1、Designation: E3160 18Standard Test Method forQuantitative Evaluation of the Antibacterial Properties ofPorous Antibacterial Treated Articles1This standard is issued under the fixed designation E3160; 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 To determine the bactericidal or bacteriostatic propertiesof porous articles treated with
3、 an active biocidal agent, samplesof porous treated materials, such as textiles or paper, areinoculated with a defined suspension of microorganisms andthen incubated. The changes in numbers of the bacterialpopulations on the treated article are compared with untreatedarticles either over designated
4、time or they are compared to theinitial bacterial population at “zero time” for the treated articleto measure antibacterial properties.1.2 This test method is used for measuring the quantitativeantibacterial activity of porous materials that have been treatedwith a biocide to inhibit the growth of b
5、acteria on the treatedmaterials. This method may also be used to measure the abilityof the treated material to inhibit the growth of a microorgan-ism. It can measure both bactericidal and bacteriostatic activity.1.3 This test method shall be performed by individualsexperienced and adept in microbiol
6、ogical procedures and infacilities suitable for the handling of the microorganisms undertest.1.4 This test method may involve 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 o
7、f the user of this standard toestablish appropriate safety, health, and environmental prac-tices and determine the applicability of regulatory limitationsprior to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization establish
8、ed in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E691 Practice for Conducting an Interlaboratory Study toDetermine t
9、he Precision of a Test MethodE1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2180 Test Method for Determining the Activity of Incor-porated Antimicrobial Agent(s) In Polymeric or Hydro-phobic MaterialsE2149 Test Method for Determining the Antimicrobial Ac-tivity of Antimic
10、robial Agents Under Dynamic ContactConditionsE2756 Terminology Relating to Antimicrobial and AntiviralAgentsE2922 Guide for The Use of Standard Test Methods andPractices for Evaluating Antibacterial Activity on Textiles2.2 AATCC (American Association of Textile Chemists andColorists) Documents:3AATC
11、C TM100 : Antibacterial Finishes on Textile Materi-als: Assessment of:2.3 ISO (International Organization for Standardization)Documents:4ISO 22196 Plastics Measurement of Antibacterial Actionon Plastic Surfaces.ISO 20743 Textiles Determination of Antibacterial Activ-ity of Antibacterial Finished Pro
12、ducts2.4 IBRG (International Biodeterioration Research Group)Documents5IBRG TEX13/005/1.0 Quantitative Method for EvaluatingBacterial Activity of Textiles and Porous Material andArticles1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Co
13、ntrol Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2018. Published July 2018. DOI: 10.1520/E3160182For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For A
14、nnual Book of ASTMStandards volume information, 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 27709-2215, http:/www.aatcc.org.4Available from International Orga
15、nization for Standardization (ISO), ISOCentral Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,Geneva, Switzerland, http:/www.iso.org.5Available from IBRG, Pale Lane, Hartley Wintney, Hants, UK RG27 8DH,http:/www.ibrg.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box
16、 C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by th
17、e World Trade Organization Technical Barriers to Trade (TBT) Committee.13. Terminology3.1 DefintionsFor definition of terms used in this testmethod, refer to, E2756 Standard Terminology Relating toAntimicrobial and Antiviral Agents.4. Summary of Test Method4.1 A liquid suspension of bacteria is appl
18、ied to porousmaterials both untreated and treated with antimicrobial fin-ishes.4.2 Samples of each treated or untreated material are inocu-lated with a specified concentration of bacteria suspended in asolution containing a defined concentration of nutrients.4.3 The inoculated materials are then inc
19、ubated under con-ditions of controlled temperature and humidity for a specifiedperiod of time.4.4 After incubation, the samples are immersed in a neu-tralizer suitable for deactivating the active substance(s) used toproduce the intended antimicrobial effect, and agitated toremove surviving organisms
20、.4.5 The number of colony forming units present in theresulting suspension is then determined using standard platecounting techniques.4.6 Changes in the number of the test organism are thencalculated in relation to the numbers present on the untreatedmaterials after a specific contact time or in rel
21、ation to thenumbers applied (inoculum count), or both.5. Significance and Use5.1 Porous articles (often textiles) are often treated withantimicrobial agents to reduce the growth of microorganismsduring use, in storage, or while waiting to be laundered, orboth. Additionally, antimicrobial agents are
22、added to reduce orcontrol the overall microbial growth on porous articles thatmay affect the materials odor, visual, chemical or physicalintegrity, or both.5.2 Antimicrobial textile test methods that measure theantimicrobial behavior of treated textiles do exist but they areoften specific for one ty
23、pe of antimicrobial agent or aredesigned to or may artificially (not expected in real life)promote the release of some specific antibacterial agents overothers. This test method is designed to be able to measure theantimicrobial activity from all common antimicrobial agentsused to treat porous artic
24、les, including textiles, without givingeither positive or negative bias to one type of chemistry orproduct over another.5.3 In an effort to avoid excessive use or abuse ofantimicrobial agents in the environment, it is important tounderstand if untreated porous articles are susceptible tomicrobial co
25、ntamination and growth. In this test method, asmall amount of nutrients is added to each test sample in orderto promote some microbial growth on susceptible test samplesbut not enough to overwhelm potential antimicrobial agentsthat may be effective in real life situations. Furthermore, lowlevels of
26、nutrients allow investigators to add soiling agents thatmay be more reflective of a specific treated products end useor expected performance.5.4 Very specific parameters are identified within thismethod to limit any variability that may be seen betweenlaboratories. Identifying and clarifying potenti
27、al variablesfound in other guides or methods used in the industry willallow for better reproducibility and repeatability between andwithin laboratories.5.5 This test method provides the foundation for conductingtests on porous antibacterial treated articles. Modifications ofthis method that simulate
28、 intended use, durability and compat-ibility of the treated article should be outlined to ensure anaccurate assessment of antimicrobial activity with each par-ticular biocide that substantiates end use claims made for thearticle. A list of these typical modifications and current testmethods for text
29、iles can be found in Guide E2922.5.6 This test method is appropriate for porous materialssuch as textiles, paper, or similar porous materials. It isintended to measure the antibacterial properties of such mate-rials. In most instances, further studies will be required tosupport and substantiate actu
30、al claims being made for theperformance of treated materials in practice or as part of aregulatory process.5.7 This test method or indicated modifications may beused to determine antimicrobial activity as indicated in 5.6 ormay be used as a routine bioassay in standard quality controlprograms.6. App
31、aratus6.1 analytical balancewith capacity and precision of 0.01to 10 g 60.001, respectively, to weigh chemicals and tocalibrate inoculum delivery volumes by pipettes.6.2 biological safety cabinetsuitable for the containmentof the test organisms used.6.3 Bunsen burnerwith a gas source and flame ignit
32、er.6.4 colony counter(optional.)6.5 dispensersfor dispensing sterile 10-ml aliquots ofdiluent/neutralizer.6.6 forcepssterile for handling treated articles.6.7 freezersa freezer at -20 62 C for the storage of mediaand additives. A second freezer at -70 C or lower to store thestocks of test organisms
33、(optional).6.8 glass rodssterile. for use in holding porous samples inplace. Rods should be no more than 40 mm in length.6.9 glovessterile, disposable, for handling test items.6.10 hot air ovenan oven at 60 6 2 C to dry clean andwrapped sterile glassware.6.11 incubatoran incubator to maintain a temp
34、erature of35 6 2 C and maintain a relative humidity of not less than80 %.6.12 inoculating loopsSterile plastic or sterilizable metalinoculating loops (10-l).6.13 magnetic stir plate and stir barslarge enough for a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.E3160 1826
35、.14 sterile (plastic) or sterilizable petri dishes10015mm for microbial growth and recovery media.6.15 pH meterhaving an accuracy of 60.1 pH units tomeasure pH of media and suspensions.Apuncture electrode ora flat membrane electrode should be used for measuring the pHof agar media.6.16 sterile or st
36、erilizable pipette and tips (electronic ornon-electronic positive displacement)100 to 1000-l pipetteand appropriate pipette tips fitted with “plungers” that candispense accurately 200-l.6.17 refrigerator4 6 2 C; for storage of media, cultureplates and reagents.6.18 sterile or sterilizable serologica
37、l pipettesreusable orsingle-use pipettes of 1.0, 5.0 and 10.0-ml capacity (optional).6.19 sterilizer (autoclave)any steam sterilizer suitable forprocessing culture media, reagents and labware; the steamsupplied to the sterilizer should be free from additives toxic tothe test organisms.6.20 sterile o
38、r sterilizable vials or tubes for dilutionsuitable to hold 30-ml easily.6.21 sterile containers for sample inoculation andincubation4 oz, screw-on lid, sterile, disposable and indi-vidually sealed specimen cups with a bottom surface diameterof 45 mm have been shown to be suitable.6.22 vortex mixerto
39、 mix the cell suspensions and neutral-izer suspensions to ensure efficient recovery of the test organ-ism(s).6.23 water bathcapable of reaching and maintaining atemperature of 45 6 2 C to keep agar media from solidifyingwhen making culture plates.6.24 incubator shakeran orbital incubator shaker capa
40、bleof agitating broth cultures of bacteria and able to maintain atemperature of 35 6 2 C.6.25 test validity control substratea suitable control ma-terial has been found to be a cellulosic filter paper such asWhatman #4 or any textile substrate shown to promote at least1 log CFU/g of bacterial growth
41、 under the parameters outlinedbelow.NOTE 1Sterilize all laboratory ware and equipment as appropriate.Sterilization can be achieved by moist heat in an autoclave, by dry heat ina hot-air oven or other appropriate, validated sterilization process. Manyof the consumable items used in this guideline can
42、 be purchasedpre-sterilized and ready for use. Sterility of all ware and equipment shouldbe confirmed prior to use.7. Reagents and Materials67.1 Sterile Tryptic Soy Broth, (TSB).7.2 Sterile Tryptic Soy Agar, (TSA).7.3 Neutralizing Broth, appropriate for the antimicrobialcompound tested (See Practice
43、 E1054).7.4 Suspension Medium, 1/500 TSB plus wetting agent(1/500 TSB). Dilute the TSB (7.1) 1:500 with distilled ordeionized water plus 0.05 % vv Triton X-100 and then adjustthe pH to a value between 6.8 and 7.2 with either sodiumhydroxide or hydrochloric acid. Sterilize by autoclaving at 12162 C.
44、If it is not used immediately after preparation, store itat 4 6 2 C.7.5 Sterile Distilled or Deionized Water.7.6 Ethyl Alcohol, for forcep sterilization.8. Test Organism8.1 Escherichia coli, American Type Culture Collection(ATCC) No. 25922.8.1.1 Cultures of the test organism should be maintainedacco
45、rding to good microbiological practice and 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-enc
46、e to monthly stock transfers. Check culture purity bymaking streak plates periodically, observing for colonies char-acteristic of Escherichia coli, Gram-staining or performingother forms of microbial identification.NOTE 2This method was developed and validated using ATCC No.25922 as the test organis
47、m. If an alternative culture is used, the resultsmust be reported as having been obtained using a modified test method. E.coli was chosen for this method due to its proven reproducibility in initiallaboratory ring tests. If the test method is modified in any way, the reportmust also include a detail
48、ed description of all modifications made,including, but not limited to: test organisms, media, buffer, bacterialconcentration, etc.9. Preparation of Bacterial Inoculum9.1 Grow a fresh overnight (18-24 h) culture of E. coli insterile 100 % TSB at 35 62 C prior to performing the test onan orbital incu
49、bator shaker allowing for maximum aeration ofculture. This culture should originate from an 18-24 h growthcoming from stock culture plates or growth on agar slants.9.2 The fresh overnight culture is then diluted with suspen-sion medium (7.4), as appropriate to obtain a bacterial concen-tration that is between 2.5 105colony forming units(CFUs)/ml and 1.0 106CFUs/mL, with a target concentrationof6105CFUs/mL. This suspension is used as the testinoculum. The test inoculum shall be used within2hofpreparation. The number of colony fo
