1、Designation: E3151 18Standard Test Method forDetermining Antimicrobial Activity and Biofilm ResistanceProperties of Tube, Yarn, or Fiber Specimens1This standard is issued under the fixed designation E3151; the number immediately following the designation indicates the year oforiginal adoption or, in
2、 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 as an in vitro, quantitativeassay to evaluate the anti
3、microbial activity of specimens withtubular geometries or small segments of yarn or fibers that havebeen treated with an antimicrobial agent. Further, the methodwas designed to provide a quantitative assessment of a speci-mens ability to resist microbial colonization and subsequentbiofilm formation
4、relative to an untreated control specimen.1.1.1 The difference in number between the planktonicmicrobial population recovered from the treated test specimenand the population recovered from the control test specimen isthe measure of the antimicrobial activity.1.1.2 The measure of the ability of the
5、treated test specimento resist biofilm development is the difference between theadherent microbial population recovered from the treated testspecimen and the adherent microbial population recoveredfrom the control test specimen.1.2 Testing is to be performed by individuals trained inmicrobiological
6、techniques under appropriately controlled con-ditions to ensure the integrity of results and personnel safety.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard may involve hazardous materials,operations, and
7、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, health, and environmental prac-tices and determine the applicability of regulatory limitationsprior to
8、 use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarrie
9、rs to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E177 Practice for Use of the Terms Precision 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 Anti-mi
10、crobial AgentsE2756 Terminology Relating to Antimicrobial and AntiviralAgents3. Terminology3.1 For definitions of terms used in this test method, refer toTerminology E2756.4. Summary of Test Method4.1 The control and treated test specimens are placed intoindividual650mmculture tubes containing suspe
11、nsions ofa known biofilm-producing strain of Staphylococcus epider-midis (ATCC 359843) at a specific titer and incubated at 35 62 C with mild agitation for 24 6 2h.4.2 After the contact time, each specimen is transferred toan individual centrifuge tube containing a sterile bufferedsaline rinse solut
12、ion, and the tube is sealed and carefullyinverted several times to remove any non-adhered or looselyadhered bacteria.4.3 The specimens are then transferred to new centrifugetubes containing low concentrations of a surfactant dispersed ina neutralizing agent demonstrated to deactivate the antimicro-b
13、ial agent with which the test specimen is treated.4.4 These tubes are sealed, vortexed, and sonicated tosuspend any bacteria adhered to the surface of the specimensand to disaggregate any biofilm clumps present.4.5 The population of planktonic bacteria within the testinoculum exposed to each test sp
14、ecimen and the re-suspended1This 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 Feb. 1, 2018. Published May 2018. DOI: 10.
15、1520/E3151-18.2For 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.3ATCC is a registered trademark and ATCC 35984
16、is a trademark of AmericanType Culture Collection, Manassas, VA.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization estab
17、lished in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1adherent bacteria harvested from the surface of each testspecimen are enumerated using standard microbiol
18、ogical tech-niques.4.6 The efficacy of the antimicrobial treatment versus theplanktonic bacteria recovered in the neutralized inocula sus-pension and the adherent bacteria recovered from the surface ofthe specimens is the percent and log10reductions calculated asthe difference between populations fr
19、om treated specimens andthose from the controls.5. Significance and Use5.1 Although a number of standardized tests currently existfor assessing the antimicrobial activity of treated polymers andtextiles, these are optimized for specimens that readily absorbthe test inoculum or that have a flat surfa
20、ce on which theinoculum can be placed, and their use for specimens withtubular geometries or for small quantities (less than 0.5 g) ofyarns or fibers requires significant manipulation of the speci-men.5.2 To adapt these methods for evaluating tubes, fiber, andyarn specimens requires distorting tubul
21、ar specimens to createa flat surface or using unacceptably large quantities of fiber oryarn specimens. Rendering a test specimen having tubulargeometry to a flat surface will limit its surface area availablefor exposure during the test and may require dissection of thespecimen, which unacceptably al
22、ters it from its original state.Testing of treated fiber and yarn specimens using availablestandardized methods typically requires large quantities ofmaterial (greater than 0.5 g) that may not be available. In bothcases, such manipulations may result in misleading results thatdo not reflect the anti
23、microbial efficacy of an unmodifiedspecimen.5.3 This method provides an environment in which theinoculum remains in intimate contact with the surfaces of thesetypes of test specimens, exposing both the intra- and extralu-minal surfaces of tubular specimens without significantmodification, and requir
24、ing only small quantities of fibers oryarns to perform testing.5.4 Classical antimicrobial test methods generally quantifythe population or concentration of microorganisms that surviveexposure to specimens treated with an antimicrobial agentwithout distinguishing whether the surviving microorganisms
25、were in a planktonic or adhered/biofilm state.5.4.1 The phenotypic behavior of bacteria in the biofilmstate differs substantially from when they are in the planktonicstate, especially with respect to susceptibility to disinfectants,sanitizers, and antimicrobial agents. Therefore, evaluating theabili
26、ty of a materials surface to resist bacterial colonizationmay be of equal or greater significance than its efficacy versusplanktonic bacteria.5.4.2 This method not only can assess the population of thechallenge species that survives planktonic exposure to the testspecimen, but also can then compare
27、that to the population thatsurvives in an adherent/biofilm state.5.5 This test method is a batch-based system in which testspecimens are exposed to a continuous, minimal fluid shearenvironment in the presence of the challenge inoculum. Theappropriateness of this simulated environment relative to the
28、intended end-use of the test material should be evaluated priorto testing.5.6 Although this method is designed to provide an initialassessment of the antimicrobial activity exhibited by a materialand its ability to resist microbial colonization under veryspecific test parameters, these conditions ma
29、y not be represen-tative of all environments to which the specimen may beexposed during its intended end-use. Various test parametersspecified in this method can be modified to evaluate a materialunder conditions that may better simulate end-useenvironments, but such alterations of the method must b
30、eclearly described when reporting results.6. Apparatus6.1 Autoclave (steam sterilizer), any suitable for processingculture media, reagents, and labware.6.2 Biological safety cabinet.6.3 Incubator, any capable of maintaining a temperature of35 6 2 C.6.4 pH meter, any capable of measuring to 0.2 units
31、.6.5 Vortex mixer.6.6 Orbital shaker, any capable of maintaining 100 rotationsper minute (rpm).6.7 Refrigerator, any capable of maintaining 4 6 2 C forstorage of media, culture plates, and reagents.6.8 Ultrasonic cleaner, any capable of a watt density outputof 100 to 133 watts per gallon at a freque
32、ncy of 42 kHz 6 6%.6.9 Timer (stopwatch), one that displays hours, minutes, andseconds.6.10 Dissecting forceps, fine tip.6.11 Scissors or Razor blade.6.12 Pipette pumps, 1- to 10-mL and 1- to 25-mL capacity.6.13 Serological pipettes, sterile reusable or single-use pi-pettes of 10.0- and 25-mL capaci
33、ty.6.14 Pipette and appropriate sterile pipette tips, variablevolume, positive displacement, 20- to 200-L volume range.6.15 Pipette and appropriate sterile pipette tips, variablevolume, positive displacement, 100- to 1000-L volume range.6.16 Petri dishes, sterile, 15 100 mm.6.17 Microcentrifuge tube
34、s, sterile, 1.7 mL.6.18 Centrifuge tubes with caps, sterile, 15 mL.6.19 Centrifuge tubes with caps, sterile, 50 mL.6.20 Culture tubes, sterile,650mm.6.21 Culture tubes and closures, sterile, any with a mini-mum volume capacity of 10 mLand a minimum diameter of 16mm. Recommended size is 16 125-mm bor
35、osilicate glass.6.22 Inoculating loops, sterile, 4-mm ring diameter.6.23 Water absorbent laboratory wipe, sterile.6.24 Sealing film, paraffin or equivalent.E3151 1826.25 Dilution vessels.7. Reagents and Materials7.1 Reagent grade chemicals shall be used in all tests.Unless otherwise indicated, it is
36、 intended that all reagents shallconform to the specifications of the committee on AnalyticalReagents of the American Chemical Society, where suchspecifications are available.4Other grades may be used, pro-vided it is pure enough to be used without lessening theaccuracy of the determination.7.2 Grow
37、th Media:7.2.1 Liquid Growth Medium, Tryptic Soy Broth (TSB),sterile (See Annex A1).7.2.2 Solid Growth and Plating Medium, Tryptic Soy Agar(TSA), sterile (See Annex A1).7.2.3 Inoculation Medium, 1/500 Tryptic Soy Broth.7.2.3.1 Aseptically add 1.0 mL of TSB (7.2.1) to 499 mL ofdistilled or deionized
38、water (7.6).7.2.3.2 Adjust the pH to a value between 6.8 and 7.2 usingeither sodium hydroxide or hydrochloric acid.7.2.3.3 Sterilize by autoclaving.7.2.3.4 If not used immediately after preparation, the inocu-lation medium can be stored at 4 6 2 C for no longer than 7days.7.3 Phosphate Buffered Sali
39、ne, sterile.7.4 Neutralizing Solution, appropriate for neutralizing theactive antimicrobial agent in the treated test specimen (SeeTestMethod E1054).7.5 Modified Neutralizing Solution, Neutralizing Solution(7.4) with 1 % by volume Polysorbate 80.7.6 Distilled or Deionized Water, sterile.7.7 Dilution
40、 Fluid or Diluent, sterile water, sterile saline,sterile buffered phosphate diluents or equivalent.8. Test Organism8.1 Staphylococcus epidermidis, American Type CultureCollection (ATCC) No. 35984.38.2 Cultures of the test organism shall be maintained usingappropriate microbiological practices.NOTE 1
41、To ensure the most consistent and accurate results, the purityof the cultures should be checked regularly using standard microbiologicalspeciation techniques.NOTE 2Additional challenge species can be substituted to evaluate thebreadth of a materials antimicrobial activity versus species to which itm
42、ay be exposed. If an alternative challenge species is used in testing, itmust be identified in the final report, along with any accommodativemodifications made to the method (that is, changes to culture media,buffers, etc.). The test report also must indicate that ASTM MethodE3151, modified, was use
43、d. The precision statistics reported in thismethod will not apply.9. Preparation of Bacterial Inoculum9.1 Grow a fresh 18- to 24-h shake culture of S. epidermidis(8.1) in sterile broth growth medium (7.2.1)at356 2 C priorto performing the test.9.1.1 Prepare these cultures from an 18- to 24-h growthf
44、rom stock culture plates or agar slants.9.2 Dilute this stock suspension of bacteria appropriatelyusing inoculation medium (7.2.3) to achieve a final bacterialtiter between 1.0 105colony forming units (CFU)/mLand 5.0105CFU/mL with the target being 3.0 105CFU/mL.9.3 Centrifuge the diluted stock suspe
45、nsion to harvest thebacterial cells.9.3.1 Centrifuge for 5 min at 15294 g.9.4 Remove at least 90 % of the resulting supernatant.9.5 Reconstitute the pelletized cells in a volume of theinoculation medium (7.2.3) equal to the volume of supernatantremoved.9.5.1 This bacterial suspension is the test ino
46、culum.9.5.2 The test inoculum is to be used within2hofpreparation.9.6 Verify the titer of bacteria in the test inoculum byperforming serial dilutions and utilizing a validated microbialenumeration technique (for example, pour plate, spread plate,spiral plate, or membrane filtration).9.6.1 Report the
47、 bacterial titer in the test inoculum in thefinal report. If the bacterial titer of the test inoculum does notfall within the range of 1.0 105CFU/mL to 5.0 105CFU/mL, the test is considered invalid and the specimen mustbe retested.NOTE 3Alternative inoculum bacterial titers and inoculum media canbe
48、substituted to better simulate the end-use conditions. If so, allmodifications must be documented in the test report and the test reportmust indicate that ASTM Method E3151, modified, was used. Theprecision statistics reported in this method will not apply.NOTE 4A nonionic surfactant may be added to
49、 the test inoculum toimprove the wetting of hydrophobic specimens. Surfactants should beshown through prior testing not to cause a change in the bacterialpopulation at the intended use-concentration. If used, the chemical nameof the surfactant and its final concentration in the test inoculum must bedocumented in the test report. The test report also must indicate thatASTM Method E3151, modified, was used. The precision statisticsreported in this method will not apply.NOTE 5In Steps 9.2 and 9.3, the stock suspension can be centrifugedprior to dilution to
