1、Designation: E2180 07 (Reapproved 2012)Standard Test Method forDetermining the Activity of Incorporated AntimicrobialAgent(s) In Polymeric or Hydrophobic Materials1This standard is issued under the fixed designation E2180; the number immediately following the designation indicates the year oforigina
2、l adoption or, in 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.INTRODUCTIONPolymeric materials such as vinyl pool liners, shower curtains, an
3、d various medical devices aretreated frequently with incorporated or bound antimicrobial agents. Practices G21 is used to determinethe ability of polymer materials to resist microbial attack or staining (see also Practice E1428);however, none of the methods permit quantitative evaluations of incorpo
4、rated antimicrobial activity.2These antimicrobials typically require contact with the microbial cell for maximal activity. Whenaqueous based bacterial inoculum suspensions are applied onto a preservative-treated plastic or otherhydrophobic material, the surface tension of the polymer often causes th
5、e inocula suspension to dome.Bacteria within the drops of inoculum may not contact the treated surface if the challenged surfacedoes not dry, or upon drying, cells may become layered. This test standard involves an agar slurryinoculum vehicle that provides a relatively uniform contact of the inocula
6、 with antimicrobial-treatedhydrophobic 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 assessingan
7、tibacterial activity; 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
8、 of the inoculum 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 metaboli
9、cally than planktonic 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
10、 low water-soluble 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 thro
11、ugh testing both non-washed and washedsamples over a time span.1.5 Knowledge of microbiological techniques is requiredfor these procedures.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard does not purport to
12、 address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard 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:3E1054 Tes
13、t Methods for Evaluation of Inactivators of An-timicrobial AgentsE1428 Test Method for Evaluating the Performance ofAntimicrobials in or on Polymeric Solids Against Staining1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents
14、and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2012. Published June 2012. Originallyapproved in 2001. Last previous edition approved in 2007 as E2180 07. DOI:10.1520/E2180-07R12.2Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assess
15、ment of the antimicro-bial activity of an insoluble quaternary amine complex in plastics,” J. Industr.Microbiol, Vol 8, No. 2, 1991, pp. 8389.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards vol
16、ume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.by Streptoverticillium reticulum (A Pink Stain Organism)G21 Practice for Determining Resistance of Synth
17、etic Poly-meric 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 vehicle, nthe carrier solution used totransport bacterial cells to a test surface. Samples includesaline, nutrie
18、nt 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 antimicrobial agent.4. Summary of Test Method4.1 This method involves inoculation of a molten (45C)agar slurry
19、 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).4.3 After the specified contact time (24 h commonly used),surviving microorganisms are recovered via elution
20、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 sonica-tion, vortexing, and/or manual extraction, that is, stomacher).4.4 Serial dilutions are made, then pour or s
21、pread 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 Bacterial colonies from each dilution series are countedand recorded.4.6 Calculation of percent reduction of bacteri
22、a 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 as plastics, epoxy resins, as well as other hard surfaces.5.2 The aqueous based bacterial inoculum remains in c
23、lose,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 anon-treated control is determined.5.3 The hydrophobic substrate may be repeatedly testedover time for assessm
24、ent 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 equivalent sterileequipment for extraction.6.5 Pipetters, (1000 L) positive displacement.6.6 Pipette Tips, sterile.6.7
25、 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 Bath, 47 Khz, cleaning non-cavitating.6.13 Vortex Mixer.6.14 pH Meter.6.15 Hot Plate, with stirrer.6.16 Spectroph
26、otometer, 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 appropriate broth.7.1.2 Tryptic Soy Agar, or appropriate agar.7.1.3 Neutralizing Broth, appropriate for the antimi
27、crobialcompound tested.(See Practice E1054.)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 appropriate dilution buffer (such asphosphate buffer or Butterfields buffer).7.2 Test OrganismsSpecific organisms are r
28、ecommendedbut 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 bacteria Pseudomonas aeruginosaATCC 15442 or Klebsiella pneumoniae ATCC 4352.7.2.3 Other microorganisms such as yeas
29、t 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 cultures (three transfers) at aspecified temperature dependent upon the optimal temperaturefor the test organism in t
30、ryptic 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 NaCl and0.3 g agar-agar in 100 mL of deionized water. Heat withstirring on a hot plate until the agar dissolve
31、s. 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 cm square samples of the treated andcontrol test materials (sample minimum of triplicates for “0” hcontrols, in
32、cubation 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 to 1-53108cells/mLwitha spectrophotometer or cell counting chamber.8.7 Dip a cotton swab into sterile 0.85 % sa
33、line (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.E2180 07 (2012)28.8 Place 1.0 mL of standardized culture (1-53108cells/mL) into the 100 mL agar slurry equilibrated at 45 6 2C. Thefinal concentra
34、tion 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 to the sample will aid in formation of a filmno more than 1 mm in depth. If the inoculum volume and/or
35、surface 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 should be reported.NOTE 2For substrates found to be extremely hydrophobic, addition ofnon-inhibitory levels
36、 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 agarslurry has also been shown to aid in the dispersion of the inoculum overan extremely hydrophobic test sur
37、face.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 the temperature in which the test substrate willbe utilized for the specified exposure period (usually
38、 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 beaccomplished with open reservoirs of certain saturated salt inwater solutions.48.11 Make serial dilutions (as de
39、scribed 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 specified contact time, aseptically re-move the incubation period control samples and incubationperiod treated
40、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 inoculum.8.13 Place the specimen cups containing the recovered testsamples into a non-cavitating sonic bath
41、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 testsurface may be imprint cultured onto tryptic soy agar followingsonication and vortexing in order to dete
42、rmine 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 determined in 8.11).8.16 Spread or pour plate each dilution into tryptic soy agaror other appropriate a
43、gar (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. Calculation9.1 Determine the geometric mean of the number of organ-isms recovered from the triplicate incubation p
44、eriod 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 or incubation period treated samples.9.2 Percent ReductionUse the following equation to cal-culate the
45、 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 antilog of the geometric mean of the number oforganisms recovered from the incubation period treatedsamples (
46、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 bacterialreduction; plastics; polymers; quantitative antibacterial assay4Corey, J. In Beuchat, L.R. ed., Food
47、and Beverage Mycology, AVI PublishingCo., Inc., Westport, CT, p. 52.E2180 07 (2012)3ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the
48、 validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your
49、 comments are invited 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 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. Indi
