1、Designation: E1428 99 (Reapproved 2009)E1428 15Standard Test Method forEvaluating the Performance of Antimicrobials in or onPolymeric Solids Against Staining by StreptoverticilliumreticulumStreptomyce species (A Pink Stain Organism)1This standard is issued under the fixed designation E1428; the numb
2、er immediately following the 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.INTRODUCT
3、IONWhen certain bacteria and mold species grow on the 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 thesurfac
4、e growth is removed. This test method is used for determining 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 know
5、n to cause undesirable staining in polymeric solids.1. Scope1.1 This test method is used for determining the performance of antimicrobial agents used in or on synthetic polymeric solidsagainst staining intended to assess susceptibility of vinyl and other solid polyer products as well as products tha
6、t may directlycontact vinyl to pink-staining by the actinomycete Streptoverticillium reticulum.bacteria This Streptomyces species.organism hasbeen chosen as an indicator organism, although other organisms have been known to cause undesirable staining in polymericsolids.1.2 This test method is not su
7、itable for evaluating dark-pigmented test samples.1.3 A knowledge of microbiological techniques is recommended for these procedures.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to addr
8、ess all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D3273 Test Metho
9、d for Resistance to Growth of Mold on the Surface of Interior Coatings in an Environmental ChamberD3274 Test Method for Evaluating Degree of Surface Disfigurement of Paint Films by Fungal orAlgal Growth, or Soil and DirtAccumulationE2756 Terminology Relating to Antimicrobial and Antiviral Agents3. T
10、erminology3.1 For definitions of terms used in this standard refer to Terminology E2756.3.2 Definitions:1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agentsand is the direct responsibility ofSubcommittee E35.15 on Antimicrob
11、ial Agents.Current edition approved Oct. 1, 2009Feb. 15, 2015. Published November 2009June 2015. Originally approved in 1991. Last previous edition approved in 20042009 asE1428 99(2004).(2009). DOI: 10.1520/E1428-99R09.10.1520/E1428-15.2 For referencedASTM standards, visit theASTM website, www.astm.
12、org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what chan
13、ges have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the offic
14、ial document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.1 microbially induced stainingundesirable pigmentation or disfiguration of an object due to surface colonization by certainmicroorganisms.3.2.1.1 DiscussionBoth bacteria
15、 and fungi produce metabolic pigments that can result in surface stains on susceptible objects.3.2.2 Pink stain organism, nrefers to a staining phenomena caused by a metabolic pigment produced by actinomycete bacteriaspecifically, Streptomyces species ATCC 25607 (deposited as Streptoverticillium ret
16、iculum).34. Summary of Test Method4.1 Test specimens are placed on an agar surface inoculated with Streptoverticillium reticulumspecies and incubated. Afterincubation, test specimens are rated visually by percentage of sample area stained.5. Significance and Use5.1 Methods such as D3273 Standard Tes
17、t Method for Resistance to Growth of Mold on the Surface of Interior Coatings in anEnvironmental Chamber and D3274 Standard Test Method for Evaluating the Degree of Surface Disfigurement of Paint Films byFungal or Algal Growth or Soil or Dirt Accumulation provide means for assessing mold and algal s
18、taining on paints.5.2 This test method provides a technique for evaluating antimicrobials in or on polymeric solids against staining byStreptoverticillium reticulum,Streptomyces species, bacteria and should assist in the prediction of performance of treated articlesunder actual field conditions.5.3
19、Conditioning of the specimens, such as exposure to leaching, weathering, and heat treatment, may have significant effectson performance of antimicrobials against staining. Determination of these effects is not included in this test method.6. Interferences6.1 An interference may be caused by contamin
20、ation of plates and agar by unwanted organisms that settle from the environment.6.2 Dark pigments mask observation of the pink stain.7. Apparatus7.1 Petri dishes, 100 mm diameter.NOTE 1Presterilized and disposable plastic petri dishes are available from most laboratory supply houses.7.2 Cotton swabs
21、, sterile.7.3 IncubatorIncubating equipment for this test method shall maintain a temperature of 29 6 1C.7.4 Autoclave.7.5 Sterilizer, ethylene oxide (optional).8. Reagents and Materials8.1 Yeast Malt Extract Agar (ISP Medium 2)ISP Medium 2 (Yeast Malt Extract Agar) 3Prepare this medium according to
22、manufacturers directions. Sabouraud Dextrose agar prepared per label directions may also be used (initiates robust vegetativegrowth and pigment production).8.2 InoculumStreptoverticillium reticulum Inoculum Streptomyces species ATCC 25607.25607 (deposited as Streptoverti-cillium reticulum).4 Maintai
23、n stock cultures on yeast malt extract agar. The stock may be kept for not more than 12 months atapproximately 3 to 10C. Subcultures, incubated at 29 6 1C for 7 to 14 days, shall be used for inoculation.9. Test Specimens9.1 From each test unit (Note 2), cut duplicate 0.75 in. diameter discs. If the
24、test unit is of different construction on each side,two specimens of each, two face up and two face down, shall be tested.NOTE 2A test unit is a solid in the form of plastic sheets, films, coated fabrics or similar polymeric materials.9.2 A test unit containing no biocide should be included as a pos
25、itive stain control.3 The sole source of supply of Bacto-Yeast ISP Medium 2(Yeast Malt Extract Agar, StockAgar), Catalog No. 0770-012770-010 known to the committee at this time isDifco Labs, P.O. Box 1058A, Detroit, MI 28232. BD, 1 Becton Drive Franklin Lakes, NJ 07417 or . If you are aware of alter
26、native suppliers, please providethis information toASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,1 which you mayattend.4 American Type Culture Collection, 10801 University Blvd., Manassas, VA20110. Note currentAT
27、CC nomenclature revisions lists this organism as Streptomyces speciesinstead of Streptoverticllium reticulum. Colony pigmentation should be monitored to ensure pink pigment is present.E1428 1529.3 A test unit known to inhibit staining by Streptoverticillium reticulumStreptomyces species should be in
28、cluded as a negativestaining control.9.4 Heavily-soiled specimens should be rinsed vigorously in water.9.5 If sterilization of test specimens is considered necessary, ethylene oxide is the sterilant of choice. Ethylene oxide residuemay affect test outcome. Proper aeration after sterilization is nece
29、ssary.10. Procedure10.1 InoculationPour sufficient yeast malt extract agar into suitable sterile dishes (see 5.17.1) to provide a solidified agar layerfrom 5 to 8 mm in depth. After the agar has solidified, moisten the agar surface by streaking with a cotton swab dipped in sterilewater.10.2 Add 3 mL
30、 of sterile saline or phosphate buffered saline to the stock culture plate and use the premoistened swab to harvestthe StreptoverticilliumStreptomyces cells.10.3 Streak the cell-laden swab on the agar surface by streaking in a manner which will ensure total coverage by the organism.This swabbing tec
31、hnique typically provides sufficient growth to yield measurable pink stain on positive stain controls.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 agar surface. S
32、hould the specimens tend to curl, weights (such as 58 in.sterile stainless steel nuts) may be placed on top of specimens to maintain good contact.10.5 IncubationCover the inoculated test plates containing the specimens and incubate at 29 6 1C for 14 days.NOTE 3Covered petri dishes containing yeast m
33、alt extract agar are considered to have the desired humidity.11. Interpretation of Results11.1 Observe the degree of staining on the samples by comparing with the inoculated, untreated control.11.2 If staining is not seen on the surface of the specimen, use sterile forceps to lift and turn the speci
34、men over and inspect theunderside for stain. The degree of staining is determined by the amount of sample surface stained rather than the intensity of thecolor. Any possible sample discoloration that is not pink in color should be disregarded when determining the degree of stain.However, pigmented s
35、amples that show a color change associated with pink (that is, a blue pigmented sample changing to purple)should be rated according to degree of stain. Rate the degree of stain in accordance with Table 1.11.3 Leachability of AntimicrobialsA“ zone of inhibition” may be observed where no organisms gro
36、w on the agar adjacentto the specimen. This indicates that the antimicrobial may be leaching from the specimen.12. Report12.1 Report the visual rating of stain in accordance with 9.111.2.13. Precision and Bias13.1 A precision and bias statement cannot be made for this test method at this time.TABLE
37、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 15314. Keywords14.1 antimicrobial efficacy; pink stain; plastics; pol
38、ymeric solids; Streptoverticillium reticulumStreptomyces speciesASTM 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 suc
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