1、Designation: D4576 08 (Reapproved 2013)Standard Test Method forMold Growth Resistance of Wet Blue1This standard is issued under the fixed designation D4576; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、 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 the determination of moldgrowth resistance of wet blue and wet white subject to storageand shipping requirem
3、ents and intended for use in leathermanufacturing. This test method may not be suitable toevaluate fungicides that are inactivated by proteins. Thisincludes alkyldimethylbenzyl ammonium chlorides.1.2 Conclusions about mold growth resistance are drawnfrom the results by comparing the test with a simu
4、ltaneouslyrun control of known resistance. Success or failure is deter-mined by the amount of mold growth relative to the control.1.3 To allow use of this test method by any laboratory,flexibility has been permitted in times, temperature, andhumidity of incubation, inoculum, hide sampling area, andc
5、hoice of control. These may be adjusted to fit local conditionsbut must be standardized.1.4 For mold growth resistance of wet white, the procedureis identical, substitute wet white for wet blue in the standardmethod.1.5 The values stated in SI units are to be regarded asstandard. No other units of m
6、easurement are included in thisstandard.1.6 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
7、limitations prior to use.2. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 wet bluehide or skin, or split of a hide or skin,tanned with basic chromium sulfate, containing approximately50 % moisture and having an acidic pH.2.1.2 wet whitea hide or skin, or split of a hide or skin
8、tanned with organic or non-organic tanning agents (excludingchromium or iron containing agents and vegetable extracts),containing approximately 50 % moisture.3. Summary of Test Method3.1 Wet blue test specimens are surrounded by but notcovered with agar, inoculated, and incubated.3.2 After various i
9、ncubation periods, mold growth is ratedas a percentage of the wet blue surface covered by mold.3.3 Resistance to mold growth of the wet blue test specimenis determined by comparison with wet blue of known resis-tance characteristics (the control), that is tested simultaneously.4. Significance and Us
10、e4.1 This test method provides a technique for evaluatingmold growth resistance characteristics of wet blue, and shouldassist in the prediction of storage time before molding occurs.4.2 The degree of correlation between this test and commer-cial quantities of wet blue in storage or shipment situatio
11、ns, orboth, has not been fully determined.5. Interferences5.1 Acommon interference is contamination of plates, agar,or samples by unwanted organisms that settle in from theenvironment.5.2 Volatility and Leachability of BiocidesA “zone ofinhibition” where no mold grows on the agar adjacent to thespec
12、imen indicates that the fungicide may leach.6. Apparatus6.1 Petri Dishes, 120 mm diameter. Sterile plastic dispos-able dishes are preferred.6.2 Incubator, or location providing similar conditions be-ing free of drafts, and capable of a constant (6 2C) tempera-ture within the 26 to 30C range.6.3 Medi
13、cine droppers, disposable plastic type delivering 30to 35 drops per mL.1This test method is under the jurisdiction ofASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee D31.02 on Wet Blue.Current edition approved May 1, 2013. Published May 2013. Originallyapproved in 1986.
14、Last previous edition approved in 2008 as D4576 - 08. DOI:10.1520/D4576-08R13.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States17. Reagents and Materials7.1 Potato Dextrose Agar,2a dehydrated plating mediumused in culturing yeasts and m
15、olds from dairy products.7.2 Inoculum,3Aspergillus niger 1 106spores per mL, orother organism or a combination of organisms known to beindigenous to the storage area of the wet blue.8. Sampling, Test Specimen, and Test Units8.1 Take test specimens from equivalent hide locations (forexample, butt are
16、a) for both test and control.8.2 If unable to test immediately, hold test specimens inseparate plastic bags and keep cool.8.3 Test specimens should be a square, with a side of 25.4mm (1 in.).8.4 Use three test specimens for each test unit of wet bluesurface to be evaluated.9. Procedure9.1 Agar Prepa
17、ration:9.1.1 Agar RequirementsA split wet blue test specimenrequires about 25 mL solution and an unsplit wet blue testspecimen requires about 40 mL. Calculate number of millilitresof agar required for tests to be performed, allowing 50 mL forvitality check.9.1.2 Weigh out 3.9 g potato dextrose agar
18、for every 100 mLof agar required.9.1.3 Pour a volume of water equivalent to total millilitresof agar solution to beaker. Bring water to boiling on hot plateequipped with magnetic stirrer mechanism. While stirring,slowly add dry agar.9.1.4 Boil agar for 20 min.NOTE 1Pressure cooking for 20 min. is pr
19、eferable to open boiling.9.1.5 Cover with aluminum foil to prevent contaminationand cool to 50C before pouring.NOTE 2This temperature is critical, as 50C allows some water ofcondensation to develop on petri dish cover providing humidity for growthof fungus.9.2 Agar Plate Preparation:9.2.1 Place one
20、wet blue test specimen in center of eachpetri dish with the surface to be tested facing up.9.2.2 Carefully lift cover from each dish and pour agar justup to, but not over, the top surface of the test specimen.9.2.3 Prepare one dish with agar only (without wet blue) forevaluation of the vitality of t
21、he inoculum.9.2.4 Let agar solidify for about 20 min.9.3 Inoculation:9.3.1 Reduce working stock of 1 106spores per mL to1105by diluting 1 volume to 10 volumes with water.9.3.1.1 Use tap water, that has been freshly boiled for 20min. and cooled to room temperature, for making dilutions.9.3.1.2 Prepar
22、e only enough diluted suspension for use in a48-hour period.9.3.1.3 Keep organism stock suspensions refrigerated atabout 4C. Do not freeze.9.3.2 Use three drops of 1 105spores per milliliter perplate using a plastic disposable medicine dropper. Deposit onedrop directly on the sample and one drop to
23、either side asshown in Fig. 1.9.3.3 Let dishes set about 1 h.NOTE 3If moved too quickly the inoculum runs over the specimensurface.NOTE 4Keep work area as clean and aseptic as possible. Work in anarea of minimal air circulation while handling wet blue, pouring agar, andinoculating plates. Keep cover
24、s on petri dishes at all times except whenpouring and inoculating.9.4 Incubate up to three weeks at constant temperature in aclean location where they will not be disturbed.9.4.1 Constant temperature is more important than theprecise temperature. A temperature of 26 to 30C is acceptableand should no
25、t vary more than 6 2C.NOTE 5Storage in a clear plastic box in a boiler room may besufficient. Use separation to prevent cross contamination.NOTE 6More rapid growth occurs at higher temperature.9.5 Control wet blue specimens of known mold resistancemust be done simultaneously with test specimens.Asuc
26、cessfultest will have less mold growth than the control.NOTE 7After completion of work, test specimens should be sterilizedby autoclaving. If that is not practical, cook for 30 min. in a pressurecooker and discard in a trash container.10. Interpretation of Results10.1 The following rating scale of 0
27、 to 4 can be used whereeach number represents the degree of growth observed on thespecimen (not on the agar) at any selected period.0No growth on specimen,2The sole source of supply of a product that meets the requirements of thismethod known to the committee at this time is Potato Dextrose Agar sto
28、ck no.0013-01-4, available from Difco Labs, P.O. Box 1058A, Detroit, MI 28232. If youare aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will receive careful consideration at ameeting of the responsible technical committee,1which you m
29、ay attend.3An inoculum that meets the requirements of this method is available as ATCC(AmericanType Culture Collection) 16404, and is available from several sources forlaboratory supplies. FIG. 1 Specimen with Inoculum Locations Shown (X)D4576 08 (2013)20.5Less than 12 % of specimen surface overgrow
30、n bymold,125 % of specimen surface overgrown by mold,250 % of specimen surface overgrown by mold,375 % of specimen sulfate overgrown by mold, and4100 % of specimen surface overgrown by mold.10.1.1 Mold growth above but not touching specimen willalso be rated zero.10.2 Suggested rating periods are 3,
31、 7, and 14 days.11. Report11.1 Report of results must contain the following:11.1.1 Rating of each test specimen plate.11.1.2 Surface tested (grain, flesh, split, etc.).11.1.3 Period of incubation when rated.11.1.4 Inoculum used.11.1.5 Location of hide from which test specimens weretaken.11.1.6 Tempe
32、rature of incubation.11.1.7 Humidity of incubator, if known.11.1.8 Vitality of inoculum.11.1.9 Difference in treatment of specimen between test andcontrol.12. Precison and Bias12.1 Precision or bias of this test method for measuringmold growth resistance of wet blue are indeterminate, since theresul
33、t merely states whether there is conformance to the criteriafor success specified in the procedure, as outlined in 1.2.13. Keywords13.1 agar plate; blue stock; inoculum; mold; wet blue; wetwhiteASTM International takes no position respecting the validity of any patent rights asserted in connection w
34、ith any item mentionedin 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 tec
35、hnical committee 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 considera
36、tion at a meeting 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 H
37、arbor Drive, PO 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).D4576 08 (2013)3
