1、Designation: D 4300 01 (Reapproved 2008)Standard Test Methods forAbility of Adhesive Films to Support or Resist the Growth ofFungi1This standard is issued under the fixed designation D 4300; the number immediately following the designation indicates the year oforiginal adoption or, in the case of re
2、vision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the Department of Defense.1. Scope*1.1 These test meth
3、ods test the ability of adhesive films toinhibit or support the growth of selected fungal speciesgrowing on agar plates by providing means of testing the filmson two agar substrates, one which promotes microbial growth,and one which does not.1.2 These test methods are not appropriate for all adhesiv
4、es.The activity of certain biocides may not be demonstrated bythese test methods as a result of irreversible reaction with someof the medium constituents.NOTE 1As an example, quaternary ammonium compounds are inac-tivated by agar.1.3 A test method is included for use with low-viscosityadhesives alon
5、g with an alternative method for use withmastic-type adhesives. Also, a method approved by the gov-ernment is given.1.4 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.5 This standard does not purport to address all of thesaf
6、ety 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. These test methodsare designed to be used by persons trained in correct m
7、icro-biological techniques. Specific precautionary statements aregiven in Section 7 and in 14.3.2.2. Referenced Documents2.1 ASTM Standards:2D 907 Terminology of AdhesivesD 1286 Methods of Test for Effect of Mold Contaminationon Permanence of Adhesive Preparations and AdhesiveBonds3G21 Practice for
8、Determining Resistance of SyntheticPolymeric Materials to Fungi2.2 TAPPI Method:T487 Fungus Resistance for Paper and Paperboard43. Terminology3.1 DefinitionsMany terms in this test method are definedin Terminology D 907.3.2 Definitions of Terms Specific to This Standard:3.2.1 adhesive preparation, n
9、the adhesive as packagedfor distribution, storage, and use.3.2.2 adhesive film, nthe small portion of the adhesivepreparation, as prepared for use by the consumer, either withadditives or as received, which is cast on a substrate, cured 24h, and represents the glue line.3.2.2.1 DiscussionFor purpose
10、s of these test methods theadhesive film is the thin layer of adhesive spread on either the21-mm fiberglass disk as described in 14.2, or the adhesivelayer 3 mm thick which is cast on the tile squares as describedin 15.1.3.2.3 zone of inhibition, nthe area on an inoculated agarplate surrounding the
11、adhesive-coated disk or tile, showing areduced fungal growth or an absence thereof.3.3 Abbreviations:3.3.1 PDApotato dextrose agar.3.3.2 MSAmineral salts agar.3.3.3 ZIzone of inhibition.4. Significance and Use4.1 These test methods are designed to be used to determinethe susceptibility of the adhesi
12、ve film to biodegradation andwhether the adhesive will carry into the bond line sufficient1These test methods are under the jurisdiction of ASTM Committee D14 onAdhesives and are the direct responsibility of Subcommittee D14.30 on WoodAdhesives.Current edition approved April 1, 2008. Published April
13、 2008. Originallyapproved in 1983. Last previous edition approved in 2001 as D 4300 01.2For referenced ASTM 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 Summar
14、y page onthe ASTM website.3Withdrawn.4Available from Technical Association of the Pulp and Paper Industry (TAPPI),15 Technology Parkway South, Norcross, GA 30092, http:/www.tappi.org.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Driv
15、e, PO Box C700, West Conshohocken, PA 19428-2959, United States.anti-fungal properties to prevent growth of fungi frequentlypresent on the gluing equipment, on adherends, or in theadhesive as applied.4.2 Potato dextrose agar (PDA) provides a complete me-dium for the growth of fungi, while mineral sa
16、lts agar (MSA)lacks a carbohydrate source and provides a less favorablemedium. Use of PDA tests the adhesive film for its ability toresist the growth of fungi on its surface as well as its ability torepel a copious growth of fungi on the adjacent agar surface.Use of MSA tests the adhesive film prima
17、rily for its ability toresist the growth of fungi on its surface. When it is used, thereis a reduced possibility that the growth from the agar will bemis-read as coming from the adhesive film, since fungalgrowth on the adjacent agar will be scant.NOTE 2The method given here using the MSA is based on
18、 PracticeG21, adapted to be used with adhesives. Requirements to meet theapproval of government specifications are the use of the MSA describedin 10.2, and a mixed species of fungi described in 8.2 for the inoculum.4.3 The results obtained when using the procedures given inthis method apply only to
19、the species used for the testing. Thetest species listed in Section 8 are frequently used by labora-tories to test for antifungal properties, but they are not the onlyones which could be used. Selection of the fungal species totest against requires informed judgment by the testing labora-tory or by
20、the party requesting the tests. These methods areespecially useful when species that have been isolated fromcontaminated adhesives are used as the test species (seeSection 8) to aid in the selection of more effective fungicides.4.4 The efficacy of some biocides may change in storagedue to the chemic
21、al and thermal environment to which they aresubjected as components of certain adhesives. These testmethods are not appropriate for determining the effect offungal contamination on adhesives under water-soaking con-ditions, because they are not designed to cover the possibilityof water-soluble bioci
22、des leaching out of the bond line.4.5 These test methods are dependent upon the physiologi-cal action of living microorganisms under a reported set ofconditions. Conclusions about the resistance of the test adhe-sive to fungal attack can be drawn by comparing the results tosimultaneously run control
23、s of known resistance. See X5.2 forstatements regarding test repeatability.5. Apparatus5.1 In addition to the standard equipment found in any fullyequipped microbiological laboratory, items from the followinglist are needed for various tests. Not all items are needed foreach test.5.1.1 Chromist Labo
24、ratory Spray Unit.55.1.2 Constant Temperature Chamber, capable of beingmaintained at 35 6 0.5C (95 6 1F) or 25 6 0.5C (77 61F), or two chambers if needed simultaneously.5.1.3 Filter Disk, Glass Microfibre, 934-AM, diameter-21mm.65.1.4 Filter Disk, Sterile Whatman No. 1.65.1.5 Filter Paper Assay Disk
25、, 1.5 cm diameter, sterile.Schleicher and Schnell, Inc., or the equivalent, has been foundsatisfactory for this purpose.65.1.6 Glass Rods, 305 mm in length having a diameter of6.3 mm.5.1.7 Glove Bag, 68 cm in length and width, 38 cm inheight.75.1.8 Hemacytometer Levy Counting Chamber, cell depth-0.1
26、 mm, Newbauer rulings.65.1.9 Hood, Laminar-Flow Type, Class II Type I.85.1.10 Jar, Screw Cap, round, approximately 1 L (1 qt,mason type).5.1.11 Pipet, Pasteur.65.1.12 Petri Dishes, sterile, disposable, top-diameter of150-mm, bottom-height of 15-mm.5.1.13 Refrigerator, capable of maintaining 4 6 1C (
27、39 62F).5.1.14 Teflon Paper or Grid, pressure sensitive overlay,coated with TFE-fluorocarbon (PTFE), vinyl sheet backing, tobe used at up to 93C (200F).96. Materials6.1 Potato Dextrose Agar, Difco or equivalent.6.2 Sterile Deionized or Distilled Water.6.3 Disinfectant SolutionAmphyll, Alcide, or com
28、parableproduct.6.4 Materials for Mineral Salts Agar. (See list in 10.2.1.)6.5 Sorbitan mono-oleate polyoxyethylene.107. Precautions7.1 Assign laboratory personnel trained in correct microbio-logical techniques to run these tests. These test methodsemploy live cultures of fungi, some of which are cap
29、able ofcausing disease or allergic reaction in some humans. Useproper microbiological procedures in order to prevent contami-nation of the cultures or of the work area. Disinfect andsterilize in an approved manner all spills and all equipmentcoming into contact with the cultures. Also sterilize in a
30、napproved manner all cultures and contaminated disposableequipment before discarding. See 1.5 and 14.3.2.7.2 In addition to other precautions, the use of a Class II,Type I containment hood is highly recommended for allprocedures that would cause formation of fungal aerosols. Thistype of laminar flow
31、 hood prevents the spread of fungal sporesthroughout the laboratory and inhalation of spores by theoperator. The hoods should be monitored by a biological safetyofficer or a health physicist if they are to be used withhazardous agents. Refer to the operating manual supplied bythe manufacturer for de
32、tailed information. This warning ap-plies specifically to the use of the Chromist laboratory sprayunit listed in 5.1.1 and in the instructions in 14.3.2.5Available from Gelman Sciences, Ann Arbor, MI.6Available from laboratory supply houses.7Available from Instruments for Research and Industry, 108
33、Franklin Ave.,Cheltenham, PA, or most laboratory supply houses.8The Biogard Hood or similar equipment is available from laboratory supplyhouses.9Gelman Sciences, or most laboratory supply houses.10Available commercially as Tween 80.D 4300 01 (2008)28. Test Species of Fungi118.1 Cultures of one or mo
34、re of the following species aresuggested for use when PDA is the medium:ATCC No.8.1.1 Aspergillus niger 96428.1.2 Aspergillus flavus 96438.1.3 Penicillium pinophilum 9644 (See X1.1.6)8.1.4 Phanerochaete chrysosporium 247258.1.5 Aureobasidium pullulansVar.melanigenum 15233NOTE 3The choice of test org
35、anisms is often made from the fungalspecies listed above. Information on these and other species is given inAppendix X1.8.2 Cultures of the following species are used for thegovernment requirements described in Section 16, using MSA:ATCC No.8.2.1 Aspergillus niger 96428.2.2 Aureobasidium pullulansVa
36、r.melanigenum 152338.2.3 Chaetomium globosum 62058.2.4 Gliocladium virens 96458.2.5 Penicillium pinophilum 9644 (See X1.1.6)NOTE 4The species listed in 8.2 are used in Practice G21. Thefollowing optional species are also sometimes used: Aspergillus flavus,(ATCC No. 9643) and Aspergillus versicolor (
37、ATCC No. 11730). See 13.2and Appendix X1.8.3 Other pure cultures or mixed cultures of fungal speciesmay be used, if agreed upon between the interested parties andupon the recommendation of the testing laboratories.9. Sterilization of Equipment and Media9.1 Follow accepted microbiological practices f
38、or sterilizingequipment and media.NOTE 5Two references for sterilization methods are TAPPI T487 (see2.2) and Ref (1).1210. Preparation of Media10.1 Potato Dextrose Agar:10.1.1 Prepare sufficient agar slants and plates for culturepropagation and conducting the tests.10.1.2 Follow the instructions giv
39、en for preparation of thecommercial product. Dissolve using heat and agitation. Trans-fer an appropriate amount of the agar solution to each flaskused for pouring plates, and 10 mL per test tube. Plug flaskwith appropriate closures. Cap tubes loosely with metal,plastic, or foam caps. Autoclave for 1
40、5 min at 103 kPa and atemperature of 121C (250F). Allow the agar to cool to 48 to50C (118 to 122F) before pouring the plates, filling to anapproximate depth of 3 mm. Allow plates to solidify. Tightenthe caps on the tubes and place them in a slanted position tosolidify, making a slant of about 51 mm.
41、 Store slants and platesin refrigerator until needed.10.2 Mineral Salts AgarPrepare sufficient medium fortests as described below:10.2.1 Dissolve in 1 L of water the designated amounts ofthe following reagents:GramsPotassium phosphate (KH2PO4)0.7Magnesium sulfate (MgSO47H2O) 0.7Ammonium nitrate (NH4
42、NO3)10Sodium chloride (NaCl) 0.005Ferrous sulfate (FeSO47H2O) 0.002Zinc sulfate (ZnSO47H2O) 0.002Manganous sulfate (MnSO44H2O) 0.001Agar 15.010.2.2 Adjust the pH of the medium by the addition of0.01N NaOH solution so that after sterilization the pH isbetween 6.0 and 6.5, and sterilize by autoclaving
43、 at 103 kPa,and 121C (250F) for 15 min.10.2.3 Prepare plates as described in 10.1.2, and store in therefrigerator until needed.11. Fungal Cultures11.1 Propagation of Fungal Cultures:11.1.1 Prepare a fresh culture for each species on PDA andlabel by species and ATCC Number. Incubate at 25 6 0.5C(77 6
44、 1F) for a minimum of 10 days or until full sporulationis achieved.11.1.2 Refrigerate the cultures. Prepare new cultures eachmonth. If contamination occurs, discard the cultures andprepare new ones.11.2 Preparation of Fungal Inoculum:11.2.1 Follow the procedure in 11.1.1 to prepare freshcultures on
45、PDA slants for each species to be used to conductthe tests.11.2.2 Harvesting Fungal Cultures and DislodgingSporesTo one tube of each species of fungi, add 15 mL ofsterile distilled or deionized water, containing 0.05 % sorbitanmono-oleate polyoxyethylene. Harvest fungal cultures anddislodge spores b
46、y rubbing the growth gently with a sterileinoculating loop or by removing it with a sterile glass rod.Transfer the washings into a sterilized container containingglass beads and shake thoroughly to break up the clumps. Filterthrough sterile layered cheese cloth or sterile nonabsorbentcotton. Adjust
47、the spore level to 1.0 3 106per mL, using ahemacytometer and the procedure in Annex. Use this sporesuspension of a single species of fungi as the inoculum for thetests described in Sections 14 and 15 when using the optiongiven in 13.1.1.1.11.2.3 For a mixed culture, obtain a spore count on eachfunga
48、l species, and adjust each suspension to the level of 1 306per mL. Combine equal portions of the spore suspensionsfrom each of the species in a common sterilized container. Usethis mixed spore suspension for the tests described in Sections14, 15, and 16 when using the option given in 13.1.1.2.12. Ad
49、hesive Sample12.1 For ready-to-use liquid adhesives, obtain an approxi-mate 250-mL sample. For adhesives to be mixed at the time ofuse, obtain a sufficient sample of each component, mix inaccordance with the manufacturers instructions, and run thetests on the prepared adhesive mix. For mastics, use theadhesive as packaged for the consumer, directly from theapplicator tube.11Cultures may be purchased from theAmerican Type Culture Collection, 12301Parklawn Drive, Rockville, MD 20852.12The boldface numbers in parentheses refer to the references at
