1、Designation: D 4576 01 (Reapproved 2006)e1Standard 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 revis
2、ion. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.e1NOTEFootnote 3 was revised in July 2008.1. Scope1.1 This test method covers the determination of moldgrowth resistance of wet blue subject
3、 to storage and shippingrequirements and intended for use in leather manufacturing.This test method may not be suitable to evaluate fungicides thatare inactivated by proteins. This includes alkyldimethylbenzylammonium chlorides.1.2 Conclusions about mold growth resistance are drawnfrom the results b
4、y comparing the test with a simultaneouslyrun 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, in
5、oculum, hide sampling area, andchoice of control. These may be adjusted to fit local conditionsbut must be standardized.1.4 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-pr
6、iate 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 skin,tanned with basic chromium sulfate, containing approximately50 % moisture and
7、 having an acidic pH.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 b
8、lue test 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 pred
9、iction 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 unwant
10、ed 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 Petri Dishes, 120 mm diameter. Sterile plastic dispos-able dishes are prefer
11、red.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 drops per mL.7. Reagents and Materials7.1 Potato Dextrose Agar,2a dehydrated p
12、lating mediumused in culturing yeasts and molds from dairy products.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 eq
13、uivalent hide locations (forexample, butt area) for both test and control.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 Oct. 1, 2006. Published November 2006. Originallyapproved
14、 in 1986. Last previous edition approved in 2001 as D 4576 - 01.2A product that meets the requirements of this method is Potato Dextrose Agarstock no. 0013-01-4, available from Difco Labs, P.O. Box 1058A, Detroit,MI 28232.3An inoculum that meets the requirements of this method is available as ATCC(A
15、merican Type Culture Collection) 16404, and is available from several sources forlaboratory supplies.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.8.2 If unable to test immediately, hold test specimens inseparate plastic bags and k
16、eep 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 Preparation:9.1.1 Agar RequirementsA split wet blue test specimenrequires about 25 mL solution and an unsplit wet blue
17、 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 for every 100 mLof agar required.9.1.3 Pour a volume of water equivalent to total millilitresof agar solution to
18、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 preferable to open boiling.9.1.5 Cover with aluminum foil to prevent contaminationand cool to 50C before pouring.NO
19、TE 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 wet blue test specimen in center of eachpetri dish with the surface to be tested facing up.9.2.2 Carefully lift c
20、over 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 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
21、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.9.3.1.2 Prepare only enough diluted suspension for use in a48-hour period.9.3.1.3 Keep organism stock suspensions refriger
22、ated 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 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
23、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 covers on petri dishes at all times except whenpouring and inoculating.9.4 Incubate up to three weeks at consta
24、nt 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 not vary more than 6 2C.NOTE 5Storage in a clear plastic box in a boiler room may besufficient. Use separati
25、on 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. A successfultest will have less mold growth than the control.NOTE 7After completion of work, test specimens s
26、hould 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 a
27、gar) 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 g
28、rowth above but not touching specimen willalso be rated zero.10.2 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 ra
29、ted.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.FIG. 1 Specimen with inoculum locations shown (X)D 4576 01 (2006)e1211.1.9 Difference in treatment of specimen betw
30、een test andcontrol.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
31、 agar plate; blue stock; inoculum; mold; wet blueASTM 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
32、, 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 re
33、vision 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 yo
34、u 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).D 4576 01 (2006)e13
