1、Designation: D4576 16Standard Test Method forMold Growth Resistance of Wet Blue and Wet White1This 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. A n
2、umber 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 ofWet Blue andWetWhite subject to storageand shipping requirements an
3、d 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 simultaneou
4、slyrun 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, andchoice o
5、f control. These may be adjusted to fit local conditionsbut must be standardized.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated wit
6、h 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. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 Wet Bluehide or skin, or split of a hid
7、e 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 skintanned with organic or non-organic tanning agents (excludingchromium or iron containing agents and vegetable extracts),containing appro
8、ximately 50 % moisture.3. Summary of Test Method3.1 Wet Blue and Wet White test specimens are surroundedby but not covered with agar, inoculated, and incubated.3.2 After various incubation periods, mold growth is ratedas a percentage of the Wet Blue and Wet White surface coveredby mold.3.3 Resistanc
9、e to mold growth of theWet Blue orWetWhitetest specimen is determined by comparison with Wet Blue orWet White of known resistance characteristics (the control),that is tested simultaneously.4. Significance and Use4.1 This test method provides a technique for evaluatingmold growth resistance characte
10、ristics of Wet Blue and WetWhite, and should assist in the prediction of storage timebefore molding occurs.4.2 The degree of correlation between this test and commer-cial quantities of Wet Blue and Wet White in storage orshipment situations, or both, has not been fully determined.5. Interferences5.1
11、 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 thespecimen indicates that the fungicide may leach.6. Apparatus6.1 P
12、etri 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 Medicine droppers, disposable plastic type delivering 30to 35 dro
13、ps per mL.7. Reagents and Materials7.1 Potato Dextrose Agar,2a dehydrated plating mediumused in culturing yeasts and molds from dairy products.1This test method is under the jurisdiction ofASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee D31.02 on Wet Blue.Current editio
14、n approved June 1, 2016. Published June 2016. Originallyapproved in 1986. Last previous edition approved in 2013 as D4576 - 08(2013).DOI: 10.1520/D4576-16.2The 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
15、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
16、ay attend.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States17.2 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 and Wet White.8. Sa
17、mpling, Test Specimen, and Test Units8.1 Take test specimens from equivalent hide locations (forexample, butt 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 i
18、n.).8.4 Use three test specimens for each test unit of Wet Blueor Wet White surface to be evaluated.9. Procedure9.1 Agar Preparation:9.1.1 Agar RequirementsA split Wet Blue or Wet Whitetest specimen requires about 25 mLsolution and an unsplit WetBlue or Wet White test specimen requires about 40 mL.C
19、alculate number of millilitres of agar required for tests to beperformed, allowing 50 mL for vitality check.9.1.2 Weigh out 3.9 g potato dextrose 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 ho
20、t 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 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
21、 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 Wet Blue and Wet White test specimen incenter of each petri dish with the surface to be tested facing up.9.2.2 Carefully lift cover from each dish an
22、d 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)for evaluation of the vitality of the 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
23、 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 Prepare only enough diluted suspension for use in a48-hour period.9.3.1.3 Keep organism stock suspensions refrigerated atabout 4C. Do not fre
24、eze.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 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
25、 4Keep work area as clean and aseptic as possible. Work in anarea of minimal air circulation while handling Wet Blue or Wet White,pouring agar, and inoculating plates. Keep covers on petri dishes at alltimes except when pouring and inoculating.9.4 Incubate up to three weeks at constant temperature i
26、n 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 not vary more than 6 2C.NOTE 5Storage in a clear plastic box in a boiler room may besufficient. Use separation to prevent cr
27、oss contamination.NOTE 6More rapid growth occurs at higher temperature.9.5 Control Wet Blue and Wet White specimens of knownmold resistance must be done simultaneously with test speci-mens. A successful test will have less mold growth than thecontrol.NOTE 7After completion of work, test specimens sh
28、ould 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 to 4 can be used whereeach number represents the degree of growth observed on thespecimen (not on the ag
29、ar) 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 overgrown by mold,375 % of specimen sulfate overgrown by mold, and4100 % of specimen surface overgrown by mold.10.1.1 Mold gr
30、owth above but not touching specimen willalso be rated zero.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 16210.
31、2 Suggested rating periods are 3, 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
32、 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 andcontrol.12. Precison and Bias12.1 Precision or bias of this test method for measuringmold growth resistance of Wet Blue
33、and Wet White areindeterminate, since the result merely states whether there isconformance to the criteria for success specified in theprocedure, as outlined in 1.2.13. Keywords13.1 agar plate; blue stock; inoculum; mold; Wet Blue; WetWhiteASTM International takes no position respecting the validity
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36、. 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 shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is
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