1、Designation: E 2180 07Standard Test Method forDetermining the Activity of Incorporated AntimicrobialAgent(s) In Polymeric or Hydrophobic Materials1This standard is issued under the fixed designation E 2180; the number immediately following the designation indicates the year oforiginal adoption or, i
2、n 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.INTRODUCTIONPolymeric materials such as vinyl pool liners, shower curtains, and various medic
3、al devices aretreated frequently with incorporated or bound antimicrobial agents. Practices G21 is used todetermine the ability of polymer materials to resist microbial attack or staining (see also PracticeE 1428); however, none of the methods permit quantitative evaluations of incorporated antimicr
4、obialactivity.2These antimicrobials typically require contact with the microbial cell for maximal activity.When aqueous based bacterial inoculum suspensions are applied onto a preservative-treated plastic orother hydrophobic material, the surface tension of the polymer often causes the inocula suspe
5、nsion todome. Bacteria within the drops of inoculum may not contact the treated surface if the challengedsurface does not dry, or upon drying, cells may become layered. This test standard involves an agarslurry inoculum vehicle that provides a relatively uniform contact of the inocula with antimicro
6、bial-treated hydrophobic surfaces.1. Scope1.1 This test method is designed to evaluate (quantitatively)the antimicrobial effectiveness of agents incorporated or boundinto or onto mainly flat (two dimensional) hydrophobic orpolymeric surfaces. The method focuses primarily on assessingantibacterial ac
7、tivity; however, other microorganisms such asyeast and fungal conidia may be tested using this method.1.2 The vehicle for the inoculum is an agar slurry whichreduces the surface tension of the saline inoculum carrier andallows formation of a “pseudo-biofilm,” providing more evencontact of the inocul
8、um with the test surface.NOTE 1This test method facilitates the testing of hydrophobic sur-faces by utilizing cells held in an agar slurry matrix. This test method, aswritten, is inappropriate to determine efficacy against biofilm cells, whichare different both genetically and metabolically than pla
9、nktonic cells usedin this test.1.3 This method can confirm the presence of antimicrobialactivity in plastics or hydrophobic surfaces and allows deter-mination of quantitative differences in antimicrobial activitybetween untreated plastics or polymers and those with boundor incorporated low water-sol
10、uble antimicrobial agents. Com-parisons between the numbers of survivors on preservative-treated and control hydrophobic surfaces may also be made.1.4 The procedure also permits determination of “shelf-life”or long term durability of an antimicrobial treatment whichmay be achieved through testing bo
11、th non-washed and washedsamples over a time span.1.5 Knowledge of microbiological techniques is requiredfor these procedures.1.6 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 standard to establish app
12、ro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E 1054 Test Methods for Evaluation of Inactivators ofAntimicrobial AgentsE 1428 Test Method for Evaluating the Performance ofAntimicrobials in or o
13、n Polymeric Solids Against Stainingby Streptoverticillium reticulum(A Pink Stain Organism)G21 Practice for Determining Resistance of Synthetic1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee
14、E35.15 on Antimicrobial Agents.Current edition approved Nov. 1, 2007. Published November 2007. Originallyapproved in 2001. Last previous edition approved in 2001 as E 2180 01.2Price, D.L., A.D. Sawant, and D.G. Ahearn. 1991. Assessment of the antimi-crobial activity of an insoluble quaternary amine
15、complex in plastics. J. Industr.Microbiol. Vol 8 (No.2):83-89.3For 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
16、.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Polymeric Materials to Fungi3. Terminology3.1 Definitions:3.1.1 agar slurry, na semi-gelatinous liquid formed when3 g/L agar-agar is added to a 0.85 % saline solution.3.1.2 inoculum ve
17、hicle, nthe carrier solution used totransport bacterial cells to a test surface. Samples includesaline, nutrient broth, tryptic soy broth, agar slurry, or otherbuffers that maintain bacterial viability.3.1.3 neutralizing recovery broth, nliquid growth mediaused to inactivate the effects of the test
18、antimicrobial agent.4. Summary of Test Method4.1 This method involves inoculation of a molten (45C)agar slurry with a standardized culture of bacterial cells.4.2 Athin layer of the inoculated agar slurry (0.5-1.0 mL) ispipetted onto the test and untreated control material (triplicatesamples minimum)
19、.4.3 After the specified contact time (24 h commonly used),surviving microorganisms are recovered via elution of the agarslurry inoculum from the test substrate into neutralizing brothand extracted via methods that provide complete removal ofthe inoculum from the test article (examples include sonic
20、a-tion, vortexing, and/or manual extraction, that is, stomacher).4.4 Serial dilutions are made, then pour or spread plates aremade of each dilution. Agar plates and dilution broths areincubated for 48 6 2 h at a specified temperature dependentupon the optimal temperature for test organism.4.5 Bacter
21、ial colonies from each dilution series are countedand recorded.4.6 Calculation of percent reduction of bacteria from treatedversus untreated samples is made.5. Significance and Use5.1 This method can be used to evaluate effectiveness ofincorporated/bound antimicrobials in hydrophobic materialssuch a
22、s plastics, epoxy resins, as well as other hard surfaces.5.2 The aqueous based bacterial inoculum remains in close,uniform contact in a “pseudo-biofilm” state with the treatedmaterial. The percent reduction in the surviving populations ofchallenge bacterial cells at 24 h versus those recovered from
23、anon-treated control is determined.5.3 The hydrophobic substrate may be repeatedly testedover time for assessment of persistent antimicrobial activity.6. Apparatus6.1 Erlenmeyer Flask, 250 mL.6.2 Petri Dishes, (15 3 100 mm), sterile.6.3 Colony Counter.6.4 Specimen Cups, (120 mL), sterile or equivale
24、nt sterileequipment for extraction.6.5 Pipetters, (1000 L) positive displacement.6.6 Pipette Tips, sterile.6.7 Test Tubes,163 100 mm.6.8 Incubator, set at required temperature (25-35 6 2C).6.9 Autoclave.6.10 Water Bath, capable of maintaining water at 45 6 2C.6.11 Sterile Cotton Swabs.6.12 Sonic Bat
25、h, 47 Khz, cleaning non-cavitating.6.13 Vortex Mixer.6.14 pH Meter.6.15 Hot Plate, with stirrer.6.16 Spectrophotometer, set at 600 nm.6.17 Sterile Cuvettes.6.18 Test Materials, sterile if specified by interested parties.6.19 Cell Counting Chamber.7. Reagents7.1 Media:7.1.1 Tryptic Soy Broth, or appr
26、opriate broth.7.1.2 Tryptic Soy Agar, or appropriate agar.7.1.3 Neutralizing Broth, appropriate for the antimicrobialcompound tested.(See Practice E 1054.)7.1.4 Agar-agar.7.1.5 NaCl.7.1.6 Sterile Deionized Water.7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mL in 16 3100 mm test tubes or appropri
27、ate dilution buffer (such asphosphate buffer or Butterfields buffer).7.2 Test OrganismsSpecific organisms are recommendedbut choice of organism should be relevant to the environmentin which the product will perform.7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC6538.7.2.2 Gram-negative bacte
28、ria Pseudomonas aeruginosaATCC 15442 or Klebsiella pneumoniae ATCC 4352.7.2.3 Other microorganisms such as yeast or fungal conidiamay also be tested using this procedure. Exposure periods maybe modified (up to 96 h) to address more resistant microorgan-isms.8. Procedure8.1 Grow 18 h bacterial cultur
29、es (three transfers) at aspecified temperature dependent upon the optimal temperaturefor the test organism in tryptic soy or appropriate broth. Thesecultures should originate from 18-24 h growth coming fromstock culture plates or growth on agar slants.8.2 Prepare the agar slurry by dissolving 0.85 g
30、 NaCl and0.3 g agar-agar in 100 mL of deionized water. Heat withstirring on a hot plate until the agar dissolves. One agar slurryshould be prepared for each organism tested.8.3 Sterilize the agar slurry by autoclaving for 15 min at 1216 2C, 15 psi., then equilibrate at 45 6 2C.8.4 Prepare 3.0 3 3.0
31、cm square samples of the treated andcontrol test materials (sample minimum of triplicates for “0” hcontrols, incubation period treated samples and incubationperiod control samples for each test organism).8.5 Place each sample into a sterile 15 3 100 mm petri dish.8.6 Adjust bacterial broth cultures
32、to 1-53108cells/mLwitha spectrophotometer or cell counting chamber.8.7 Dip a cotton swab into sterile 0.85 % saline (with orwithout a non-inhibitory levels of a surfactant) and pre-wet thetest sample. This will aid in dispersing the agar slurry evenlyon the sample.E21800728.8 Place 1.0 mL of standar
33、dized culture (1-53108cells/mL) into the 100 mL agar slurry equilibrated at 45 6 2C. Thefinal concentration should be 1-53106cells/mL in the moltenagar slurry.8.9 Pipet 0.5-1.0 mL of inoculated agar slurry onto the testand control samples. Slow, gentle application at a low angle ofincidence relative
34、 to the sample will aid in formation of a filmno more than 1 mm in depth. If the inoculum volume and/orsurface area of the sample are modified, the inoculum volumeshould be adjusted to provide an agar slurry 1 mm in depthover the entire sample surface. The inoculum volume andsurface area tested shou
35、ld be reported.NOTE 2For substrates found to be extremely hydrophobic, addition ofnon-inhibitory levels of a surfactant to the agar slurry may also aid insurface wetting properties.Alternatively, the slurry may be cooled to 25 62C then applied to the polymer surface. Application of pre-cooled agarsl
36、urry has also been shown to aid in the dispersion of the inoculum overan extremely hydrophobic test surface.8.10 Allow the agar slurry inoculum to gel and then placethe samples in an incubator at a specified temperature depen-dent upon the optimal temperature for the test organism or onewhich mimics
37、 the temperature in which the test substrate willbe utilized for the specified exposure period (usually 24 6 2 h).Low humidity in the incubator can cause drying of the agarslurry inoculum on the samples, therefore relative humiditywithin the incubator should be at or above 75 %. This can beaccomplis
38、hed with open reservoirs of certain saturated salt inwater solutions.48.11 Make serial dilutions (as described in 8.12-8.16)oftheagar slurry recovered immediately from “0” h control samplesand spread or pour plate each dilution to determine cfu/mLrecoverable at time “0 h.”8.12 Following the specifie
39、d contact time, aseptically re-move the incubation period control samples and incubationperiod treated samples from the petri dishes to 120 mLspecimen cups or other suitable container containing a suffi-cient volume of neutralizing broth to form an initial 1:10 or1:100 dilution of the original inocu
40、lum.8.13 Place the specimen cups containing the recovered testsamples into a non-cavitating sonic bath and sonicate for 1min.8.14 Sonication should be followed by 1 min of vigorousmechanical vortexing. This should facilitate the completerelease of the agar slurry from the sample. Note that the tests
41、urface may be imprint cultured onto tryptic soy agar followingsonication and vortexing in order to determine release effi-ciency of the inoculum from the treated surface.8.15 Perform serial dilutions of the initial neutralizing brothsufficient to include one dilution beyond the original inoculum(as
42、determined in 8.11).8.16 Spread or pour plate each dilution into tryptic soy agaror other appropriate agar (for example, Sabourauds agar forfungi) and incubate plates at an optimal temperature for the testorganism for 48 6 2h.8.17 Count and record colony numbers for each dilutionplate.9. Calculation
43、9.1 Determine the geometric mean of the number of organ-isms recovered from the triplicate incubation period controland incubation period treated samples by the following equa-tion:geometric mean 5Log10X11 Log10X21 Log10X3!3(1)where:X = number of organisms recovered from the incubationperiod control
44、 or incubation period treated samples.9.2 Percent ReductionUse the following equation to cal-culate the percent reduction:% reduction 5a2b! 3 100a(2)where:a = the antilog of the geometric mean of organisms recov-ered from the incubation period control samples (asdetermined in 9.1), andb = the antilo
45、g of the geometric mean of the number oforganisms recovered from the incubation period treatedsamples (as determined in 9.1).10. Precision and Bias10.1 A precision and bias statement has not been developedfor this method at this time.11. Keywords11.1 agar slurry; hydrophobic surface; percent bacteri
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48、ed either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a
49、 fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).4Corey, J. In Beuchat, L.R. ed. Food and Beverage Mycology AVI PublishingCo. Inc. Westpo
