1、Designation: D5590 00 (Reapproved 2010)Standard 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 indic
2、ates 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 covers an accelerated meth
3、od fordetermining 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 an
4、y, 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. Referenced Documents2.1 ASTM Standards:2D822 Practice for Filtered Open-Flame Carbon-Arc E
5、xpo-sures of Paint and Related CoatingsD3273 Test Method for Resistance to Growth of Mold onthe Surface of Interior Coatings in an EnvironmentalChamberD3456 Practice for Determining by Exterior Exposure Teststhe Susceptibility of Paint Films to Microbiological AttackD4141 Practice for Conducting Bla
6、ck Box and Solar Con-centrating Exposures of CoatingsD4587 Practice for Fluorescent UV-Condensation Expo-sures of Paint and Related CoatingsD5031 Practice for Enclosed Carbon-Arc Exposure Tests ofPaint and Related CoatingsG21 Practice for Determining Resistance of Synthetic Poly-meric Materials to F
7、ungi3. Summary of Test Method3.1 This test method outlines a procedure to (1) prepare asuitable specimen for testing, (2) inoculate the specimen withthe proper fungal species, (3) expose the inoculated samplesunder the appropriate conditions for growth, and (4) provide aschedule and guidelines for v
8、isual growth ratings. This testmethod is not designed to include all the necessary proceduresto maintain the proper microbiological techniques required toprovide the most accurate results.4. Significance and Use4.1 Defacement of paint and coating films by fungal growth(mold, mildew) is a common phen
9、omenon, and defacement byalgal growth can also occur under certain conditions. It isgenerally known that differences in the environment, lighting,temperature, humidity, substrate pH, and other factors inaddition to the coating composition affect the susceptibility ofa given painted surface. This tes
10、t method attempts to provide ameans to comparatively evaluate different coating formulationsfor their relative performance under a given set of conditions.It does not imply that a coating that resists growth under theseconditions will necessarily resist growth in the actual applica-tion.NOTE 1It is
11、hoped that a ranking of relative performance would besimilar to that ranked from outdoor exposures. However, this test methodshould not be used as a replacement for exterior exposure (that is, PracticeD3456) since many other factors, only a few of which are listed will affectthose results.NOTE 2Seve
12、ral companies have reported reasonable correlation ofresults from this test with actual use when testing film-forming, pigmentedcoatings. Round-robin testing of this test method versus exterior exposureis planned.1This test method is under the jurisdiction of ASTM Committee D01 on Paintand Related C
13、oatings, Materials, and Applications and is the direct responsibility ofSubcommittee D01.28 Biodeterioration.Current edition approved June 1, 2010. Published July 2010. Originally approvedin 1994. Last previous edition approved in 2005 as D5590 00 (2005). DOI:10.1520/D5590-00R10.2For referenced ASTM
14、 standards, visit the ASTM website, www.astm.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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohoc
15、ken, PA 19428-2959, United States.4.2 Familiarity with microbiological 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 m
16、aintaining aconstant temperature between 25 and 30C, relative 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
17、 it for a minimum of15 min. An autoclave is not necessary 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
18、into ten 216 by 28-mm (8.5 by1.1-in. strips).5.8 Atomizer 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. (see Practice G21, 6.3.
19、)5.12 Nutrient-Salts Solution, (see 5.11 without agar).5.13 Counting Chamber (Hemocytometer).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 theCommi
20、ttee on Analytical Reagents 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 in
21、dicated, referencesto 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
22、. Preparation of the Fungal Spore Inocula7.1 Fungal CulturesUse the following test fungi in pre-paring the inocula:5,6,7,8Fungi ATCC #5MYCO #7Aspergillus niger 6275 .Penicillium funiculosum 11797 391Aureobasidium pullulans59348 .NOTE 3Other organisms may be of specific interest for certainapplicatio
23、ns or geographical areas. Such other pure cultures, or isolatedwild strains, may be used as agreed upon by the parties involved. Theseorganisms were selected based on the historical data from use in TestMethod D3273.7.2 Maintain stock cultures of these fungi separately on anappropriate medium such a
24、s potato dextrose 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 sus
25、pension.7.3 Prepare 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 lo
26、osen the spores.Carefully aspirate the water and spore suspension with a sterilepasteur pipet (trying to avoid obtaining mycelia).7.4 Check the collected spore suspension under the micro-scope for mycelial contamination and make a note of therelative populations of spores versus mycelial forms.7.5 D
27、ilute the spores suspension with sterile nutrient saltssolution such that the resultant spore suspension contains 0.8 to1.2 by 104spores/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 andus
28、ed 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 spore suspension may be prepared fresh each day ormay be held in the refrigerator at 3 to 10C (37 to 50F) for not
29、more than 4 days.3Pre-prepared plates are 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 tes
30、ting of reagents notlisted by the American 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.5The sole source of supply of Aspergillus niger
31、 and Aureobasidium pullulansstrains known to the committee at this time is the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, MD, 20852.6If you are aware of alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive
32、careful consider-ation at a meeting of the responsible technical committee,1which you may attend.7The sole source of supply of Penicillium funiculosum strain known to thecommittee at this time is the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, MD, 20852.8Historically kno
33、wn as Pullularia pullulans.D5590 00 (2010)28. Preparation of Test Specimens8.1 A set of coatings to be tested should preferably containa positive and a negative growth control. That is, one that isknown to support fungal growth, and one that is known toinhibit growth completely.Aset of Whatman #2 (o
34、r equivalent)filter papers or the drawdown papers without coating may besuitable growth controls.8.2 Make sure to handle the disks or drawdown sectionswith sterile tongs or tweezers.NOTE 4Sterilization or aseptic handling of the test material, or both,avoids bacterial or other contamination that may
35、 interfere with the testresults.8.3 Coatings to be tested will be applied to 4.2-cm (1.65-in.)glass fiber filter paper disks, or to the 28 by 216-mm (1.1 by8.5-in.) drawdown strips. The samples are prepared for evalu-ation by brush coating strips of drawdown paperboard, or glassfilter disks with eac
36、h sample in duplicate. Take care to apply athin, even coating, with the same thickness for all coatingsamples.NOTE 5One or both sides of the substrate (drawdown strips or filterpaper) may be coated as agreed upon by the parties involved.NOTE 6With the drawdown strips, this can be conveniently accom-
37、plished by punching a hole in the top of the strip and suspending the stripfrom a drying rack with string or a twist tie. The label for each strip canbe written in the top 12.7 mm (12 in.) of the strip (near the hole) and thecoating applied below that 12.7-mm (12-in.) strip. Another 12.7-mm(12-in.)
38、area can be left uncoated at the bottom of the strip to permitholding the strip while brushing. This would still leave sufficient coatedarea for six 28 by 28-mm (1.1 by 1.1-in.) test squares from each strip. Withthe filter disks, a hole can be punched near the edge of the disk.8.4 After application,
39、 suspend the sample disks or stripsfrom drying racks and allow them to air dry for 24 to 72 h atroom temperature.8.5 If accelerated weathering, heat aging, or other precon-ditioning of samples is also to be run, prepare a separate set ofduplicate sample disks or strips. The results from these sample
40、smay be compared with those from the unweathered or uncon-ditioned samples.NOTE 7There are a variety of methods that could be used to simulateaccelerated effects of weathering (sunlight or rain, or both) on the samples.For example, a leach test that is frequently used to simulate the effects ofrainw
41、ater (an important factor for fungal growth) is outlined in Note 8.Conditioning of specimens by artificial weathering may be done accordingto one of the following practices: D822, D4141, D4587 or D5031.NOTE 8A leaching test may be conducted as follows: One of thereplicate sets is leached with distil
42、led water for 24 h, then allowed to airdry. The coated substrate can be leached by suspension for 24 h in 1-gal(4-L) containers of distilled water with a flow rate such that there are six(6) changes in 24 h (or other flow rate as agreed upon by the partiesinvolved). Differences in the integrity of t
43、he coatings after this leachingshould be noted. The test panels are then air dried for 24 h under the sameconditions as the unleached samples (see 7.4).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
44、 the center of pre-pouredagar plates. If the plates were stored in the refrigerator, allowthem to equilibrate to room temperature prior to placement ofthe samples.8.6.1 This test may be conducted on a nutritive agar plates(either PDA or Malt) alone. However, if all samples failcompletely on the nutr
45、itive agar plates, additional informationcould be obtained by repeating the samples testing usingnutrient salts agar plates (without a carbon source in the plates,growth and test conditions are less severe). This additionaltesting may be run simultaneously if agreed upon between theparties involved.
46、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. pullulans 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 t
47、he 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 thesurface is covered, but not to oversaturate the samples.Alternately, a separate sterile cotton swab m
48、ay be used to applyand evenly spread the inoculum over the surface of each testsample. Be certain that the amounts of inoculum used are thesame between each of the various samples under test. Thisshould be done using the same method by the same applicatorat the same time for all samples.9.1.5 Incuba
49、te all plates at 28C under 85 to 90 % relativehumidity for 4 weeks.10. Evaluation of Results10.1 Rate the growth weekly for four weeks according tothe following:Observed Growth on Specimens RatingNone 0Traces of growth (10 %) 1Light growth (1030 %) 2Moderate growth (3060 %) 3Heavy growth (60 % to complete coverage) 4NOTE 9These ratings are for microbial growth, not coating perfor-mance, so as not to be confused with exterior evaluations that run from 10to 0. The lower growth ratings should correspond to longer time periodsof fungus-free surface under
