1、Designation: D5590 17Standard Test Method forDetermining the Resistance of Paint Films and RelatedCoatings to Fungal Defacement by Accelerated Four-WeekAgar Plate Assay1This standard is issued under the fixed designation D5590; the number immediately following the designation indicates the year ofor
2、iginal 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 covers an accelerated method fordetermining
3、the relative resistance of two or more paints orcoating films to fungal growth.1.2 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with
4、 its use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accor-dance with internationally recognized pr
5、inciples 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.2. Referenced Documents2.1 ASTM Standards:2D1005 Test Method for Measure
6、ment of Dry-Film Thick-ness of Organic Coatings Using MicrometersD3273 Test Method for Resistance to Growth of Mold on theSurface of Interior Coatings in an Environmental Cham-berD3456 Practice for Determining by Exterior Exposure Teststhe Susceptibility of Paint Films to MicrobiologicalAttackD4587
7、Practice for Fluorescent UV-Condensation Expo-sures of Paint and Related CoatingsD4708 Practice for Preparation of Uniform Free Films ofOrganic CoatingsD6132 Test Method for Nondestructive Measurement of DryFilm Thickness of Applied Organic Coatings Using anUltrasonic Coating Thickness GageD6695 Pra
8、ctice for Xenon-Arc Exposures of Paint andRelated CoatingsG21 Practice for Determining Resistance of Synthetic Poly-meric Materials to Fungi3. Summary of Test Method3.1 This test method outlines a procedure to (1) prepare asuitable specimen for testing, (2) inoculate the specimen withthe proper fung
9、al species, (3) expose the inoculated samplesunder the appropriate conditions for growth, and (4) provide aschedule and guidelines for visual growth ratings. This testmethod is not designed to include all the necessary proceduresto maintain the proper microbiological techniques required toprovide th
10、e most accurate results.4. Significance and Use4.1 Defacement of paint and coating films by fungal growth(mold, mildew) is a common phenomenon, and defacement byalgal growth can also occur under certain conditions. It isgenerally known that differences in the environment, lighting,temperature, humid
11、ity, substrate pH, and other factors inaddition to the coating composition affect the susceptibility ofa given painted surface. This test method attempts to provide ameans to comparatively evaluate different coating formulationsfor their relative performance under a given set of conditions.It does n
12、ot imply that a coating that resists growth under theseconditions will necessarily resist growth in the actual applica-tion. The method is not intended to simulate or replace indooror outdoor exposure of paint films or related coatings.NOTE 1It is hoped that a ranking of relative performance would b
13、esimilar to that ranked from outdoor exposures. Paint designated for servicein exterior conditions should be pre-conditioned by laboratory acceleratedweathering prior to exposure to fungi. All pre-conditioning must bedetailed in the final report. This test method however, should not be usedas a repl
14、acement for exterior exposure (that is, Practice D3456) sincemany other factors, only a few of which are listed will affect those results.1This test method is under the jurisdiction of ASTM Committee D01 on Paintand Related Coatings, Materials, and Applications and is the direct responsibility ofSub
15、committee D01.28 on Biodeterioration.Current edition approved Dec. 1, 2017. Published March 2018. Originallyapproved in 1994. Last previous edition approved in 2010 as D5590 00 (2010)1.DOI: 10.1520/D5590-17.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer
16、 Service 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 StatesThis international standard was developed
17、 in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.14.2 Familiarity with mi
18、crobiological techniques is required.This test method should not be used by persons without at leastbasic microbiological training.5. Apparatus and Materials5.1 Balance, capable of weighing to 0.10 g.5.2 Incubator, or other device capable of maintaining aconstant temperature between 25 and 30C, rela
19、tive humidityof 85 %.5.3 Refrigerator, or other device capable of maintaining atemperature of 4 6 2C.5.4 Petri Dishes, 100 by 15 mm (3.9 by 0.6 in.).5.5 Autoclave, capable of producing 103 kPa (15 psi) ofsteam pressure at 121C and maintaining it for a minimum of15 min. An autoclave is not necessary
20、if pre-prepared mediaplates are used.5.6 Paint Brush, coarse bristle, 12 to 19 mm (12 to34 in.).5.7 Substrate, Filter Paper (Glass fiber, Grade 391, 4.2 cm(1.65 in.) or drawdown paper (unlaquered chart paper 216 by280 mm (8.5 by 11 in.), cut into ten 216 by 28-mm (8.5 by1.1-in.) strips.5.8 Atomizer
21、or Chromatography Sprayer.5.9 Sterile Glass Rods, Forceps, 250-mL Glass ErlenmeyerFlasks, Test Tubes, and other routine microbiological equip-ment.5.10 Potato Dextrose Agar (PDA) or Malt Agar.35.11 Nutrient-Salts Agar (as found in Practice G21).5.11.1 Prepare this medium by dissolving in 1 Lof water
22、 thedesignated amounts of the following reagents:Monopotassium phosphate (KH2PO4) 0.7 gMangnesium sulfate (MgSO47H2O) 0.7 gAmmonium nitrate (NH4NO3) 1.0 gSodium chloride (NaCL) 0.005 gFerrous sulfate (FeSO47H2O) 0.002 gZinc sulfate (ZnSO47H2O) 0.002 gManganese sulfate (MnSO4H2O) 0.001 gAgar 15.0 gDi
23、potassium phosphate (K2HPO4) 0.7 g5.11.2 Sterilize the test medium by autoclaving at 121C(250F) for 20 min. Adjust the pH of the medium so that aftersterilization the pH is between 6.0 and 6.5.5.12 Nutrient-Salts Solution, (see 5.11 without agar).5.13 Counting Chamber (Hemocytometer).5.14 Glass Wool
24、.5.15 Laboratory Accelerated Weathering and LeachingEquipment, if used.6. Reagents and Materials6.1 Purity of ReagentsReagent grade chemicals should beused in all tests. Unless otherwise indicated, it is intended thatall reagents should conform to the specifications of theCommittee on Analytical Rea
25、gents of the American ChemicalSociety, where such specifications are available.4Other gradesmay be used, provided they are first ascertained to be ofsufficiently high purity to permit use without decreasing theaccuracy of the determination.6.2 Purity of WaterUnless otherwise indicated, referencesto
26、water are understood to mean distilled water or water ofequal or higher purity.6.3 PDA or Malt Agar plates can be purchased prepared, orthe PDAand MaltAgar powder can be purchased and preparedaccording to the instructions using standard microbiologicaltechniques and equipment.7. Preparation of the F
27、ungal Spore Inocula7.1 Fungal CulturesUse the following test fungi in pre-paring the inocula:5,6,7,8Fungi ATCC #5NRRL6Aspergillus niger 6275 334Penicillium pinophilum711797 3647Aureobasidium pullulans89348 62100NOTE 2These organisms were selected based on the historical datafrom use in Test Method D
28、3273. Other organisms may be of specificinterest for certain applications or geographical areas. Such other purecultures, or isolated wild strains, may be used as agreed upon by theparties involved.7.2 Maintain stock cultures of these fungi separately on anappropriate medium such as potato dextrose
29、agar plates orslants. The stock culture may be kept for not more than 4months at approximately 3 to 10C (37 to 50F). Subcultureindividual fungi onto slants or plates 7 to 20 days at 28 to 30C(82 to 86F) prior to each experiment, and use these subcul-tures in preparing the spore suspension.7.3 Prepar
30、e a spore suspension of each of the test fungi bypouring into one subculture of each fungus a sterile 10-mLportion of water, or of a sterile solution containing 0.05 g/L ofa nontoxic wetting agent such as sodium dioctylsulfosuccinate.Swirl or gently agitate the slant or plate to loosen the spores.Ca
31、refully aspirate the water and spore suspension with a sterilePasteur pipet (trying to avoid obtaining mycelia).7.4 Filter the shaken or ground suspension through a thinlayer of sterile glass wool in a glass funnel into a sterile flaskin order to remove mycelial fragments.7.5 Dilute the spores suspe
32、nsion with sterile nutrient saltssolution such that the resultant spore suspension contains 0.8 to1.2 by 106spores/mL as determined with a counting chamber.7.6 Repeat this operation for each organism used in the test.The A. pullulans spores should be maintained separately and3Pre-prepared plates are
33、 available from microbiological supply companies, orthey may be prepared using standard microbiological equipment and techniques.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the America
34、n Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.5American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,MD 20852, is t
35、he current known source of the fungal strains used in this testing.6NRRL strains without a Certificate of Analysis are available from the USDAAgricultural Research Service.7Historically known as Penicillium funiculosum. It has also been re-identified asTalaromyces pinophilus by DNA barcode sequencin
36、g.8Historically known as Pullularia pullulans.D5590 172used as a separate inoculum for a separate set of plates andsamples. Blend equal volumes of the remaining organismsresultant spore suspensions to obtain the mixed spore suspen-sion.7.7 The mixed spore suspension may be prepared fresh eachday or
37、may be held in the refrigerator at 3 to 10C (37 to 50F)for not more than four days. The individual spore suspensionsmay be held in the refrigerator at 3 to 10C (37 to 50F) for notmore than fourteen days.8. Preparation of Test Specimens8.1 Aset of coatings to be tested shall contain a positive anda n
38、egative growth control. That is, one that is known to supportfungal growth, and one that is known to inhibit growthcompletely. A set of Whatman #2 (or equivalent) filter papersor the drawdown papers without coating may be suitablegrowth controls serving as a viability control for the fungalspores. A
39、 sterile agar plate can be used as one of the controls.8.2 Handle the disks or drawdown sections with steriletongs or tweezers.8.3 Coatings to be tested shall be applied to 4.2-cm (1.65-in.) glass fiber filter paper disks, or to the 28 by 216-mm (1.1by 8.5-in.) drawdown strips. The samples are prepa
40、red forevaluation by brush coating strips of drawdown paperboard orglass filter disks with each sample in triplicate. Films can alsobe prepared as instructed in Practice D4708. Take care to applya thin, even coating, with the same thickness for all coatingsamples, as specified by the paint manufactu
41、rer, unless partiesdecide otherwise. Coating thickness can be recorded by spreadrate (for example, grams/square cm) or confirmed with TestMethod D1005 or Test Method D6132, or both, as agreedbetween parties and reported in the final report.NOTE 3One or both sides of the substrate (drawdown strips or
42、 filterpaper) may be coated as agreed upon by the parties involved and shouldbe noted in the report.NOTE 4Coating thickness can be confirmed with Test Method D1005or Test Method D6132, or both, and may be reported in the final report ifperformed. Alternatively, coating thickness can be recorded by s
43、pread rate(for example, grams/square cm).8.4 After application, allow to dry or cure at conditionsagreed upon by interested parties.8.5 If accelerated weathering, heat aging, or other precon-ditioning of samples is also to be run, prepare a separate set oftriplicate sample disks or strips. The resul
44、ts from these samplesmay be compared with those from the unweathered or uncon-ditioned samples.NOTE 5There are a variety of methods that could be used to simulate,in an accelerated manner, effects of weathering (sunlight or rain, or both)on the sample. Recommended conditioning of specimens by artifi
45、cialweathering may be done per one of the following practices: Test Cycle 2in Practice D4587 or Test Cycle 6 for Practice D6695. Acceleratedweathering conditions must be specified in the report and agreed upon byinterested parties.NOTE 6A leaching test may be conducted as agreed upon byinterested pa
46、rties.8.6 If the drawdown strips are being used, cut them intoroughly 28-mm (1.1-in.) squares. Place these specimensquares, or the coated filter disks, on the center of pre-pouredagar plates. If the plates were stored in the refrigerator, allowthem to equilibrate to room temperature prior to placeme
47、nt ofthe samples.8.6.1 This test may be conducted on a nutritive agar plates(either PDA or Malt Agar) alone. However, if all samples failcompletely on the nutritive agar plates, additional informationcould be obtained by repeating the samples testing usingnutrient salts agar plates (without a carbon
48、 source in the plates,growth and test conditions are less severe). This additionaltesting may be run simultaneously if agreed upon between theparties involved.9. Procedure9.1 Inoculation of the Test Specimens:9.1.1 The A. niger and P. funiculosum may be testedtogether on the same plates. The A. pull
49、ulans must be testedseparately to ensure its survival.9.1.2 Combine an equal portion of the A. niger and P.funiculosum spore suspensions.9.1.3 Run a count of the spores using a counting chamber toconfirm the inoculum count for each test (see 7.5).9.1.4 Apply a thin coat of fungal suspension to eachspecimen using a sterile atomizer or pipet, making sure theentire surface of the sample and the agar plate are covered, butnot to oversaturate the samples. Alternately, a separate sterilecotton swab may be used to apply and evenly spread theinoculum over th
