1、Designation: E2149 13aStandard Test Method forDetermining the Antimicrobial Activity of AntimicrobialAgents Under Dynamic Contact Conditions1This standard is issued under the fixed designation E2149; 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 the antimicro-bial activity of non-leaching, ant
3、imicrobial-treated specimensunder dynamic contact conditions. This dynamic shake flasktest was developed for routine quality control and screeningtests in order to overcome difficulties in using classicalantimicrobial test methods to evaluate substrate-bound antimi-crobials. These difficulties inclu
4、de ensuring contact of inocu-lum to treated surface (as in AATCC 100), flexibility ofretrieval at different contact times, use of inappropriatelyapplied static conditions (as in AATCC 147), sensitivity, andreproducibility.1.2 This test method allows for the ability to evaluate manydifferent types of
5、 treated substrates and a wide range ofmicroorganisms. Treated substrates used in this test methodcan be subjected to a wide variety of physical/chemical stressesor manipulations and allows for the versatility of testing theeffect of contamination due to such things as hard water,proteins, blood, se
6、rum, various chemicals, and other contami-nants.1.3 Surface antimicrobial activity is determined by compar-ing results from the test sample to controls run simultaneously.1.4 The presence of an antimicrobial that requires neutral-ization is determined by the post-test.1.5 Proper neutralization of al
7、l antimicrobials must beconfirmed using Test Methods E1054.1.6 This test method should be performed only by thosetrained in microbiological techniques.1.7 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 This standard may inv
8、olve hazardous materials,operations, and equipment. This standard does not purport toaddress all of the safety concerns, if any, associated with itsuse. It is the responsibility of the user of this standard toestablish appropriate safety and health practices and deter-mine the applicability of regul
9、atory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Anti-microbial Agents2.2 AATCC Documents:3AATCC 147 Antibacterial Activity Assessment of TextileMaterials: Parallel Streak MethodAATCC 100 Antibacterial Finishes on Fabrics3
10、. Summary of Test Method3.1 The antimicrobial activity of a substrate-bound, non-leaching antimicrobial agent is dependent upon direct contactof microbes with the active chemical agent. This test deter-mines the antimicrobial activity of a treated specimen byshaking samples of surface-bound material
11、s in a concentratedbacterial suspension for a one hour contact time. The suspen-sion is serially diluted both before and after contact andcultured. The number of viable organisms from the suspensionis determined and the percent reduction (or log10reduction) iscalculated by comparing retrievals from
12、appropriate controls.4. Significance and Use4.1 Chemically bonded, antimicrobial agents are not free todiffuse into their environment under normal conditions of use.This test method ensures good contact between the bacteria andthe treated fiber, fabric, or other substrate, by constant agitationof th
13、e test specimen in a challenge suspension during the testperiod.1This test method is 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 Oct.
14、1, 2013. Published December 2013. Originallyapproved in 2001. Last previous edition approved in 2013 as E2149 13. DOI:10.1520/E2149-13A.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume in
15、formation, 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Consh
16、ohocken, PA 19428-2959. United States14.2 The metabolic state of the challenge species can directlyaffect measurements of the effectiveness of particular antimi-crobial agents or concentrations of agents. The susceptibility ofthe species to particular biocides could be altered depending onits life s
17、tage (cycle). One-hour contact time in a buffer solutionallows for metabolic stasis in the population. This test methodstandardizes both the growth conditions of the challengespecies and substrate contact times to reduce the variabilityassociated with growth phase of the microorganism.4.3 Leaching o
18、f an antimicrobial is dependent upon the testconditions being utilized and the ultimate end use of theproduct. For example, water soluble antimicrobials will beprone to removal from the test surface using the methoddescribed in Section 13 but insoluble compounds will not. It isfor this reason that t
19、he use of the term leaching throughout thisdocument is limited to only the testing conditions describedherein. To determine if a compound is immobilized in allconditions or during the end use of the product additionaltesting may be required.4.4 This test method cannot determine if a compound isleach
20、ing into solution or is immobilized on the substrate. Thistest method is only intended to determine efficacy as describedin 4.5 and subsequent portions of the method.4.5 This test method is intended to evaluate antimicrobialagents that are not removed from the surface by the aqueoustesting condition
21、s, as evaluated by Section 13. If an antimicro-bial agent that is shown to be removed from the surface bySection 13 is utilized in this test methodology, controls must beincluded such that appropriate neutralization steps are includ-ing during recovery and enumeration.4.6 The test is suitable for ev
22、aluating stressed or modifiedspecimens, when accompanied by adequate controls.NOTE 1Stresses may include laundry, wear and abrasion, radiationand steam sterilization, UV exposure, solvent manipulation, temperaturesusceptibility, or similar physical or chemical manipulation.5. Definitions5.1 Immobili
23、zed: The antimicrobial remains on the surfaceof the article throughout the test as determined by the absenceof bactericidal activity in Section 13. A neutralizer does notneed to be included for this type of antimicrobial5.2 Leaching: Removal of the antimicrobial from the sur-face by the test conditi
24、ons being utilized, resulting in aconcentration high enough to cause bactericidal activity asdefined in Section 13. A valid neutralizer must be utilized forthis type of antimicrobial6. Apparatus6.1 Air displacement pipettes, Eppendorf or equivalent, 100to 1000 L with disposable tips.6.2 Analytical b
25、alance, to weigh chemicals and substratesand to standardize inoculum delivery volumes by pipettes.6.3 Glassware:6.3.1 Contact Flask, 250 mL Erlenmeyer flask, capped,autoclavable.6.3.2 Test tubes, 18 150 mm rimless bacteriological testtubes used for growing test organisms and for serial dilution.6.4
26、Incubator, capable of maintaining a temperature of 35 62C.6.5 Shaker, wrist action, capable of aggressive agitation ofbacteria and substrate solutions.6.6 Spectrophotometer, capable of measuring an absorbanceof 475 nm.6.7 Sterile serological pipettes, capable of 50 and 10 mLcapacity.6.8 Sterilizer,
27、any suitable steam sterilizer producing theconditions of sterility.6.9 Vortex mixer, to vortex dilution tubes during serialdilutions.6.10 Water bath, for short term storage of liquefied agarmedia, capable of maintaining 45 to 50C.7. Reagents7.1 Buffer SolutionThe following solution is preparedfrom r
28、eagent-grade chemicals. For buffer stock solution(0.25M KH2PO4): Prepare a fresh stock solution at least onceevery 6 months as follows: Weigh 34 6 0.1 g of potassiumdihydrogen phosphate into a 1000 mL beaker. Add 500 mL ofdistilled water.Adjust pH to 7.2 6 0.1 with a dilute solution ofNaOH. Dilute t
29、o 1000 mL; transfer to a flask and store at 4C.For working buffer solution (0.3mM KH2PO4): Prepare a freshsolution at least once every 2 months as follows: Transfer 1 60.01 mL of stock buffer solution with a sterile pipette to flaskcontaining 800 mL of distilled water. Cap, sterilize and store atroo
30、m temperature.7.2 Media:7.2.1 Tryptic Soy Broth, prepared according to manufactur-ers directions.7.2.2 Plate Count Agar, prepared according to manufactur-ers directions.7.3 Wetting Agent SurfactantAgents must be shown byprior testing at the intended use concentration not to cause areduction or incre
31、ase in bacterial numbers. DC Q2-52114at0.01 % final dilution of working buffer solution has beenshown to be effective.8. Test Organism8.1 Escherichia coli, American Type Culture Collection No.25922.8.1.1 Cultures of the test organism should be maintainedaccording to good microbiological practice and
32、 checked forpurity on a routine basis. Consistent and accurate testingrequires maintenance of a pure, uncontaminated test culture.Avoid contamination by use of good sterile technique in platingand transferring. Avoid mutation or reversion by strict adher-ence to monthly stock transfers. Check cultur
33、e purity by4The sole supplier of DC Q2-5211 known to the committee at this time is DowCorning, Midland, MI. If you are aware of alternative suppliers, please provide thisinformation to ASTM International Headquarters. Your comments will receivecareful consideration at a meeting of the responsible te
34、chnical committee,1whichyou may attend.E2149 13a2making streak plates periodically, observing for colonies char-acteristic of Escherichia coli, and Gram-staining.8.1.2 Alternative organisms can be substituted depending onthe end use of the product. However, the precision and biasstatement has been d
35、eveloped using Escherichia coli ATCC25922. There is are no data to support a precision and biasstatement for other organisms at this point. Use of alternateorganisms shall be included in the report, in addition to anyother modification of media, buffer, bacterial concentration,etc.9. Parameters9.1 S
36、urface preparation or conditioning must be specified.Prior manipulation of the specimen may be required in order todemonstrate maximum activity in a desired time frame andmust be reported and compared to identically handled controls.9.2 The weight, size, and material of construction of speci-men mus
37、t be specified.9.3 Specimens should be prepared such that they canmaximize agitation and are reflective of a recordable ratio ofsurface area to test titer.10. Preparation of Bacterial Inoculum10.1 Grow a fresh 18 h shake culture of Escherichia coli insterile Tryptic Soy Broth at 35 6 2C prior to per
38、forming thetest.10.2 Dilute the culture with the sterile buffer solution untilthe solution has an absorbance of 0.28 6 0.02 at 475 nm, asmeasured spectrophotometrically. This has a concentration of1.5-3.0 108CFU/mL. Dilute appropriately into sterile buffersolution to obtain a final concentration of
39、1.5-3.0 105CFU/mL. This solution will be the working bacterial dilution.11. Test Specimen11.1 Preparation of Test Specimen:11.1.1 Fabric and PaperSamples are selected on weightbasis and weighed to 1.0 6 0.1 g.11.1.2 Powder and Granular MaterialWeigh to 1.0 60.1 g. The material must settle after shak
40、ing so that nospecimen interferes with the retrieval and counting techniques.11.1.3 Other Solids (Surface Treatment)Reduce the solidin size to fit into the flask or use a sterile wide-mouth bottle.Use a specimen that gives 4 in.2(25.8 cm2) of treated surfacearea. Specimen may also be selected on wei
41、ght basis, 60.1 g,at the discretion of the investigator. Care must be exercisedduring shaking not to break the flask or bottle. The untreatedspecimen of the solid must not absorb the solution. If appro-priate to the nature of the test specimen, it can be mounted asa seal for the test container so th
42、at only the treated surface is indirect contact with the inoculum.NOTE 2Solids anticipated in this part of the method are plastics, glassbeads or chips, ceramics, metal chips, or similar hard surfaces. Samplemass can vary from 0.5g-2.0g depending on sample composition.However, the precision and bias
43、 statement has been developed using a onegram sample of a textile material only. There are no data to support aprecision and bias statement for other sample masses or sample types atthis point. Include in the report the use of alternate sample mass or type,in addition to any other modifications of t
44、emperature, method of dynamiccontact, etc.12. Procedure for Determining Antimicrobial Activity12.1 Prepare the specimen to be tested as described inSection 11. One treated piece of each specimen is required.One untreated piece of each specimen of identical compositionis highly recommended for each s
45、eries of specimen tested.12.2 Prepare one sterile 250 mLscrew-cap Erlenmeyer flaskfor each treated and untreated specimen, and one “inoculumonly” sample for the series being run. Add 50 6 0.5 mL ofworking dilution of bacterial inoculum prepared in 10.2 to eachflask.12.3 Determine bacterial concentra
46、tion of solution at the“0” time by performing serial dilutions and standard platecount techniques from the “inoculum only” sample flask12.4 Place the test and control specimen in their individualflasks. No series of test flasks should be large enough to requiremore than 5 min, post-contact, between
47、the first and last serialdilution.12.5 Place the series of flasks on the wrist-action shaker.Shake at maximum stroke for 1 h 6 5 min. Immediately serialdilute and plate each sample out in triplicate, as was done forthe “0” contact time subgroup (12.3).NOTE 3Residual bacterial retention in/on specime
48、n could be testedusing appropriate retrieval techniques such as agar imprint tests or bufferextraction and plate count.NOTE 4Filter solutions in which samples have degraded duringshaking. Whatman filter paper Type 1 has been found to be appropriate forthis step. Contents of the “inoculum only” flask
49、 must be treated in thesame manner.12.5.1 Alternative contact times can be used depending onthe end use of the product. However, the precision and biasstatement has been developed using a one hour contact time.There are no data to support a precision and bias statement forother contact times at this point. Use of alternate contact timesshall be included in the report, in addition to any othermodifications of temperature, method of dynamic contact, etc.12.6 Allow all the Petri dishes from both subsets to incubatefor at 35 6 2C for 24 h.12.7 Count the colonies
