1、Designation: D 4576 08Standard Test Method forMold Growth Resistance of Wet Blue1This standard is issued under the fixed designation D 4576; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in par
2、entheses 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 requirements and intende
3、d 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 simultaneouslyrun co
4、ntrol 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, andchoice of control
5、. 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 This standard does not purport to address all of thesafety concerns, if any, associated with its
6、 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. Terminology2.1 Definition of Term Specific to This Standard:2.1.1 wet bluehide or skin, or split of a hide or sk
7、in,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 skintanned with organic or non-organic tanning agents (excludingchromium or iron containing agents and vegetable extracts),containing approximatel
8、y 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 incubation 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 t
9、est specimenis determined by comparison with wet blue of known resis-tance characteristics (the control), that is tested simultaneously.4. Significance and Use4.1 This test method provides a technique for evaluatingmold growth resistance characteristics of wet blue, and shouldassist in the predictio
10、n 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 situations, orboth, has not been fully determined.5. Interferences5.1 A common interference is contamination of plates, agar,or samples by unwanted or
11、ganisms that settle in from theenvironment.5.2 Volatility and Leachability of BiocidesA “zone ofinhibition” where no mold grows on the agar adjacent to thespecimen indicates that the fungicide may leach.6. Apparatus6.1 Petri Dishes, 120 mm diameter. Sterile plastic dispos-able dishes are preferred.6
12、.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 Medicine droppers, disposable plastic type delivering 30to 35 drops per mL.7. Reagents and Materials7.1 Potato Dextrose Agar,2a dehydrated platin
13、g mediumused in culturing yeasts and molds from dairy products.1This test method is under the jurisdiction of ASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee D31.02 on Wet Blue.Current edition approved Sept. 1, 2008. Published October 2008. Originallyapproved in 1986. L
14、ast previous edition approved in 2006 as D 4576 - 01(2006)1.2The sole source of supply of a product that meets the requirements of thismethod known to the committee at this time is Potato Dextrose Agar stock no.0013-01-4, available from Difco Labs, P.O. Box 1058A, Detroit, MI 28232. If youare aware
15、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 may attend.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
16、 PA 19428-2959, United States.7.2 Inoculum,3Aspergillus niger 1 3 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, b
17、utt area) 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 Aga
18、r Preparation: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 dextros
19、e agar 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
20、. is preferable 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 Pla
21、ce one 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 vitali
22、ty of the inoculum.9.2.4 Let agar solidify for about 20 min.9.3 Inoculation:9.3.1 Reduce working stock of 1 3 106spores per mL to1 3 105by 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.
23、3.1.2 Prepare 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 3 105spores per milliliter perplate using a plastic disposable medicine dropper. Deposit onedrop directly on the sample an
24、d one drop to 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 plat
25、es. Keep covers 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 acceptabl
26、eand should not 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
27、specimens. A successfultest 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 r
28、ating scale of 0 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,0.5Less than 12 % of specimen surface overgrown bymold,125 % of specimen surface overgrown by mold,250 % of specimen surface over
29、grown 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, 7, and 14 days.3An inoculum that meets the requirements of this method is availabl
30、e as ATCC(American Type Culture Collection) 16404, and is available from several sources forlaboratory supplies. FIG. 1 Specimen with Inoculum Locations Shown (X)D457608211. Report11.1 Report of results must contain the following:11.1.1 Rating of each test specimen plate.11.1.2 Surface tested (grain
31、, 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 Temperature 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 an
32、dcontrol.12. Precison and Bias12.1 Precision or bias of this test method for measuringmold growth resistance of wet blue are indeterminate, since theresult merely states whether there is conformance to the criteriafor success specified in the procedure, as outlined in 1.2.13. Keywords13.1 agar plate
33、; blue stock; inoculum; mold; wet blue; wetwhiteASTM 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 such patent rights,
34、 and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for rev
35、ision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you
36、 shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor 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).D4576083
