1、Designation: E1428 99 (Reapproved 2009)Standard Test Method forEvaluating the Performance of Antimicrobials in or onPolymeric Solids Against Staining by Streptoverticilliumreticulum (A Pink Stain Organism)1This standard is issued under the fixed designation E1428; the number immediately following th
2、e designation indicates the year oforiginal 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.1. Scope1.1 This test method is used
3、 for determining the perfor-mance of antimicrobial agents used in or on synthetic poly-meric solids against staining by the actinomycete Streptover-ticillium reticulum. This organism has been chosen as anindicator organism, although other organisms have been knownto cause undesirable staining in pol
4、ymeric solids.1.2 This test method is not suitable for evaluating dark-pigmented test samples.1.3 A knowledge of microbiological techniques is recom-mended for these procedures.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard
5、.1.5 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 appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Summary of
6、Test Method2.1 Test specimens are placed on an agar surface inoculatedwith Streptoverticillium reticulum and incubated. After incuba-tion, test specimens are rated visually by percentage of samplearea stained.3. Significance and Use3.1 This test method provides a technique for evaluatingantimicrobia
7、ls in or on polymeric solids against staining byStreptoverticillium reticulum, and should assist in the predic-tion of performance of treated articles under actual fieldconditions.3.2 Conditioning of the specimens, such as exposure toleaching, weathering, and heat treatment, may have significanteffe
8、cts on performance of antimicrobials against staining.Determination of these effects is not included in this testmethod.4. Interferences4.1 An interference may be caused by contamination ofplates and agar by unwanted organisms that settle from theenvironment.4.2 Dark pigments mask observation of the
9、 pink stain.5. Apparatus5.1 Petri dishes, 100 mm diameter.NOTE 1Presterilized and disposable plastic petri dishes are availablefrom most laboratory supply houses.5.2 Cotton swabs, sterile.5.3 IncubatorIncubating equipment for this test methodshall maintain a temperature of 29 6 1C.5.4 Autoclave.5.5
10、Sterilizer, ethylene oxide (optional).6. Reagents and Materials6.1 Yeast Malt Extract Agar (ISP Medium 2)2Prepare thismedium according to manufacturers directions.6.2 InoculumStreptoverticillium reticulum ATCC25607.3Maintain stock cultures on yeast malt extract agar. Thestock may be kept for not mor
11、e than 12 months at approxi-mately 3 to 10C. Subcultures, incubated at 29 6 1C for 7 to14 days, shall be used for inoculation.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimi
12、crobial Agents.Current edition approved Oct. 1, 2009. Published November 2009. Originallyapproved in 1991. Last previous edition approved in 2004 as E1428 99(2004).DOI: 10.1520/E1428-99R09.2The sole source of supply of Bacto-Yeast Malt ExtractAgar, Stock No. 0770-01known to the committee at this tim
13、e is Difco Labs, P.O. Box 1058A, Detroit, MI28232. If you are aware of alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive careful consider-ation at a meeting of the responsible technical committee,1which you may attend.3American Type
14、Culture Collection, 10801 University Blvd., Manassas, VA20110.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.7. Test Specimens7.1 From each test unit (Note 2), cut duplicate 0.75 in.diameter discs. If the test unit is of different c
15、onstruction oneach side, two specimens of each, two face up and two facedown, shall be tested.NOTE 2Atest unit is a solid in the form of plastic sheets, films, coatedfabrics or similar polymeric materials.7.2 A test unit containing no biocide should be included asa positive stain control.7.3 A test
16、unit known to inhibit staining by Streptoverticil-lium reticulum should be included as a negative stainingcontrol.7.4 Heavily-soiled specimens should be rinsed vigorously inwater.7.5 If sterilization of test specimens is considered necessary,ethylene oxide is the sterilant of choice. Ethylene oxide
17、residuemay affect test outcome. Proper aeration after sterilization isnecessary.8. Procedure8.1 InoculationPour sufficient yeast malt extract agar intosuitable sterile dishes (see 5.1) to provide a solidified agar layerfrom 5 to 8 mm in depth. After the agar has solidified, moistenthe agar surface b
18、y streaking with a cotton swab dipped insterile water.8.2 Add 3 mL of sterile saline or phosphate buffered salineto the stock culture plate and use the premoistened swab toharvest the Streptoverticillium cells.8.3 Streak the cell-laden swab on the agar surface bystreaking in a manner which will ensu
19、re total coverage by theorganism. This swabbing technique typically provides suffi-cient growth to yield measurable pink stain on positive staincontrols.8.4 Use sterile forceps to place the sterile test specimens,two to a plate, on the agar surface. There shall be good contactestablished between the
20、 test specimen and the inoculated agarsurface. Should the specimens tend to curl, weights (such as58in. sterile stainless steel nuts) may be placed on top ofspecimens to maintain good contact.8.5 IncubationCover the inoculated test plates containingthe specimens and incubate at 29 6 1C for 14 days.N
21、OTE 3Covered petri dishes containing yeast malt extract agar areconsidered to have the desired humidity.9. Interpretation of Results9.1 Observe the degree of staining on the samples bycomparing with the inoculated, untreated control.9.2 If staining is not seen on the surface of the specimen, usester
22、ile forceps to lift and turn the specimen over and inspect theunderside for stain. The degree of staining is determined by theamount of sample surface stained rather than the intensity ofthe color. Any possible sample discoloration that is not pink incolor should be disregarded when determining the
23、degree ofstain. However, pigmented samples that show a color changeassociated with pink (that is, a blue pigmented sample chang-ing to purple) should be rated according to degree of stain. Ratethe degree of stain in accordance with Table 1.9.3 Leachability of AntimicrobialsA“ zone of inhibition”may
24、be observed where no organisms grow on the agaradjacent to the specimen. This indicates that the antimicrobialmay be leaching from the specimen.10. Report10.1 Report the visual rating of stain in accordance with 9.1.11. Precision and Bias11.1 A precision and bias statement cannot be made for thistes
25、t method at this time.12. Keywords12.1 antimicrobial efficacy; pink stain; plastics; polymericsolids;Streptoverticillium reticulumASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard a
26、re 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 five years andif not
27、 revised, either reapproved or withdrawn. Your 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 y
28、ou 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,Unite
29、d 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).TABLE 1 Degree of Stain RatingObserved Stain on Specimens RatingNo Stain 0Trace of Stain (less than 10 % coverage) 1Slight Stain (10 to 30 % coverage) 2Moderate Stain (30 to 50 % coverage) 3Heavy Stain (50 % to complete coverage) 4E1428 99 (2009)2
