1、Designation: E 2149 01Standard Test Method forDetermining the Antimicrobial Activity of ImmobilizedAntimicrobial Agents Under Dynamic Contact Conditions1This standard is issued under the fixed designation E 2149; the number immediately following the designation indicates the year oforiginal adoption
2、 or, in the case of revision, the year of last revision. 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 the resistance ofnon-leaching anti
3、microbial treated specimens to the growth ofmicrobes under dynamic contact conditions. This dynamicshake flask test was developed for routine quality control andscreening tests in order to overcome difficulties in usingclassical antimicrobial test methods to evaluate substrate-bound antimicrobials.
4、These difficulties include ensuring con-tact of inoculum to treated surface (as in AATCC 100),flexibility of retrieval at different contact times, use of inap-propriately applied static conditions (as in AATCC 147),sensitivity, and reproducibility. This test also allows for theversatility of testing
5、 contamination due to such things as hardwater, proteins, blood, serum, various chemicals, and othercontaminates or physical/chemical stresses or manipulations ofthe specimens of interest.1.2 Surface antimicrobial activity is determined by compar-ing results from the test sample to simultaneously ru
6、n controls.1.3 The presence of a leaching antimicrobial is both pre-and post-determined by the presence of a zone of inhibition.1.4 This test method should be performed only by thosetrained in microbiological techniques.1.5 This standard may involve hazardous materials, opera-tions, and equipment. T
7、his 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 regulatory limitations prior to use.2. Referenced Document
8、s2.1 ASTM Standards:E 1054 Practices for Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products2.2 Other Documents:AATCC Test Method 147-1998 Antibacterial Activity As-sessment of Textile Materials: Parallel Streak Method.American Associati
9、on of Textile Chemists and Colorists,RTP, NCAATCC Test Method 100-1999 Antibacterial Finishes onFabrics, Evaluation of American Association of TextileChemists and Colorists, RTP, NC3. Summary of Test Method3.1 Immobilized antimicrobial agents, such as surfacebonded materials, are not free to diffuse
10、 into their environmentunder normal conditions of use. Test methods such as AATCC147 that are directly dependent on the ready leachability of theantimicrobial agent from the treated fabric are inappropriate forevaluating immobilized antimicrobial agents. The followingtest method ensures good contact
11、 between the bacteria and thetreated fiber, fabric, or other substrate by constant agitation ofthe test specimen in a bacterial suspension during the testperiod. The test is suitable for evaluating stressed or modifiedspecimens when accompanied by adequate controls.NOTE 1Stresses may include laundry
12、, wear and abrasion, radiationand steam sterilization, UV exposure, solvent manipulation, temperaturesusceptibility, or similar physical or chemical manipulation.4. Significance and Use4.1 The antimicrobial activity of a substrate-bound antimi-crobial is dependent upon direct contact of microbes wit
13、h theactive chemical agent. This test determines the antimicrobialactivity of treated specimen by shaking samples of surfacebound materials in a concentrated bacterial suspension for aone hour contact time or other contact times as specified by theinvestigator. The suspension is serially diluted bot
14、h . beforeand after contact and cultured. The number of viable organismsin the suspension is determined and the percent reduction iscalculated based on initial counts or on retrievals from appro-priate untreated controls.NOTE 2This method is intended for those surfaces having a percentreduction acti
15、vity of 50 % to 100 % for the specified contact time.5. Apparatus5.1 Sterilizer,5.2 Incubator,1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntimicrobialAgents.Current edition approved June 10, 2001. Publishe
16、d August 2001.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.3 Spectrophotometer,5.4 Shaker, Wrist ActionA Wrist Action Shaker is recom-mended but other means of agitation such as reciprocal actionshakers may be satisfactory for r
17、outine testing. Shaker mustensure good agitation. Rotary shakers are unacceptable.5.5 Water Bath,5.6 Vortex Mixer,5.7 Glassware,5.7.1 Contact Flask, 250 ml Erlenmeyer flask, capped,autoclavable.5.7.2 Dilution Vessels,5.7.3 Pipettes,5.8 Agar, bore 8-mm diameter.6. Reagents6.1 Buffer SolutionFor test
18、specimen which might alterthe pH of the system, Sorensens Phosphate Buffer (pH 6.8) isrecommended.2Other appropriate buffers must be shown not tocause a reduction or increase in bacterial numbers by priortesting at the intended use concentration. For all other samples,the following solution is recom
19、mended and is prepared fromreagent grade chemicals. For buffer stock solution (0.25MKH2PO4): Prepare a fresh solution at least once every 6 monthsas follows: Weigh 34 6 0.1 g of potassium dihydrogenphosphate into a 100 ml beaker. Add 500 ml of distilled water.Adjust pH to 7.2 6 0.1 with a dilute sol
20、ution of NaOH. Diluteto 1000 ml; transfer to a flask and store at 4C. For workingbuffer solution (0.3mM KH2PO4): Prepare a fresh solution atleast once every 2 months as follows: Transfer 1 ml 6 0.01 mlof stock buffer solution, with a sterile pipette to flask contain-ing 800 ml of distilled water. Ca
21、p and sterilize.6.2 Media:6.2.1 Nutrient Broth (Difco Laboratories, Detroit, MI orequivalent) or media appropriate of organism selected.6.2.2 Tryptone Glucose Extract Agar (Difco Laboratories,Detroit, MI or equivalent) or media appropriate for organismselected.6.3 Wetting Agent SurfactantAgents must
22、 be shown not tocause a reduction or increase in bacterial numbers by priortesting at the intended use concentration.NOTE 3Dow Corning, Midland, MI Q2-5211 at 0.01 % final dilutionor equivalent can be used.7. Test Organism7.1 Klebsiella pneumoniae, American Type Culture Collec-tion No. 4352. Other o
23、rganisms may be used at the discretionof the investigator.7.1.1 Cultures of the test organism should be maintainedaccording to good microbiological practice and checked forpurity, on a routine basis. Consistent and accurate testingrequires maintenance of a pure, uncontaminated test culture.Avoid con
24、tamination 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 and observing for a singlespecies characteristic type of colonies.NOTE 4A glossiness in the br
25、oth culture of Klebsiella pneumoniae isa sign of reversion and the culture should not be used.8. Parameters8.1 Aerobic organisms and/or contact times used must bespecified.8.2 Surface preparation or conditioning must be specified.Prior manipulation of the specimen in order to demonstratemaximum acti
26、vity in desired time frame must be recorded andcompared to identically handled controls.8.3 The weight, size, and material of construction of speci-men must be specified.8.4 Specimens should be prepared such that they canmaximize agitation and are reflective of a recordable ratio ofsurface area to t
27、est titer.8.5 Wetting agent surfactants must be used with highlyhydrophobic specimen.9. Preparation of Bacterial Inoculum9.1 Grow a fresh 18 hour shake culture of KlebsiellapneumoniaeKlebsiella pneumoniae in sterile nutrient broth foreach series of samples. If other organisms are specified, theyshou
28、ld be prepared in the same manner, unless other media anddifferent calibration techniques are specified.9.2 Dilute the culture with the sterile buffer solution untilthe solution has an absorbance of 0.28 6 0.01 at 475 nm, asmeasured spectrophotometrically. This has a concentration of1.5-3.0 3 108CFU
29、/ml. Dilute appropriately into sterile buffersolution to obtain a final concentration of 1.5-3.0 3 105CFU/ml. This solution will be the working bacterial dilution.NOTE 5For other organisms, adjust final concentration to 1.5-3.0 3105CFU/ml by appropriate methods.10. Test Specimen10.1 Preparation of T
30、est Specimen:10.1.1 Fabric and PaperSamples are selected on weightbasis at the discretion of the investigator and weighed to 6 0.1g.NOTE 6Weight, usually between 0.5 and 2.0 g, must ensure strongagitation during contact period. Specimen should be cut into small enoughportions to ensure maximum agita
31、tion and must be identical in sizebetween treated and untreated controls. Clumping of specimen negativelyaffects reproducibility.10.1.2 Powder and Granular MaterialWeigh to 6 0.1 g.The material must settle after shaking so that no specimeninterferes with the retrieval and counting techniques.10.1.3
32、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 9 sq. in. (58 cm2) of treated surfacearea. Specimen may also be selected on weight basis at thediscretion of the investigator, weigh to 6 0.1 g. Care must beexer
33、cised during shaking not to break the flask or bottle. Theuntreated specimen of the solid must not absorb the solution.The test specimen may be mounted as a seal for the testcontainer so that only the treated surface is in direct contactwith the inoculum.2Clinical Chemistry: Principle and Technics,
34、Second Edition 1974, Table A-3e,p 1592.E2149012NOTE 7Solids anticipated in this part of the method are plastics, glassbeads or chips, ceramics, metal chips, or similar hard surfaces.11. Procedure for Determining Antimicrobial Activity11.1 Prepare the antimicrobial bonded surface specimen tobe tested
35、 as in Section 10. One treated piece of each specimenis required. One untreated piece of each specimen of identicalcomposition is required for each series of specimen tested.11.2 Prepare one sterile 250 ml screw-cap Erlenmeyer flaskfor each treated and untreated specimen in addition to one“inoculum
36、only” sample. If a series is being run, prepare oneflask for each specimen and one for each type of specimen asa control. Add 50 6 0.1 ml of working dilution of bacterialinoculum prepared in 9.2 to each flask.11.3 Cap the flasks and place them on a wrist-action shaker.Shake them at maximum speed for
37、 1 min. 6 5 sec. Each flaskis considered to be a “0” contact time. Determine bacterialconcentration of solution at the “0” time by performing serialdilutions and standard plate count techniques. No series of testflasks should be large enough to require more that 5 minutesbetween the first and last s
38、erial dilution after contact.NOTE 8For extra assurance against effects of solution retained anti-microbial, neutralizer containing dilution buffer can be used. See E 1054.11.4 As soon as the “0” contact time sub-samples have beenprepared, place the test and control specimen in their individualflasks
39、. Recap the flasks and place them on the wrist-actionshaker. Shake at maximum stroke for 1 hr. 6 5 min. unless adifferent contact time is specified. Immediately transfer 1 60.01 ml from each flask to a test tube, serial dilute and plate outin duplicate as was done for the “0” contact time subgroup.N
40、OTE 9The investigator may run the actual shake flask portion of theprocedure at any desired temperature consistent with the test organism,and/or reflective of the temperatures anticipated for the end-use of thespecimen, and good microbiological procedures.NOTE 10Residual bacterial retention in/on sp
41、ecimen could be testedusing appropriate retrieval techniques such as agar imprint tests or bufferextraction and plate count.11.5 Allow all the petri dishes from both subsets to incubatefor 24 to 48 hrs.11.6 Count the colonies in petri dish. Record the values,average the duplicate petri dish numbers
42、and convert theaverage to colony forming units per milliliter (CFU/ml) ofbuffer solution in the flask. If the duplicate counts of anysample do not agree within 15 %, discard that sample andrepeat the test.NOTE 11The presence of the original test organism may be confirmedby Gram stains and colony mor
43、phology.11.7 Calculate the percent reduction of the organisms result-ing from contact with the specimen using the followingformula. Results can be presented in either percent reductionwhen measuring CFU/ml or as a death rate constant whencalculating mean log10density of bacteria.Reduction, % CFU/ml!
44、5B AB3 100Death Rate Constant mean log10density!5B Awhere:A = CFU per milliliter (or mean log10density of bacteria)for the flask containing the treated substrate after thespecified contact time, andB = “0” contact time CFU per milliliter (or mean log10density of bacteria) for the flask used to deter
45、mine “A”before the addition of the treated substrate.11.8 The counts for the flask containing the inoculum onlycontrol after specified contact time (C) and counts for the flaskcontaining the untreated control after specified contact time (D)should be within 15 %. If they are not, calculate the perce
46、ntreduction of organisms from treated sample (A) directlycompared to the untreated control (D). Repeat above formulareplacing D for B and report accordingly.where:C = CFU per milliliter (or mean log10density of bacteria)for the flask containing the inoculum only control afterspecified contact time,
47、andD = CFU per milliliter (or mean log10density of bacteria)for the flask containing the untreated substrate after thespecified contact time.NOTE 12If no untreated fabric control is available and the counts forthe flask containing the inoculum only control after specified contact time(C) are not wit
48、hin 15 % of original count, the test must be repeated.11.9 Record and report the value to the nearest half percentor one-tenth mean log10density of bacteria if a specific deathrate constant is desired.12. Procedure for Determining Presence of LeachingAntimicrobial12.1 Analysis of specimen (Pre-Test)
49、:12.1.1 Measure presence or absence of zone of inhibitionusing AATCC 147-1998.12.2 Analysis of Supernatant (Post-Test):12.2.1 Inoculate Tryptone Glucose Extract agar plate oragar appropriate for the test organism with confluent lawn oforganisms (1 3 105CFU/ml) and allow to dry.12.2.2 Bore 8 mm diameter hole in center of inoculated agarplate and remove plug.12.2.3 Prepare the specimen to be tested as in Section 10.One treated and one untreated piece of each specimen isrequired.12.2.4 Prepare two sterile 250 ml flasks containing 50 mlsterile buffer solution.12.2.5
