1、Designation: E1428 15aStandard Test Method forEvaluating the Performance of Antimicrobials in or onPolymeric Solids Against Staining by Streptomyce species (APink Stain Organism)1This standard is issued under the fixed designation E1428; the number immediately following the designation indicates the
2、 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.INTRODUCTIONWhen certain bacteria and mold species grow on the
3、surface of flexible or “plasticized” polymers,metabolites such as pigments in the case of certain bacteria and melanin (dark stains from fungalgrowth) cause undesirable stains on the polymer surface. Theses stains may persist even after thesurface growth is removed. This test method is used for dete
4、rmining the performance of antimicrobialagents used in or on synthetic polymeric solids against pink-staining by the actinomycete,Streptomyces species. This organism has been chosen as an indicator organism, although otherorganisms have been known to cause undesirable staining in polymeric solids.1.
5、 Scope1.1 This test method is intended to assess susceptibility offlat two dimensional vinyl films and other solid polymerproducts as well as products that may directly contact vinyl topink-staining by the actinomycete bacteria Streptomyces spe-cies. This test method may not be suitable for highly t
6、exturedor porous substrates.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 i
7、n thisstandard.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.
8、2. Referenced Documents2.1 ASTM Standards:2D3273 Test Method for Resistance to Growth of Mold on theSurface of Interior Coatings in an Environmental Cham-berD3274 Test Method for Evaluating Degree of Surface Dis-figurement of Paint Films by Fungal or Algal Growth, orSoil and Dirt AccumulationE2756 T
9、erminology Relating to Antimicrobial and AntiviralAgents3. Terminology3.1 For definitions of terms used in this standard refer toTerminology E2756.3.2 Definitions:3.2.1 microbially induced stainingundesirable pigmenta-tion or disfiguration of an object due to surface colonization bycertain microorga
10、nisms.3.2.1.1 DiscussionBoth bacteria and fungi produce meta-bolic pigments that can result in surface stains on susceptibleobjects.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agentsand is the direct respon-sibility of Subco
11、mmittee E35.15 on Antimicrobial Agents.Current edition approved May 1, 2015. Published June 2015. Originallyapproved in 1991. Last previous edition approved in 2015 as E1428 15. DOI:10.1520/E1428-15A.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Servic
12、e at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.2 Pink stain organism, nrefers to a stainin
13、g phenom-ena caused by a metabolic pigment produced by actinomycetebacteria specifically, Streptomyces species ATCC 25607 (de-posited as Streptoverticillium reticulum).34. Summary of Test Method4.1 Test specimens are placed on an agar surface inoculatedwith Streptoverticillium species and incubated.
14、 Afterincubation, test specimens are rated visually by percentage ofsample area stained.5. Significance and Use5.1 Methods such as D3273 Standard Test Method forResistance to Growth of Mold on the Surface of InteriorCoatings in an Environmental Chamber and D3274 StandardTest Method for Evaluating th
15、e Degree of Surface Disfigure-ment of Paint Films by Fungal or Algal Growth or Soil or DirtAccumulation provide means for assessing mold and algalstaining on paints.5.2 This test method provides a technique for evaluatingantimicrobials in or on polymeric solids against staining byStreptomyces specie
16、s, bacteria and should assist in the predic-tion of performance of treated articles under actual fieldconditions.5.3 Conditioning of the specimens, such as exposure toleaching, weathering, and heat treatment, may have significanteffects on performance of antimicrobials against staining.Determination
17、 of these effects is not included in this testmethod.6. Interferences6.1 An interference may be caused by contamination ofplates and agar by unwanted organisms that settle from theenvironment.6.2 Dark pigments mask observation of the pink stain.6.3 If contaminants from the test specimen overgrow the
18、Actinomycete inoculum and block the production of pinkpigment or direct specimen contact, the test is to be declaredinvalid and steps to address contaminants taken.7. Apparatus7.1 Petri dishes, 100 mm diameter.NOTE 1Presterilized and disposable plastic petri dishes are availablefrom most laboratory
19、supply houses.7.2 Cotton swabs, sterile.7.3 IncubatorIncubating equipment for this test methodshall maintain a temperature of 29 6 1C.7.4 Autoclave.7.5 Sterilizer, ethylene oxide (optional).8. Reagents and Materials8.1 ISP Medium 2 (Yeast Malt Extract Agar)3Prepare thismedium according to manufactur
20、ers directions. SabouraudDextrose agar prepared per label directions may also be used(initiates robust vegetative growth and pigment production).8.2 Inoculum Streptomyces species ATCC 25607 (depos-ited as Streptoverticillium reticulum).4Maintain stock cultureson yeast malt extract agar. The stock ma
21、y be kept for not morethan 12 months at approximately 3 to 10C. Subcultures,incubated at 29 6 1C for 7 to 14 days, shall be used forinoculation.9. Test Specimens9.1 From each test unit (Note 2), cut duplicate 0.75 in.diameter discs. If the test unit is of different construction oneach side, two spec
22、imens 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.9.2 A test unit containing no biocide should be included asa positive stain control.9.3 A test unit known to inhibit staining by
23、 Streptomycesspecies should be included as a negative staining control.9.4 Heavily-soiled specimens should be rinsed vigorously inwater.9.5 If sterilization of test specimens is considered necessary,ethylene oxide is the sterilant of choice. Ethylene oxide residuemay affect test outcome. Proper aera
24、tion after sterilization isnecessary.10. Procedure10.1 InoculationPour sufficient yeast malt extract agarinto suitable sterile dishes (see 7.1) to provide a solidified agarlayer from 5 to 8 mm in depth. After the agar has solidified,moisten the agar surface by streaking with a cotton swabdipped in s
25、terile water.10.2 Add 3 mL of sterile saline or phosphate buffered salineto the stock culture plate and use the premoistened swab toharvest the Streptomyces cells.10.3 Streak the cell-laden swab on the agar surface bystreaking in a manner which will ensure total coverage by theorganism. This swabbin
26、g technique typically provides suffi-cient growth to yield measurable pink stain on positive staincontrols.10.4 Use sterile forceps to place the sterile test specimens,two to a plate, on the agar surface. There shall be good contactestablished between the test specimen and the inoculated agarsurface
27、. Should the specimens tend to curl, weights (such as58in. sterile stainless steel nuts) may be placed on top ofspecimens to maintain good contact.3The sole source of supply of ISP Medium 2(Yeast Malt Extract Agar), CatalogNo. 2770-010 known to the committee at this time is BD, 1 Becton Drive Frankl
28、inLakes, NJ 07417 or . If you are aware of alternative suppliers, pleaseprovide this information to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.4American Type Culture Collection, 10801 Univ
29、ersity Blvd., Manassas, VA20110. Note current ATCC nomenclature revisions lists this organism as Strepto-myces species instead of Streptoverticllium reticulum. Colony pigmentation shouldbe monitored to ensure pink pigment is present.E1428 15a210.5 IncubationCover the inoculated test plates contain-i
30、ng the specimens and incubate at 29 6 1C for 14 days.NOTE 3Covered petri dishes containing yeast malt extract agar areconsidered to have the desired humidity.11. Interpretation of Results11.1 Observe the degree of staining on the samples bycomparing with the inoculated, untreated control.11.2 If sta
31、ining is not seen on the surface of the specimen,use sterile forceps to lift and turn the specimen over and inspectthe underside for stain. The degree of staining is determined bythe amount of sample surface stained rather than the intensityof the color. Any possible sample discoloration that is not
32、 pinkin color should be disregarded when determining the 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 1.11.
33、3 Leachability of AntimicrobialsA“ zone of inhibition”may be observed where no organisms grow on the agaradjacent to the specimen. This indicates that the antimicrobialmay be leaching from the specimen.12. Report12.1 Report the visual rating of stain in accordance with11.2.13. Precision and Bias13.1
34、 A precision and bias statement cannot be made for thistest method at this time.14. Keywords14.1 antimicrobial efficacy; pink stain; plastics; polymericsolids; Streptomyces speciesASTM International takes no position respecting the validity of any patent rights asserted in connection with any item m
35、entionedin 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 committ
36、ee and must be reviewed every five years andif not 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 meet
37、ing 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, P
38、O 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).
39、Permission rights to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 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 15a3
