1、Designation: E2406 09Standard Test Method forEvaluation of Laundry Sanitizers and Disinfectants for Usein High Efficiency Washing Operations1This standard is issued under the fixed designation E2406; the number immediately following the designation indicates the year oforiginal adoption or, in the c
2、ase 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.1. Scope1.1 This test method is designed to evaluate sanitizing/disinfectant laundry detergents/addit
3、ives for use in high effi-ciency (HE) automatic clothes washing operations that typi-cally utilize very low wash water volumes. This test method isdesigned to provide testing with representative vegetativebacteria but can also be designed to accommodate the testing offungi and viruses.NOTE 1Test Met
4、hod E2274 is the recommended method to evaluatesanitizing/disinfectant laundry detergent/additives for use in traditionalhigh wash water volume automatic clothes washing operations.1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 The values stated in SI units are to be
5、 regarded asstandard. No other units of measurement are included in thisstandard.NOTE 2In 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 all of thesafety concerns, if any, associated with its use
6、. 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:2D1193 Specification for Reagent WaterE177 Practice for Use of the Terms Prec
7、ision 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 An-timicrobial AgentsE2274 Test Method for Evaluation of Laundry Sanitizersand Disinfectants2.2 AATCC Standard:AATCC
8、70-1997 Water Repellency; Tumble Jar DynamicAbsorption Test32.3 EPA Standard:DIS/TSS 13 LaundryAdditivesDisinfection and Sanitiza-tion, U.S. Environmental Protection Agency, Office ofPesticide Programs, April 198043. Terminology3.1 Definitions:3.1.1 active antimicrobial ingredienta substance added t
9、oa 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 microorganisms.3.1.3 carrier count controlprocedure used to determinethe initial number of microorganisms on a fabri
10、c 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 test formulation, vehicle control or productcontrol for use in the test procedure.3.1.5 diluted product solu
11、tiontest formulation, vehiclecontrol, or product control diluted to use concentration.3.1.6 neutralizationa process that results in quenching theantimicrobial activity of a test formulation. This may beachieved by dilution of the test formulation(s) to reduce the1This test method is under the jurisd
12、iction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 1, 2009. Published December 2009. Originallyapproved in 2004. Last previous edition approved in 2004 as E2406 04. DOI:
13、10.1520/E2406-09.2For referenced ASTM standards, visit the ASTM website, www.astm.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 American Association of Text
14、ile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.4Available from United States Environmental Protection Agency (EPA), ArielRios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:/www.epa.gov.1Copyright ASTM International, 100 Barr H
15、arbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.concentration 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
16、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 surfactant.3.1.8 product controla formulation with or without anactive ingredien
17、t(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 test formula-tion.3.1.11 wash waterthe liquid contained in the exposurecha
18、mber previously exposed to either uninoculated fabric orfabric inoculated with the challenge microorganism.4. Summary of Test Method4.1 Under simulated laundry conditions, sets of inoculatedfabric swatches are placed into low volumes of diluted productsolution and agitated. After a specified contact
19、 time, the washwater and the test fabric are individually cultured eitherquantitatively (sanitizer efficacy) or qualitatively (disinfectantefficacy).NOTE 3See appropriate regulatory guidance document for the mini-mum number of replicates required to make a specific claim.5. Significance and Use5.1 T
20、he procedure in this test method is used to evaluate theeffectiveness of a test reagent (antimicrobial agent/activeingredient) or formulation to reduce or completely kill bacterialpopulations on contaminated fabrics and in wash water follow-ing a single wash under simulated low wash volume condi-tio
21、ns. (See Table 1.)6. Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec.6.2 IncubatorAny incubator that can maintain the opti-mum temperature 62C for growth of the challenge microor-ganism(s).6.3 SterilizerAny suitable steam sterilizer producing theconditions of sterilit
22、y.6.4 Timer (Stop-clock)Any device that can be read forminutes and seconds.6.5 Exposure ChamberContainer with closure that canwithstand sterilization. Dimension and volume capacity shouldbe consistent for use in Test Method E2274.NOTE 4Standard lids may form a vacuum seal when steam sterilized.To av
23、oid, prior to sterilization place a piece of paper between lid and jar.6.6 Stainless Steel SpindlesSpindles 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
24、. long).They are shaped so that vertical sections form 150 anglewhere the free ends of the 2 outer horizontal extensions aresharpened to a point. This will be used as scaffolding for initialwrapping of fabric ballast.6.7 AgitatorTumbling device intended to rotate ExposureChamber through 360 vertical
25、 orbit of 4 to 8 in. diameter at 45to 60 rpm or a comparable tumbling devices such as Launder-ometer or Tumble Jar described in AATCC 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
26、 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 ReagentsAmembrane or cartridge filtration system (0.22 m pore diam-eter). Required for sterilizing heat-sensitive solutions.6.15 Membrane Filt
27、ration 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 100 by 15 mm. Required forperforming standard plate counts and used in preparation ofcontaminated fabric carri
28、ers.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.TABLE 1 Typical Use PatternsUsage PatternFabric:Wash WaterRatioWash WaterVolumeSpindle WireRequirementD
29、eep Fill Top Load Washers $ 1:5-15 75-225 mL RetainHigh Efficiency/Horizontal FrontLoad Washers1:5 75mL OmitFIG. 1 Stainless Steel Spindle Schematic(Top View and Side View)E2406 092NOTE 5Other test fabrics/blends may be used at the discretion of theinvestigator.7.4 Dilution Fluid, AOAC Phosphate buf
30、fer dilution wateror 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 distilled, equivalent to orbetter than Type 3, see Specification D1193.7.5.2 AOAC Synthetic Hard Wat
31、er.7.5.3 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 wetting agent with HLB(hydrophilic-lipophilic balance) value of approximately 13.Prepare solution containin
32、g 0.5 % nonoxynol-10 class ofethoxylated alkyl phenols, for example Tergitol NP-10 orTriton X-100 and 0.5 % Na2CO3.7.7 Neutralizing BrothsGrowth media appropriate for thechallenge microorganism containing chemical agents to sup-press antibacterial activity. Alternatively, the neutralizingbroths may
33、be of sufficient volume to reduce the concentrationof the antimicrobials to below active levels. See step 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 microorganisms,
34、as applicable.7.9 Culture Media:7.9.1 Nutrient Agar A.7.9.2 Nutrient Agar B.7.9.3 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 microorganism(s).7
35、.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).7.10.1 Add 0.
36、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 filter,aliquote a
37、nd store at either 4 6 2C or 20 6 2C for no longerthan 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 materials may vary amon
38、g 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 testing.8. Fabric a
39、nd 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 wetting agent are rem
40、oved (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 secure onto the o
41、uter 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 strip end.Apply additional staples to the 6th
42、and 7th folds along onehorizontal side of the fabric bundle to create “pockets” that willsecure individual fabric swatches during tumbling. Fabricwrapped spindles may be sterilized in individual exposurechambers. Alternatively, fabric wrapped spindles may besterilized separately from exposure chambe
43、rs. 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 approximately 1 by 1.5 in. will be cutfrom the remaining scoured fabric. A nontoxic permanentmarker may be used to place a mark on th
44、e 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 exposure chamber, prepare at least 3 fabriccarriers and 1 fabric wrapped spindle for each active testf
45、ormulation/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 three consecutive transfers
46、prior to use in testing.9.2 On the day prior to testing, transfer the cells into Frenchsquare bottles containing 20 mL Nutrient Agar B. Incubate 18to24hat356 2C, agar side down.9.3 Remove growth from the French square bottles usingthree-mL dilution fluid and five sterile glass beads to suspendgrowth
47、. 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.NOTE 9The initial inoculum concentration for different challengemicroorganisms may vary and should be determined from carrier andwash water nu
48、mbers 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 active test formulationand product control (at least 1 L) according to manufacturerE2406 093instructions, diluted in synthetic water with
49、 appropriate hard-ness and pre-equilibrated to target test temperature.NOTE 10Fabric to wash water ratios based on usage pattern must beconsidered in this step (see DIS/TSS 13).NOTE 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 three hours prior to use and maintainsolution at test temperature. Some active ingredients mayrequire preparation and use in less than three hours.11. Procedure11.1 Inoculate three steril
