1、Designation: D7819 12 (Reapproved 2016)Standard Test Method forEnumeration of Yeast and Mold on Fresh (Uncured) Hidesand Skins1This standard is issued under the fixed designation D7819; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, the year of last revision. 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 enumeration of yeast andmold on fresh (uncured) hides and skins. This test
3、method isapplicable to uncured hides and skins.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility
4、 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. Referenced Documents2.1 ASTM Standards:2D6715 Practice for Sampling and Preparation of Fresh orSalt-Preserved (Cured) Hides and Skins for Che
5、mical andPhysical TestsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE177 Practice for Use of the Terms Precision and Bias inASTM Test Methods3. Summary of Test Method3.1 Samples of uncured hides and skins are serially dilutedand plated on agar conta
6、ining an antibiotic solution. The platesare incubated at 20 25C for 5 days.4. Significance and Use4.1 This test method enumerates yeast and mold. Yeast andmold have been known to cause damage to hides and skins.5. Apparatus5.1 Incubator, 20 25C.5.2 Colony counter (not mandatory, but highly recom-men
7、ded).5.3 Sterile pipets.5.4 Stomacher, for mixing initial dilution. (If stomacher isunavailable, hand-mix.)5.5 Balance.5.6 Sterile petri dishes.5.7 Autoclave (sterilizer). (Check the effectiveness of ster-ilization weekly. For example, place spore suspensions or stripsof Bacillus stearothermophilus
8、(commercially available) insideglassware for a full autoclave cycle. Follow manufacturersdirections for sterilization of specific media.)5.8 pH meter.5.9 Waterbath, 45 6 1C.5.10 Stomacher bags, or sterile, sealable quart plastic bag(e.g. Food storage type, sterile bag).5.11 Cutting tool, sterile (e.
9、g. scalpel blade and forcep, asneeded for cutting cured hides and skins).5.12 Vortex mixer, for mixing dilution tubes (optional).5.13 Autoclave thermometer, or equivalent for monitoringautoclave temperature.6. Reagents and Materials6.1 Butterfields Phosphate Stock Solution: Dissolve 34 gKH2PO4(Potas
10、sium Phosphate monobasic) in 500 mL DIwater. Adjust the pH to 7.2 6 0.1 with 1N 6N NaOH. Bringvolume to 1 L with DI water. Sterilize for 15 min at 121C.NOTE 1Typical autoclave setting is 120 124C at 15 psi. (See 5.7.)6.2 Butterfields Phosphate Diluent (BPD): Take 1.25 mLofButterfields Phosphate Stoc
11、k solution (6.1) and bring to 1 Lwith DI water. Dispense into 1-litre bottles and 9-mL dilutiontubes. Sterilize for 15 min at 121C. (See Note 1.)6.3 Potato Dextrose Agar (PDA).1This test method is under the jurisdiction ofASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee
12、D31.02 on Wet Blue.Current edition approved Sept. 1, 2016. Published October 2016. Originallyapproved in 2012. Last previous edition approved in 2012 as D7819 12. DOI:10.1520/D7819-12R16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at servicea
13、stm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.4 Antibiotic solution (Chloramphenicol)3 (needed toinhibit
14、 bacterial growth on agar).6.5 Distilled or deionized water.6.6 NaOH, 1N 6N.6.7 Bacillus stearothermophilus spore suspensions or strips(commercially available), or equivalent.7. Hazards7.1 All reagents and chemicals should be handled with care.Before using any chemical, read and follow all safety pr
15、ecau-tions and instructions on the manufacturers label or MSDS(Material Safety Data Sheet).8. Sampling8.1 The specimen shall be sampled in accordance withPractice D6715, and placed in sterile containers.9. Preparation of Potato Dextrose Agar and AntibioticSolution9.1 Prepare the antibiotic stock (10
16、 000 ppm) solution bydissolving1gofchloramphenicol in 100 mL sterile deionizedor distilled water. Store this stock solution in a dark location at5C for up to two months.9.2 Suspend 39 g of Potato Dextrose Agar in 1 litre ofdeionized or distilled water and heat to boiling to dissolvecompletely.9.3 Ad
17、d 10 mL of chloramphenicol stock solution per litreof agar to give a concentration of 100 ppm. Sterilize in theautoclave for 15 min at 121C. (See Note 1.) Cool to 45 6 1Cin a waterbath. Once medium has been tempered, it can be heldfor 2 3 h before use, provided the water level in the waterbathis 2 3
18、 cm above the surface of the agar. Final pH of the agar:5.6 6 0.2.10. Procedure10.1 Using a sterile scalpel, aseptically obtain a 20 6 0.1 gspecimen that includes both the flesh side and the hair side.Weigh it into a sterile bag.Add 180 g of BPD (6.2) diluent intothe same sterile bag. Stomach or han
19、d-massage for 1 min. Thisprovides a 1:10 dilution.10.2 Prepare the following sample dilutions: 10-2,10-3,10-4,10-5,10-6, and 10-7(see Fig. 1).Example: To obtain a 10-2dilution, mix the 10-1dilution andpipet 1 mL of that 10-1dilution into a 9-mL dilution tube.NOTE 2When transferring the aliquots betw
20、een the tubes, the analystmust use a different pipet or pipet tip for each transfer.10.3 Pipet 1 mL of each dilution into the appropriate,separate petri dishes.10.4 Pour prepared agar (9.3) that has been previouslytempered to 45 6 1C into the dish.NOTE 3Add agar within 1 2 min after adding dilution
21、to avoidadherence of sample to bottom of dish. Do not pour agar directly on thesample. Replace the cover.10.5 Swirl the plate gently in a figure-eight motion to evenlydistribute the sample.10.6 Allow agar to solidify.10.7 Incubate at 20 25C for 5 days (a cabinet at roomtemperature is acceptable for
22、use).3The sole source of supply known to the committee at this time is Sigma-Aldrich, Cat. # C0378 (25 g). If you are aware of alternative suppliers, pleaseprovide this information to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible tec
23、hnical committee,1which you may attend.FIG. 1 PlatingD7819 12 (2016)2NOTE 4Do not stack plates higher than 3, and do not invert the plates.Let plates remain undisturbed until time for counting. Moving the platescould dislodge spores, thus creating extraneous growths that are not partof the original
24、colony.10.8 Following incubation, count only those plates that have10 150 colonies.NOTE 5Estimated counts can be made on plates with 150 colonies:report as estimated counts. In making such counts, the standard 15 100mm petri dish is considered to have an area of about 56 cm2, therefore, usea factor
25、of 56 when estimating the count. Example: 1 mLof a 10-4dilutionwas plated and the plate has an average count of 5 colonies per cm2.Therefore, the estimated count for that plate is556=280, and theestimated count for that dilution is 280 10,000 = 2,800,000. Estimatedcounts can also be made on plates w
26、ith 10 colonies: report as estimatedcounts.10.9 Record each plates dilution and count on the work-sheet. Record the yeast count as A, and record the mold as B.NOTE 6Yeast colonies will appear as 2 3 mm in diameter with asatin-like or matte finish. Most are opaque white, but they may bepigmented (ora
27、nge or pink), sometimes yellow. Most produce a fermentedfruity or bakery aroma. They are convex or conical (raised off the surface)in shape.NOTE 7Mold colonies will have a whiskery or cotton tuft-likeappearance and may tend to spread over the surface of the agar. They areusually gray, brown, blue-gr
28、een and green in color, and sometimesbecome dark gray or even black. Count from the underside of the platewhen mold overgrowth has occurred. If mold colonies are present, do notopen the plates. Tape them shut before proper disposal.NOTE 8If mainly yeasts are present, plates with 150 colonies areusua
29、lly countable. However, if substantial amounts of mold are present,depending on the type of mold, the upper countable limit may be loweredat the discretion of the analyst.11. Calculation of Results for Yeast and Mold11.1 Calculate by using the following formula:Yeast 5 A 3 C (1)where:A = number of c
30、olonies counted in step 10.9, andC = dilution factor (see Table 1).Mold 5 B 3 C (2)where:B = number of colonies counted in step 10.9, andC = dilution factor (see Table 1).12. Report12.1 Report the results from 11.1 as Yeast per g of sampleand Mold per g of sample, respectively.NOTE 9When requested,
31、the counts for yeast and mold may becombined as one count, and reported as “Yeast the details aregiven in an ASTM Research Report.413.1.1 Repeatability Limit (r)Two test results obtainedwithin one laboratory shall be judged not equivalent if theydiffer by more than the “r” value for that material; “
32、r”istheinterval representing the critical difference between two testresults for the same material, obtained by the same operatorusing the same equipment on the same day in the samelaboratory.13.1.1.1 Repeatability limits are listed in Tables 2 and 3.13.1.2 Reproducibility Limit (R)Two test results
33、shall bejudged not equivalent if they differ by more than the “R” valuefor that material; “R” is the interval representing the criticaldifference between two test results for the same material,obtained by different operators using different equipment indifferent laboratories.13.1.2.1 Reproducibility
34、 limits are listed in Tables 2 and 3below.13.1.3 The above terms (repeatability limit and reproduc-ibility limit) are used as specified in Practice E177.13.1.4 Any judgment in accordance with statements 13.1.1and 13.1.2 would normally have an approximate 95 % prob-ability of being correct, however t
35、he precision statistics ob-tained in this ILS must not be treated as exact mathematicalquantities which are applicable to all circumstances and uses.The limited number of materials tested and laboratories report-ing results guarantees that there will be times when differencesgreater than predicted b
36、y the ILS results will arise, sometimeswith considerably greater or smaller frequency than the 95 %probability limit would imply. The repeatability limit and thereproducibility limit should be considered as general guides,and the associated probability of 95 % as only a rough indicatorof what can be
37、 expected.13.2 BiasAt the time of the study, there was no acceptedreference material suitable for determining the bias for this testmethod, therefore no statement on bias is being made.13.3 The precision statement was determined through sta-tistical examination of 28 test results, from two laborator
38、ies, ona single uncured hide material.4Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D31-1018.TABLE 1 Dilution FactorTest Tube Plated (10.3) Dilution Factor10-210010-31,00010-410,00010-5100,00010-61,000,00010-710,000,000TABLE 2
39、 Colony Forming Units per gramAvgARepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibilityLimitxsrsRrRFresh(uncured)hide68643 14807 15379 41461 43062AThe average of the laboratories calculated averages.D7819 12 (2016)314. Keywords14.1 hides; mold; skins; yeastA
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41、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 revision of this standard or for additional standard
42、sand 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 shouldmake your views known to the ASTM Committe
43、e 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 o
44、r 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 Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 3 Log Yeast and MoldAvgARepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibilityLimitxsrsRrRFresh(uncured)hide4.826 0.100 0.103 0.280 0.290AThe average of the laboratories calculated averages.D7819 12 (2016)4
