1、Designation: E2149 10Standard Test Method forDetermining the Antimicrobial Activity of ImmobilizedAntimicrobial Agents Under Dynamic Contact Conditions1This standard is issued under the fixed designation E2149; the number immediately following the designation indicates the year oforiginal adoption o
2、r, in the case 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-le
3、aching, antimicrobial-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 difficu
4、lties include ensuring contact of inocu-lum to treated surface (as in AATCC 100-2004), flexibility ofretrieval at different contact times, use of inappropriatelyapplied static conditions (as in AATCC 147-2004), sensitivity,and reproducibility.1.2 This test method allows for the ability to evaluate m
5、anydifferent types of 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 wate
6、r,proteins, blood, serum, 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 a leaching antimicrobial is determinedboth pre- and post-test.1.5 This test method shoul
7、d be performed only by thosetrained in microbiological techniques.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard may involve hazardous materials, opera-tions, and equipment. This standard does not purport
8、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 regulatory limitations prior to use.2. Referenced Documents2.1 AATCC Documents:2AATCC 14
9、7-2004 Antibacterial Activity Assessment ofTextile Materials: Parallel Streak MethodAATCC 100-2004 Antibacterial Finishes on Fabrics3. Summary of Test Method3.1 The antimicrobial activity of a substrate-bound, non-leaching antimicrobial agent is dependent upon direct contactof microbes with the acti
10、ve chemical agent. This test deter-mines the antimicrobial activity of a treated specimen byshaking samples of surface-bound materials 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 viabl
11、e organisms from the suspensionis determined and the percent reduction (or log10reduction) iscalculated by comparing retrievals from appropriate controls.4. Significance and Use4.1 Immobilized, as chemically bonded, antimicrobialagents are not free to diffuse into their environment undernormal condi
12、tions of use. Textile test methods, such asAATCC 147-2004, that are directly dependent on the readyleachability of the antimicrobial agent from the treated fabricare inappropriate for evaluating immobilized antimicrobialagents. This test method ensures good contact between thebacteria and the treate
13、d fiber, fabric, or other substrate, byconstant agitation of the test specimen in a challenge suspen-sion during the test period.4.2 The metabolic state of the challenge species can directlyaffect measurements of the effectiveness of particular antimi-crobial agents or concentrations of agents. The
14、susceptibility ofthe species to particular biocides could be altered depending onits life stage (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 time
15、s to reduce the variabilityassociated with growth phase of the microorganism.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 Antimicrobial Agents.Current edition approved May 1, 20
16、10. Published May 2010. Originallyapproved in 2001. Last previous edition approved in 2001 as E2149 01, which waswithdrawn in 2010 and reinstated in May 2010. DOI: 10.1520/E2149-10.2Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park,
17、NC 27709, http:/www.aatcc.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.3 Liquid analysis of antimicrobial activity of non-leachingagents provides the ability to completely wet-out a testsubstrate. With the use of wetting-age
18、nt surfactants, falsenegatives observed when comparing hydrophobic and hydro-philic substrates can be reduced.4.4 This test method is not intended for directly comparingthe activities of leaching and non-leaching antimicrobialagents. In liquid environments, leaching biocides may releasethe active in
19、gredient at differential rates. Furthermore, residualantimicrobial activity of leaching biocides may be present inserial dilution and may exert additional activity after desiredcontact time, unless adequately sequestered at end of test.Controls for both of these factors are not included in this test
20、method; therefore, screening protocols are introduced to iden-tify the presence of leaching biocides.4.5 The test is suitable for evaluating stressed or modifiedspecimens, when accompanied by adequate controls.NOTE 1Stresses may include laundry, wear and abrasion, radiationand steam sterilization, U
21、V exposure, solvent manipulation, temperaturesusceptibility, or similar physical or chemical manipulation.5. Apparatus5.1 Agar bore, 8-mm diameter.5.2 Air displacement pipettes, Eppendorf or equivalent, 100to 1000 L with disposable tips.5.3 Analytical balance, to weigh chemicals and substratesand to
22、 standardize inoculum delivery volumes by pipettes.5.4 Glassware:5.4.1 Contact Flask, 250 mL Erlenmeyer flask, capped,autoclavable.5.4.2 Test tubes,183 150 mm rimless bacteriological testtubes used for growing test organisms and for serial dilution.5.5 Incubator, capable of maintaining a temperature
23、 of 35 62C.5.6 Shaker, wrist action, capable of aggressive agitation ofbacteria and substrate solutions.5.7 Spectrophotometer, capable of measuring an absorbanceof 475 nm.5.8 Sterile serological pipettes, capable of 50 and 10 mLcapacity.5.9 Sterilizer, any suitable steam sterilizer producing thecond
24、itions of sterility.5.10 Vortex mixer, to vortex dilution tubes during serialdilutions.5.11 Water bath, for short term storage of liquefied agarmedia, capable of maintaining 45 to 50C.6. Reagents6.1 Buffer SolutionThe following solution is preparedfrom reagent-grade chemicals. For buffer stock solut
25、ion(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 to 1000 mL; transfer to a flask and store at 4
26、C.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 atroom temperature.6.2 Media:6.2.1 Tryptic Soy Bro
27、th, prepared according to manufactur-ers directions.6.2.2 Plate Count Agar, prepared according to manufactur-ers directions.6.3 Wetting Agent SurfactantAgents must be shown byprior testing at the intended use concentration not to cause areduction or increase in bacterial numbers. DC Q2-52113at0.01 %
28、 final dilution of working buffer solution has beenshown to be effective.7. Test Organism7.1 Escherichia coli, American Type Culture Collection No.25922.7.1.1 Cultures of the test organism should be maintainedaccording to good microbiological practice and checked forpurity on a routine basis. Consis
29、tent 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 culture purity bymaking streak plates periodically,
30、 observing for colonies char-acteristic of Escherichia coli, and Gram-staining.NOTE 2Original method, ASTM E2149-01, specified Klebsiellapneumoniae as test organism. Escherichia coli is used in this test methodas it is easier to handle and is a more universally accepted test typeorganism.8. Paramete
31、rs8.1 Surface 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.8.2 The weight, size, and material of construction of speci-
32、men must be specified.8.3 Specimens should be prepared such that they canmaximize agitation and are reflective of a recordable ratio ofsurface area to test titer.9. Preparation of Bacterial Inoculum9.1 Grow a fresh 18 h shake culture of Escherichia coli insterile Tryptic Soy Broth at 35 6 2C prior t
33、o performing thetest.9.2 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 3 108CFU/mL. Dilute appropriately into sterile buffersolution to obtain a final concentrati
34、on of 1.5-3.0 3 105CFU/mL. This solution will be the working bacterial dilution.3The sole source of supply of the apparatus known to the committee at this timeis Dow Corning, Midland, MI. If you are aware of alternative suppliers, pleaseprovide this information to ASTM International Headquarters. Yo
35、ur comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.E2149 10210. Test Specimen10.1 Preparation of Test Specimen:10.1.1 Fabric and PaperSamples are selected on weightbasis and weighed to 1.0 6 0.1 g.10.1.2 Powder and Granular Materia
36、lWeigh to 1.0 60.1 g. The material must settle after shaking so that nospecimen interferes with the retrieval and counting techniques.10.1.3 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
37、treated surfacearea. Specimen may also be selected on weight 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
38、, it can be mounted asa seal for the test container so that only the treated surface is indirect contact with the inoculum.NOTE 3Solids anticipated in this part of the method are plastics, glassbeads or chips, ceramics, metal chips, or similar hard surfaces.11. Procedure for Determining Antimicrobia
39、l Activity11.1 Prepare the specimen to be tested as described inSection 10. One treated piece of each specimen is required.One untreated piece of each specimen of identical compositionis highly recommended for each series of specimen tested.11.2 Prepare one sterile 250 mL screw-cap Erlenmeyer flaskf
40、or 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 9.2 to eachflask.11.3 Determine bacterial concentration of solution at the “0”time by performing serial dilutions and standard plate co
41、unttechniques from the “inoculum only” sample flask11.4 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 the first and last serialdilution.11.5 Place the series of flasks on the wrist-actio
42、n 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 (11.3).NOTE 4Residual bacterial retention in/on specimen could be testedusing appropriate retrieval techniques such as agar imprint tests o
43、r bufferextraction and plate count.NOTE 5Filter 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 must be treated in thesame manner.11.6 Allow all the Petri dishes from both subsets
44、 to incubatefor at 35 6 2C for 24 h.11.7 Count the colonies in Petri dish. Record the values,average the triplicate Petri dish numbers and convert theaverage to colony-forming units per millilitre (CFU/mL).NOTE 6The presence of the original test organism may be confirmedby Gram stains and colony mor
45、phology.NOTE 7When the number of colony-forming units per mL is less than30 on the lowest dilution plate, report the recovered CFU/mL as “30”,determine the percent reduction and report the reduction as “greater than”the percent found.11.8 Calculate percent or log bacterial reduction.11.8.1 If the av
46、erage CFU/mL values for the untreatedcontrol and the “inoculum only” flask agree within 15 % afterspecified contact time, or if an untreated control is notavailable, calculate percent reduction of the organisms result-ing from treated sample (A) directly compared to “inoculumonly” flask after specif
47、ied contact time (B) using the followingformula. Results can be presented in either percent reductionwhen measuring CFU/mL or as Log10bacterial reduction whencalculating mean log10density of bacteria.Reduction, % CFU/mL!5B AB3 100Log10bacteria reduction 5 Log10B! Log10A!where:A = CFU per millilitre
48、for the flask containing the treatedsubstrate after the specified contact time, andB = CFU per millilitre for the “inoculum only” flask afterthe specified contact time.11.8.2 If the untreated control (if present) and the “inoculumonly” flask do not agree within 15 %, calculate the percentreduction o
49、f organisms from treated sample (A) directly com-pared to the untreated control (C).Reduction, % CFU/mL!5C AC3 100Log10bacteria reduction 5 Log10C! Log10A!where:A = CFU per millilitre for the flask containing the treatedsubstrate after the specified contact time, andC = CFU per millilitre for the flask containing the untreatedsubstrate after the specified contact time.11.9 Record and report the value to the nearest one-hundredth percent or log10reduction of bacteria. Whetherreduction calculations are based on values from an untreatedcontrol or inocul
