3.7.7 Neutralizing Subculture MediaAneutralizing mediumcapable of supporting the growth
35、of the test organism (fordisinfection testing) following exposure to the test material inaccordance with Test Methods E1054. Alternatively, the neu-tralizing broths may be of sufficient volume to reduce theconcentration of the antimicrobials to below active levels. See12.8.7.8 Challenge Microorganis
36、ms (DIS/TSS 13):7.8.1 Klebsiella pneumoniae, ATCC 4352.7.8.2 Staphylococcus aureus, ATCC 6538.7.8.3 Pseudomonas aeruginosa, ATCC 15442.7.8.4 Other microorganisms, as applicable.7.9 Culture Media:7.9.1 Nutrient Agar A.67.9.2 Nutrient Agar B.67.9.3 Media suitable for identification of microorganism(s)
37、used in the study.7.9.4 Soybean casein digest medium or other suitablemedia, with or without specific neutralizers, for recovery of thechallenge microorganism(s).7.10 Organic Soil Loadwhen an organic soil load is to beincorporated in the suspension of the challengemicroorganism(s), defibrinated heat
38、-inactivated animal serummay be used or a mixture of the following stock solutions inphosphate buffer dilution water (pH 7.2) may be used (see 7.4):7.10.1 Add 0.5 g of tryptone to 10 mL phosphate buffer.7.10.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mLof phosphate buffer.7.10.3 Add 0.04 g of b
39、ovine mucin to 10 mL of phosphatebuffer.7.10.4 Prepare the solutions separately and sterilize bypassage through a 0.22 m pore diameter membrane filter,apportioned and stored at either 4 6 2C or -20 6 2C for nolonger than 3 months.7.10.5 To obtain a 500 L inoculum of the challengemicroorganism, add t
40、o 340 L of the microbial suspension 25L, 100 L, and 35 L of BSA, mucin and Tryptone stocksolutions, respectively.NOTE 6The quality of the above materials may vary among manu-facturers or product lots. Therefore, preliminary screening of such items isrecommended to ensure compatibility with the test
41、microorganism(s).NOTE 7The investigator should confirm the appropriate organic soilusage with the appropriate regulatory agency prior to initiating testing.8. Test Microorganisms (810,2400)8.1 Klebsiella pneumoniae, ATCC 4352.8.2 Staphylococcus aureus, ATCC 65388.3 Pseudomonas aeruginosa, ATCC 15442
42、8.4 Other microorganisms, as applicable.9. Preparation of Test Microorganisms9.1 Subculture microorganism(s) on Nutrient Agar Athrough at least one daily transfer, incubating at 35 6 2C.9.2 On the day prior to testing, wash the slant and transferthe cells into French square bottles containing 20 mL
43、ofsolidified Nutrient Agar B. Incubate 18 to 24 h at 35 6 2C,agar side down.9.3 Remove growth from the French square bottles usingthree-mL dilution fluid and five sterile glass beads to suspendgrowth. The cultures will be standardized to yield approxi-mately 108colony forming units (CFU) per mL of S
44、. aureusand 109CFU/mL of K. pneumoniae and P. aeruginosa.NOTE 8The initial inoculum concentration for the and other challengemicroorganisms may vary and should be determined from carrier andwash water numbers control recovery (see Section 12).9.4 A soil load may be added to each inoculum (see 7.10)1
45、0. Fabric and Spindle Preparation10.1 Scour test fabric by boiling approximately 300 g ofmaterial for1hin3Lofdistilled or deionized water containing1.5-g sodium carbonate and 1.5-g nonionic wetting agent.Rinse fabric, first in boiling water and then in cold water, untilall visual traces of wetting a
46、gent are removed (that is, foam-ing). Remove as much water as possible from fabric.10.2 Air dry for at least 24 h at ambient room temperatureensuring the material is completely dry.10.3 Cut scoured dry fabric into strips 2 in. (5 cm) wide andweighing 15 6 0.1 g each. For cotton fabrics, pierce one e
47、ndof the 15-g test fabric strip and secure onto the outer horizontalextension of a stainless steel spindle. Wind the strip around thethree horizontal extensions with sufficient tension to obtain 126Offcial Methods of Analysis of the AOAC International , AOAC, Washington,DC, Chapter 6: Disinfectants.
48、E2274 163but not 13 laps while using the entire 15 6 0.1 g of fabric.Staples, a pin, or autoclaveable fabric tag may be used tosecure the fabric. Fabric wrapped spindles may be sterilized inindividual exposure chambers. Alternatively, fabric wrappedspindles may be sterilized separately from exposure
49、 chambers.Ensure fabric on spindles and exposure chambers are dry priorto testing.NOTE 9Fabric may be purchased in pre-cut strips and then scoured.10.4 Fabric carriers of approximately 1 by 1.5 in. will be cutfrom the remaining scoured fabric. Nontoxic permanentmarker may be used to place a mark on the edge of each carrier.Alternatively, attach a pin to the short side of each carrier.Place fabric carriers in glass petri dishes and sterilize. Ensuredryness of fabric prior to testing.10.5 For each challenge microorganism, prepare at leas
