1、Designation: E2274 09Standard 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 top-loadingautomatic clothes
3、washing operations. This test method isdesigned predominantly to provide testing with representativevegetative bacteria but can also be designed to accommodatethe testing of fungi and viruses.NOTE 1This test method does not evaluate sanitizing/disinfectantlaundry detergent/additives for use in front
4、-loading, low water volumeautomatic clothes washing operations.1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.NOTE 2In this method, metric units ar
5、e used for all applications,except for distance in which case inches are used.1.4 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 deter
6、mine the applica-bility of regulatory limitations prior to use.2. Referenced 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
7、 Test MethodE1054 Test Methods for Evaluation of Inactivators of An-timicrobial Agents2.2 Other Documents:AATCC Test Method 70-1997 Water Repellency; TumbleJar Dynamic Absorption Test3DIS/TSS 13 LaundryAdditivesDisinfection and Sanitiza-tion, U.S. Environmental Protection Agency, Office ofPesticide
8、Programs, April 198043. Terminology3.1 Definitions:3.1.1 active antimicrobial ingredienta substance added toa formulation intended specifically for the inhibition or inac-tivation of microorganisms.3.1.2 antimicrobial agent(s)an active ingredient designedto suppress the growth or action of microorga
9、nisms.3.1.3 carrier count controlprocedure used to determinethe initial number of microorganisms on a fabric carrierfollowing the inoculation and drying procedure.3.1.4 diluentsterile deionized water, sterile distilled wateror sterile synthetic AOAC hard water that may be used toprepare the active t
10、est formulation, vehicle control or productcontrol for use in the test procedure.3.1.5 diluted product solutiontest formulation, vehiclecontrol, or product control diluted to use concentration.3.1.6 neutralizationa process that results in quenching theantimicrobial activity of a test formulation. Th
11、is may beachieved by dilution of the test formulation(s) to reduce theconcentration of the antimicrobials, or through the use ofchemical agents, called neutralizers, to suppress antibacterialactivity.3.1.7 numbers controlin assessing sanitizer level perfor-mance, procedure used to determine the numb
12、er of microor-ganisms remaining on the fabric carriers and in the wash waterfollowing the test procedure in the presence of the diluent. Thismay also be performed using diluent or phosphate bufferdilution water with surfactant.3.1.8 product controla formulation with or without anactive ingredient(s)
13、 used for comparison to the test formula-tion.3.1.9 test formulationa formulation containing an antimi-crobial agent(s).1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobia
14、l Agents.Current edition approved Nov. 1, 2009. Published December 2009. Originallyapproved in 2003. Last previous edition approved in 2003 as E2247 03. DOI:10.1520/E2274-09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For
15、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.4Available from United States Environm
16、ental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:/www.epa.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.10 vehicle controlthe test formulation without theactive ingredie
17、nt(s) used for comparison to the test formula-tion.3.1.11 wash waterthe liquid contained in the exposurechamber previously exposed to either uninoculated fabric orfabric inoculated with the challenge microorganism.4. Summary of Test Method4.1 Under simulated laundry conditions, sets of inoculatedfab
18、ric 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 qualitatively (disinfectant efficacy).NOTE 3See appropriate regulatory guidance document for t
19、he 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 ingredient)or formulation in the reduction or complete kill of the bacterialpopulation in fabr
20、ic and wash water following a single wash.6. Apparatus6.1 Colony Counter, any of several types may be used, forexample, Quebec.6.2 Incubator, any incubator that can maintain the optimumtemperature, 62C, for growth of the challenge microorgan-ism(s).6.3 Sterilizer, any suitable steam sterilizer produ
21、cing theconditions of sterility.6.4 Timer (Stop-clock), any device that can be read forminutes and seconds.6.5 Exposure Chamber, container with closure that canwithstand sterilization. Should be large enough to hold a singlestainless steel spindle yet allow diluted product solution tocompletely cont
22、act the entire fabric spindle during the tum-bling period.NOTE 4Standard lids may form a vacuum seal when steam sterilized.To avoid, prior to sterilization place a piece of paper between lid and jar.6.6 Stainless Steel spindles, Spindles are fabricated from asingle continuous piece of stainless stee
23、l wire, (116 in. diameterand bent to contain 3 horizontal extensions, 2 in. longconnected by 2 vertical sections approximately 2 in. long.)They are 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
24、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 comparable tumbling devices such as, launderometer ortumble jar described in AATCC Test Method 70-1997.6.8 Micropipettor (and Pipet Tips), suitable to deliv
25、er 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 accommodate 15 6 0.1 gof test fabric.6.13 Sterile Glass Beads,3to4mm.6.14 Filter Sterilization System for Media and Reagents,amembrane or cartridge filtr
26、ation system (0.22 m pore diam-eter). Required for sterilizing heat-sensitive solutions.6.15 Membrane Filtration System for Capture of the TestOrganism(s), sterile 47 mm diameter membrane filters (0.45m pore diameter) and holders for such filters.7. Reagents and Materials7.1 Petri Dishes, sterile 10
27、0 by 15 mm. Required forperforming standard plate counts and used in preparation ofcontaminated 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 brighte
28、ners.NOTE 5Other test fabrics/blends may be used at the discretion of theinvestigator.7.4 Dilution Fluid, AOAC Phosphate buffer dilution water5or other suitable diluent containing appropriate neutralizers forserial dilution of test samples.7.5 Water for Dilution of Formulations under Test:7.5.1 Wate
29、r, sterile, deionized or distilled, equivalent to orbetter than Type 3, see Specification D1193.7.5.2 AOAC Synthetic Hard Water.57.5.3 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
30、 Alkaline nonionic wetting 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% Na2CO3.5Offcial Methods of Analysis of the AOAC International , AOAC,
31、 Washington,DC, 16th ed, Chapter 6: Disinfectants, 1995.FIG. 1 Stainless Steel Spindel Schematic (top view and sideview).E2274 0927.7 Neutralizing Brothsgrowth media appropriate for thechallenge microorganism containing chemical agents to sup-press antibacterial activity. Alternatively, the neutrali
32、zingbroths may be of sufficient volume to reduce the concentrationof the antimicrobials to below active levels. See 11.8.7.8 Challenge Microorganisms (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 microo
33、rganisms, as applicable.7.9 Culture Media:7.9.1 Nutrient Agar A.57.9.2 Nutrient Agar B.57.9.3 Media suitable for identification of microorganism(s)used in the study.7.9.4 Soybean casein digest medium or other suitable me-dia, with or without specific neutralizers, for recovery of thechallenge microo
34、rganism(s).7.10 Organic Soil Loadwhen an organic soil load is to beincorporated in the suspension of the challenge microorgan-ism(s), defibrinated heat-inactivated animal serum may be usedor a mixture of the following stock solutions in phosphatebuffer dilution water (pH 7.2) may be used (see 7.4):7
35、.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 bovine mucin to 10 mL of phosphatebuffer.7.10.4 Prepare the solutions separately and sterilize bypassage through a 0.22 m pore diameter membrane filte
36、r,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 to 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 mater
37、ials may vary among manu-facturers or product lots. Therefore, preliminary screening of such items isrecommended to ensure compatibility with the test microorganism(s).NOTE 7The investigator should confirm the appropriate organic soilusage with the appropriate regulatory agency prior to initiating t
38、esting.8. Fabric and Spindle Preparation8.1 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 wet
39、ting agent are removed (that is, foam-ing). Remove as much water as possible from fabric.8.2 Air dry for at least 24 h at ambient room temperature.8.3 Cut scoured dry fabric into strips 2 in. (5 cm) wide andweighing 15 6 0.1 g each. For cotton fabrics, pierce one endof the 15-g test fabric strip and
40、 secure onto the outer horizontalextension of a stainless steel spindle. Wind the strip around thethree horizontal extensions with sufficient tension to obtain 12but not 13 laps while using the entire 15 6 0.1 g of fabric.Staples or a pin may be used to secure the fabric. Fabricwrapped spindles may
41、be sterilized in individual exposurechambers. Alternatively, fabric wrapped spindles may besterilized separately from exposure chambers. Ensure drynessof fabric on spindles and exposure chambers prior to testing.NOTE 8Fabric may be purchased in pre-cut strips and then scoured.8.4 Fabric carriers of
42、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 p
43、rior to testing.8.5 For each challenge microorganism, prepare at least 3fabric carriers and 1 fabric wrapped spindle for each active testformulation/product and control/numbers control.9. Preparation of Challenge Microorganisms9.1 Subculture microorganism(s) on Nutrient Agar Athrough at least three
44、daily transfers, incubating at 35 6 2C.If only one daily transfer is missed, it is not necessary to repeatthe three consecutive transfers prior to use in testing.9.2 On the day prior to testing, transfer the cells into Frenchsquare bottles containing 20 mL of solidified Nutrient Agar B.Incubate 18 t
45、o 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. aureusand 109CFU/mL of K. pneumoniae and
46、 P. aeruginosa.5NOTE 9The initial inoculum concentration for different 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).10. Preparation of Test Sample10.1 Prepare a
47、sufficient volume of diluted active testformulation and product control (at least 1 L) according tomanufacturer instructions, using diluent pre-equilibrated to testtemperature.NOTE 10 Fabric to wash-water ratios based on usage patterns must beconsidered in this step (see DIS/TSS 13 and Table 1.)NOTE
48、 11When appropriate use AOAC hard water in preparation oftest product (see 7.5.2).10.2 Using diluent at test temperature, prepare test productdilution no more than 3 h prior to use and maintain solution attest temperature. Some active ingredients may require prepa-ration and usage in less than 3 h.1
49、1. Procedure11.1 Inoculate three sterile fabric carriers (in a single sterilePetri dish) with 0.01 to 0.03 mL of prepared inoculum percarrier. Disperse the inoculum over an approximate 1- by 1.5in. area of each carrier, avoiding the marker, staple, or safetypin. Dry the carriers in a 35 6 2C incubator until the carriersTABLE 1 Typical Use PatternsUsage Pattern Fabric: Wash-water RatioHome or coin-operated laundering 1:10Industrial laundering 1:5E2274 093are visibly dry, but not longer than 30 min. Use swatches(carriers) within1hofdryin
