1、Designation: E2180 07 (Reapproved 2017)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 hydrophobicsurfaces by utilizing cells held in an agar slurry matrix. This test method,as written, is inappropriate to determine efficacy against biofilm cells,which are different both genetically and metabolica
9、lly than planktoniccells used in 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 l
10、ow 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 throug
11、h 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 a
12、ddress 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.1.8 This international standard was developed in accor
13、-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 TechnicalBarriers to Trade (TBT) Committee.1This test method is under the
14、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 April 1, 2017. Published April 2017. Originallyapproved in 2001. Last previous edition approved in 2
15、012 as E2180 07(2012).DOI: 10.1520/E2180-07R17.2Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assessment of the antimicro-bial activity of an insoluble quaternary amine complex in plastics,” J. Industr.Microbiol, Vol 8, No. 2, 1991, pp. 8389.Copyright ASTM International, 100 Barr Harbor Drive, PO
16、 Box 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 b
17、y the World Trade Organization Technical Barriers to Trade (TBT) Committee.12. Referenced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE1428 Test Method for Evaluating the Performance ofAntimicrobials in or on Polymeric SolidsAgainst Staining
18、by Streptomyce species (A Pink Stain Organism)G21 Practice for Determining Resistance of Synthetic 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 carr
19、ier 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 antimicrobial ag
20、ent.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).4.3 After the s
21、pecified 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 includesonication, vortexing,
22、 and/or manual extraction, that is, stom-acher).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 Bacterial colonies from
23、 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 as plastics, epoxy
24、 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 anon-treated cont
25、rol 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, (15 100 mm), sterile.6.3 Colony Counter.6.4 Specimen Cups, (120 mL), sterile or equivalent sterileequipment
26、 for extraction.6.5 Pipetters, (1000 L) positive displacement.6.6 Pipette Tips, sterile.6.7 Test Tubes, 16 100 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
27、 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 appropriate broth.7.1.2
28、 Tryptic Soy Agar, or appropriate agar.7.1.3 Neutralizing Broth, appropriate for the antimicrobialcompound 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 16 100 mm test tubes or appropriate dilution buffer (
29、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 bacteria Pseudomonas aerug
30、inosaATCC 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.3For referenced ASTM standards, visit the ASTM website, www.ast
31、m.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.E2180 07 (2017)28. Procedure8.1 Grow 18 h bacterial cultures (three transfers) at aspecified temperature dependent upon the
32、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 NaCl and0.3 g agar-agar in 100 mL of deionized water. Heat withst
33、irring 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 3.0 cm square samples of the treated andcontrol test materials (sample m
34、inimum 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 15 100 mm petri dish.8.6 Adjust bacterial broth cultures to 1-5108cells/mL witha spectrophotometer or cell counting chamber.8.7
35、 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.8.8 Place 1.0 mLof standardized culture (1-5108cells/mL)into the 100 mL agar slurry equilibrated at 45 6
36、2C. The finalconcentration should be 1-5106cells/mL in the molten agarslurry.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 i
37、noculum 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 should be reported.NOTE 2For substrates found to be extremely hydrophobic, addition of
38、non-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 agarslurry has also been shown to aid in the dispersion of the inoculum overan extremely
39、 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 the temperature in which the test substrate willbe utilized for the specified exp
40、osure 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 beaccomplished with open reservoirs of certain saturated salt inwater solutions.48.11 Make se
41、rial dilutions (as described in 8.12 8.16)ofthe agar slurry recovered immediately from “0” h controlsamples and spread or pour plate each dilution to determinecfu/mL recoverable at time “0 h.”8.12 Following the specified contact time, aseptically re-move the incubation period control samples and inc
42、ubationperiod 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 inoculum.8.13 Place the specimen cups containing the recovered testsamples into a non-
43、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 testsurface may be imprint cultured onto tryptic soy agar followingsonication and vort
44、exing 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 determined in 8.11).8.16 Spread or pour plate each dilution into tryptic soy agar
45、or 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. Calculation9.1 Determine the geometric mean of the number of organ-isms recovered from the t
46、riplicate incubation period controland incubation period treated samples by the following equa-tion:geometric mean 5Log10X11Log10X21Log10X3!3(1)where:X = number of organisms recovered from the incubationperiod control or incubation period treated samples.9.2 Percent ReductionUse the following equati
47、on to cal-culate the percent reduction:% reduction 5a 2 b! 3100a(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 pe
48、riod 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.4Corey, J. In Beuchat, L.R. ed., Food and Beverage Mycology, AVI PublishingCo., Inc., Westport, CT, p. 52.E2180 07 (2017)311. Keywords11.1 agar slur
49、ry; hydrophobic surface; percent bacterialreduction; plastics; polymers; quantitative antibacterial assayASTM 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 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
