1、Designation: E3031 15Standard Test Method forDetermination of Antibacterial Activity on Ceramic Surfaces1This standard is issued under the fixed designation E3031; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last re
2、vision. 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 method is designed to quantitatively evaluate theantibacterial activity of glazed ceramic surfaces that have beenspecifi
3、cally designed to contain an antibacterial treatment aspart of the glaze. This test method is meant to compare theefficacy of one ceramic surface to another ceramic surfaceusing the stated conditions and is not meant to be extrapolatedto other conditions.1.2 Knowledge of microbiological techniques i
4、s requiredfor this test.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 does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this st
5、andard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Stu
6、dy toDetermine the 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 MaterialsE2756 Terminology Relating to Antimicrobial and Antivira
7、lAgents2.2 ISO Standard:3ISO 22196 Measurement of Antibacterial Activity on Plas-tics and Other Non-porous Surfaces3. Terminology3.1 For definitions of terms used in this test method refer toTerminology E2756.4. Summary of Test Method4.1 This test method is used for evaluating the antibacterialeffec
8、t of antimicrobials incorporated into a ceramic glaze. Thisstandard does not seek to imitate all possible real worldscenarios but to provide a standardized method to comparemultiple antimicrobial technologies that can be incorporated orcoated on a ceramic surface. The inherent nature of the ceramict
9、ile allows for desiccation, therefore each ceramic specimen isequilibrated to the testing environment for 18- 24 h. Once thetiles are equilibrated, bacteria are inoculated onto the surfacefollowed by a 24-h exposure time. Bacteria are recovered in aneutralizer broth and enumerated according to a val
10、idatedmethod. Log reductions are calculated for a treated versus anuntreated sample.5. Significance and Use5.1 Current solid surface test methodologies, such as theTest Method E2180 and ISO 22196, do not take into accountthe complexities associated with a ceramic surface. Thisincludes, but is not li
11、mited to, differing chemistries incorpo-rated into the glaze and desiccation due to water absorptionthrough the bisque body. Each point will be elaborated below:5.1.1 The glaze composition of ceramic tiles can varybetween manufacturers, lots, and product lines. Some glazechemistries such as tin, sil
12、ver and copper can negatively impactthe testing conditions. Therefore, an untreated tile from thesame lot is not always suitable for comparison. The control tileproposed herein is capable of supporting growth over theindicated time frame and nutrient level (see Section 9).5.1.2 Desiccation is a comm
13、on problem when testing tilesurfaces. This can be overcome by pre-hydrating the tile byplacing the specimen on a moistened wipe and allowing1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility o
14、f Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 15, 2015. Published December 2015. DOI:10.1520/E3031152For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume informat
15、ion, refer to the standards Document Summary page onthe ASTM website.3Available from International Organization for Standardization (ISO), ISOCentral Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,Geneva, Switzerland, http:/www.iso.org.Copyright ASTM International, 100 Barr Harbo
16、r Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1incubation for 18 to 24 h before beginning the test. Thisreduces the number of false positive results and more accu-rately measures the ability of the antimicrobial to inhibitgrowth.5.2 This test method utilizes a low inoculum loa
17、d andrequires growth on the control substrate to demonstrate a validtesting environment. In addition, while some antimicrobialsdemonstrate activity against static cultures, others requiregrowth of the bacteria to maintain activity. A low inoculumlevel will allow for both types of antimicrobials to b
18、e examinedwith the same testing conditions.6. Apparatus6.1 Incubatorcapable of maintaining a temperature of 356 2C and 75% RH.6.2 Pipettercontinuously adjustable between 100 L and1000 L.6.3 Sterilizerany suitable steam sterilizer with conditionsthat produce sterility of samples.6.4 Petri dishsterile
19、 150 mm by 15 mm for holding thesamples6.5 Culture tubes and closuresany with a volume capacityof 10 mL and a minimum diameter of 16 mm. Recommendedsize is 16 mm by 125 mm borosilicate glass with a threadedopening.6.6 Cover film25 mm by 25 mm sterile polyethylene orother suitable material that does
20、not impact bacterial growth.6.7 Large Water Absorbent Laboratory wipeto facilitatepre-hydration of samples similar to a KimwipesKimtech4delicate task wiper 30 cm by 30 cm.6.8 Vortex mixerto provide a homogenous bacterial sus-pension prior to inoculation of samples and prior to theenumeration techniq
21、ue that will be used.6.9 Plastic screw top jar150 ml capacity that has anopening large enough to insert the sample as a vessel forrecovery.6.10 Wrist action shakerto recover bacteria from samples.6.11 Petri dish100 mm by 15 mm for enumeration.6.12 Shaking incubatorcapable of maintaining 35 6 2C.7. R
22、eagents and Materials57.1 Dilution fluid or diluentsterile Butterfields bufferedphosphate.7.2 Growth medium.7.2.1 Overnight culturebrain heart infusion broth pre-pared according to the manufacturers instruction.7.2.1.1 Alternative media may be used for overnight cultureof the organism, such as trypt
23、ic soy broth, but details shall beincluded in the final report.7.2.2 Inoculation broth1:500 dilution of nutrient broth asdefined below:7.2.2.1 Prepare nutrient broth by dissolving 3.0 g of meat(beef) extract, 10.0 g peptone, and 5.0 g of sodium chloride in1000 mL of distilled or deionized water.7.2.
24、2.2 Dilute the nutrient broth with distilled or deionizedwater to a 500-fold volume and adjust the pH to a valuebetween 6.8 and 7.2 with sodium hydroxide or hydrochloricacid.7.2.2.3 Sterilize by autoclaving at 120C for 30 min.7.3 Solid growth mediatryptic soy agar plates.7.4 Sterile deionized watero
25、r equivalent.7.5 NeutralizerA neutralizer should be selected that hasbeen shown to effectively neutralize the active according toTest Methods E1054.8. Culture Preparation8.1 Escherichia coli American Type Culture Collection,ATCC No. 8739 is the organism to be utilized for this test.Grow a fresh 18 6
26、 1 h culture in sterile brain heart infusionbroth at 35 6 2C and shaking at 110 r/min prior to beginningthe test. Dilute this suspension appropriately in the inoculationbroth described in 7.2.2 to obtain 1-5 104CFU/mL. This willbe the working bacterial stock solution.9. Untreated Control Specimen9.1
27、 Control tiles suitable for testing purposes may beprepared from glaze ingredients that are free of elements thatcontribute to antimicrobial activity. One example of a productthat meets this criterion is F-524.6However, glazed tiles aregenerally acceptable as controls if they can be shown to meetthe
28、 following criterion:9.1.1 Can support 1.5 log growth under the test conditionsgiven herein as calculated in 12.4.9.1.1.1 If a control tile, as described above, is not availablethen the use of borosilicate glass squares, cut to the samedimensions as described in 10.1, can be substituted as controlsp
29、ecimens. Glass squares shall meet performance specifica-tions indicated in 9.1.1.10. Sample Preparation10.1 Prepare five (5) replicates of each specimen, measuring50 mm by 50 mm 6 1mm2(see Section 9). Wipe testspecimens to remove any debris from processing, place in asterilization pouch/container an
30、d autoclave for at 120C for 1h.NOTE 1If the active ingredient is affected by autoclaving, then other4Kimwipe is a registered trademark of Kimberly-Clark Dallas TX, USA5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testin
31、g of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.6The sole source of supply of the of the control t
32、iles (F-524) suitable for testingpurposes prepared from glaze ingredients that are free of elements that contribute toantimicrobial activity and known to the committee at this time is Fusion Ceramics,Inc. (Carrollton, Ohio USA). If you are aware of alternative suppliers, pleaseprovide this informati
33、on to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.E3031 152types of sterilization can be used.10.2 While test specimens are being sterilized, fold andplace two large 1-ply laboratory wipes
34、(30 cm by 30 cm) intoa 150 by 15 mm sterile petri dish. Fold in such a way to get 18layers in a 10 cm by 10 cm square. Moisten with steriledeionized water until the wipe is saturated.10.3 Remove a sterile test specimen (10.1) from steriliza-tion pouch aseptically, place onto the wipe in the petri di
35、sh(10.2). Visually monitor the dish during preparation to preventexcess water from accumulating in the dish.10.4 Incubate the dish containing the sample at 35 6 2Cwith 75% RH for 18 to 24 h.11. Procedure11.1 Inoculation and Incubation:11.1.1 Remove test specimens from the incubator and pro-ceed to 1
36、1.1.211.1.2 Pipette 100 L of the prepared bacterial stock solu-tion (8.1) onto each pre-hydrated test specimen (see Section10). The test specimen will remain on the moistened wipe forthe duration of the test. Addition of water may be necessary ifthe saturated wipe has become dry.11.1.3 Enumerate the
37、 inoculum by spread or pour plate.11.1.4 Place a 25 mm by 25 mm polyethylene film on top ofthe inoculum to ensure even contact with the surface. Makesure that no inoculum leaves the surface of the ceramic tile.11.1.4.1 In accordance with 6.6, the cover film to be utilizedshould not affect bacterial
38、growth or absorb water. If the testspecimen size is increased, the volume of inoculum and coverfilm shall be increased proportional to the surface area of thesample (a ratio of 1L:6.25 mm2).11.1.5 Place each petri dish containing inoculated samplesin the incubator at 35 6 2C with 75% RH for 24 6 1h.
39、11.2 Recovery:11.2.1 After the specified incubation time, remove the testspecimen or control from the petri dish and loosen the coverfilm. Note any desiccation that is observed for each sample.11.2.2 Place the film and ceramic test specimen into a sterile150-mL plastic screw top jar containing 100 m
40、L of neutralizer.Shake for 1 min on a wrist-action shaker set to the maximumspeed.NOTE 2Alternative vessels and volumes may be utilized but theirdescription will be included in the report. In addition, alternative recoverytechniques, such as vortex and sonication, may also be utilized. Use ofother r
41、ecovery methods should be noted in the test report.FIG. 1 Flowchart of Testing OperationE3031 15311.2.3 Recover culturable organisms from appropriate dilu-tions by use of spread- or pour plate, spiral plate, or by othervalid microbial enumeration methods.11.2.4 Incubate plates at 35 6 2C for 24 h.11
42、.2.5 Count and record colony numbers for each dilution.12. Calculation or Interpretation of Results12.1 For each test specimen, determine the number of viablebacteria per specimen:N 5 C 3 D 3 V! (1)where:N = is the number of viable bacteria recovered from testspecimen;C = is the average plate count;
43、D = is the dilution factor for the plates counted;V = is the volume, in ml, of neutralizer added to thespecimen;If no colonies were recovered in any of the agar plates for adilution series, then record the number of colonies counted as“ Log10Control the treatment isconsidered bactericidal13.3 If Log
44、 reduction 0.5 the sampleis considered to display partial inhibition.14. Precision and Bias14.1 The precision of this test method is based on anintra-laboratory study of E3031 Test Method for Determinationof Antibacterial Activity on Ceramic Surface, conducted in2014. A single laboratory participate
45、d in this study, testing theLog10reduction of Escherichia coli on ceramics treated withtwo different inhibitors. Every “test result” represents theaverage of five determinations. The laboratory reported tenreplicate test results for each ceramic material, as well as twogrowth control samples. Except
46、 for the use of only onelaboratory, Practice E691 was followed for the design andanalysis of the data; the details are given in ASTM ResearchReport No. RR:E35-1010.714.1.1 Repeatability (r)The difference between repetitiveresults obtained by the same operator in a given laboratoryapplying the same t
47、est method with the same apparatus underconstant operating conditions on identical test material withinshort intervals of time would in the long run, in the normal andcorrect operation of the test method, exceed the followingvalues only in one case in 20.14.1.1.1 Repeatability can be interpreted as
48、maximumdifference between two results, obtained under repeatabilityconditions that is accepted as plausible due to random causesunder normal and correct operation of the test method.14.1.1.2 Repeatability limits are listed in Table 1 and Table2 below.14.1.2 Reproducibility (R)The difference between
49、twosingle and independent results obtained by different operatorsapplying the same test method in different laboratories usingdifferent apparatus on identical test material would, in the longrun, in the normal and correct operation of the test method,exceed the following values only in one case in 20.14.1.2.1 Reproducibility can be interpreted as maximumdifference between two results, obtained under reproducibilityconditions that is accepted as plausible due to random causesunder normal and correct operation of the test method.14.1.2.2 Repro
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