1、Designation: E 2274 03Standard Test Method forEvaluation of Laundry Sanitizers and Disinfectants1This standard is issued under the fixed designation E 2274; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、 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 test method is designed to evaluate sanitizing/disinfectant laundry detergents/additives for use in top-loadingautomatic cloth
3、es 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 fr
4、ont-loading, low water volumeautomatic clothes washing operations.1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 In this method metric units are used for all applications,except for distance in which case inches are used.1.4 This standard does not purport to address
5、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 Documents2.1 ASTM Standards:2D 1193 Specificati
6、on for Reagent WaterE 1054 Practices for Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products2.2 Other Documents:TheAmericanAssociation ofTextile ChemistsAnd Colorists(AATCC) Test Method 70-1997 Water Repellency;Tumble Jar Dynamic Absorpt
7、ion TestOfficial Methods of Analysis of AOAC Internation-al AOAC, Washington, D.C., Chapter 6: Disinfectants,17th ed., 2000.DIS/TSS 13 LaundryAdditivesDisinfection and Sanitiza-tion, U.S. Environmental Protection Agency, Office ofPesticide Programs, April 1980Federal Standard 40 CFR Part 160, Good L
8、aboratory Prac-tice Standards3Canadian Pest Management Regulatory Standards TradeMemorandum T-1-2153. 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 agen
9、t(s)an active ingredient designedto suppress the growth or action of microorganisms.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 wat
10、eror sterile synthetic AOAC hard water that may be used toprepare the active test formulation, vehicle control or productcontrol for use in the test procedure.3.1.5 diluted product solutiontest formulation, vehiclecontrol, or product control diluted to use concentration.3.1.6 neutralizationa process
11、 that results in quenching theantimicrobial activity of a test formulation. This 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 controli
12、n assessing sanitizer level perfor-mance, procedure used to determine the number 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 surfac
13、tant.3.1.8 product controla formulation with or without anactive ingredient(s) used for comparison to the test formula-tion.3.1.9 test formulationa formulation containing an antimi-crobial agent(s).3.1.10 vehicle controlthe test formulation without theactive ingredient(s) used for comparison to the
14、test formula-tion.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntibacterialAgents.Current edition approved Dec. 1, 2003. Published January 2004.2For referenced ASTM standards, visit the ASTM website, www.as
15、tm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washing
16、ton, DC 20401.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.11 wash waterthe liquid contained in the exposurechamber previously exposed to either uninoculated fabric orfabric inoculated with the challenge microorganism.4. Summa
17、ry 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 qualitatively (d
18、isinfectant efficacy).NOTE 2See 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 ingredient)or fo
19、rmulation in the reduction or complete kill of the bacterialpopulation in fabric 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 c
20、hallenge microorgan-ism(s).6.3 Sterilizer, any suitable steam sterilizer producing 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 sing
21、lestainless steel spindle yet allow diluted product solution tocompletely contact the entire fabric spindle during the tum-bling period.NOTE 3Standard 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 spi
22、ndles, 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 shaped so that vertical sections form 150 angles, freeends of 2 outer hori
23、zontal 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 comparable tumbling devices such as, Launderometeror Tumble Jar described in A
24、ATCC 70-1997.6.8 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 accommodate 15 6 0.1 gof test fabric.6.13 Sterile Glass Beads,3to4mm.6.14 Filter Steril
25、ization 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 the TestOrganism(s), sterile 47 mm diameter membrane filters (0.45m pore diameter) and holders for
26、such filters.7. Reagents and Materials7.1 Petri Dishes, sterile 100 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, p
27、lain-weave cotton print cloth and withoutbluing or optical brighteners.4NOTE 4Other 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 s
28、amples.7.5 Water for Dilution of Formulations under Test:7.5.1 Water, sterile, deionized or distilled, equivalent to orbetter than Type 3, see Specification D 1193.7.5.2 AOAC Synthetic Hard Water.57.5.3 All water used for preparation of test solutions shall besterile.7.6 Purity of Reagentsreagent gr
29、ade chemicals shall beused in all tests.7.6.1 Sodium carbonate.7.6.2 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% N
30、a2CO3.4Fabric #400 8-1978) obtained from Test Fabric, Inc., P.O. Box 26, West Pittson,PA 18643 or supplier of comparable specifications.5Offcial Methods of Analysis of the AOAC International, AOAC, Washington,DC, 16th 16th ed, Chapter 6: Disinfectants, 1995.FIG. 1 Stainless Steel Spindel Schematic (
31、top view and sideview).E22740327.7 Neutralizing Brothsgrowth media appropriate for thechallenge microorganism containing chemical agents to sup-press antibacterial activity. Alternatively, the neutralizingbroths may be of sufficient volume to reduce the concentrationof the antimicrobials to below ac
32、tive levels. See 11.8.7.8 Challenge Microorganisms,67.8.1 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.57.9.2 Nutrient Agar B.57.9.3 Media suitable fo
33、r 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 microorganism(s).7.10 Organic Soil Loadwhen an organic soil load is to beincorporated in the suspension of the challe
34、nge 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):7.10.1 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
35、 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 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 inocu
36、lum 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 5The quality of the above materials may vary among manu-facturers or product lots. Therefore, preliminary screening of such items isrecommende
37、d to ensure compatibility with the test microorganism(s).NOTE 6The investigator should confirm the appropriate organic soilusage with the appropriate regulatory agency prior to initiating testing.8. Fabric and Spindle Preparation8.1 Scour test fabric by boiling approximately 300 g ofmaterial for1hin
38、3Lofdistilled 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 agent are removed (that is, foam-ing). Remove as much water as possible from fabric.8.2 Air dry for at lea
39、st 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 secure onto the outer horizontalextension of a stainless steel spindle. Wind the strip around thethree horizon
40、tal 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 be sterilized in individual ExposureChambers. Alternatively, fabric wrapped spindles may besterilized separatel
41、y from Exposure Chambers. Ensure drynessof fabric on spindles and Exposure Chambers prior to testing.NOTE 7Fabric may be purchased in pre-cut strips and then scoured.8.4 Fabric carriers of approximately 1 by 1.5 in. will be cutfrom the remaining scoured fabric. Nontoxic permanentmarker may be used t
42、o 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.8.5 For each challenge microorganism, prepare at least 3fabric carriers and 1 fabric wrapped sp
43、indle 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 daily transfers, incubating at 35 6 2C.If only one daily transfer is missed, it is not necessary to repeatthe t
44、hree 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 to 24 h at 35 6 2C, agar side down.9.3 Remove growth from the French square bottles usingthree-mL dilution fluid
45、 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 P. aeruginosa.5NOTE 8The initial inoculum concentration for different challengemicroorganisms may vary and sho
46、uld 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 sufficient volume of diluted active testformulation and product control (at least 1 L) according tomanufacturer
47、 instructions, using diluent pre-equilibrated to testtemperature.NOTE 9 Fabric to wash-water ratios based on usage patterns must beconsidered in this step (see DIS/TSS 13 and Table 1.)NOTE 10When appropriate use AOAC hard water in preparation oftest product (see 7.5.2).10.2 Using diluent at test tem
48、perature, 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.11. Procedure11.1 Inoculate three sterile fabric carriers (in a single sterilePetri dish) with 0.01 to 0.03 mL of
49、 prepared inoculum per6DIS/TSS 13. Laundry AdditivesDisinfection and Sanitization. U.S. Environ-mental Protection Agency, Office of Pesticide Programs. April 1980.TABLE 1 Typical Use PatternsUsage Pattern Fabric: Wash-water RatioHome or coin-operated laundering 1:10Industrial laundering 1:5E2274033carrier. 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 carriersare visibly dry, but not longer than 30 min. Use swatches(carr
