1、Designation: D7819 12Standard 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 revision, the year of las
2、t 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 method isapplicabl
3、e 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 of the user of th
4、is 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 Salt Pre-served (Cured) Hides and Skins for Chemical and Physi-cal Tests
5、E691 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 containing an antibiotic sol
6、ution. The platesare incubated at 2025C 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, 2025C.5.2 Colony counter (not mandatory, but highly recom-mended).5.3 Sterile pipets.5
7、.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 (commercially available) in
8、sideglassware 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.g. scalpel blade and forcep
9、, 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(Potassium Phosphate monobasic) i
10、n 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 120124C at 15 psi. (See 5.7.)6.2 Butterfields Phosphate Diluent (BPD): Take 1.25 mLofButterfields Phosphate Stock solution (6.1) and bring t
11、o 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 D31.02 on Wet Blue.Current e
12、dition approved Sept. 1, 2012. Published October 2012. DOI: 10.1520/D7819-122For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onth
13、e ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.4 Antibiotic solution (Chloramphenicol3) (needed toinhibit bacterial growth on agar).6.5 Distilled or deionized water.6.6 NaOH, 1N 6N.6.7 Bacillus stearothermophilus spo
14、re 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 precau-tions and instructions on the manufacturers label or MSDS(Material Safety Data Sheet).8. Sampling8.1 The s
15、pecimen 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 (10,000 ppm) solution bydissolving1gofchloramphenicol in 100 mL sterile deionizedor distilled water. Store this st
16、ock solution in a dark location at#5C 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 Add 10 mL of chloramphenicol stock solution per litreof agar to give a concentration of 100 ppm. Sterilize in th
17、eautoclave for 15 min at 121C. (See Note 1.) Cool to 45 6 1Cin a waterbath. Once medium has been tempered, it can be heldfor 23 h before use, provided the water level in the waterbathis 23 cm above the surface of the agar. Final pH of the agar:5.6 6 0.2.10. Procedure10.1 Using a sterile scalpel, ase
18、ptically 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 hand-massage for 1 min. Thisprovides a 1:10 dilution.10.2 Prepare the following sample dilutions: 10-2,10-3,10-4,10
19、-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 intoa9mLdilution tube.NOTE 2When transferring the aliquots between the tubes, the analystmust use a different pipet or pipet tip for each transfer.10.3 Pipet 1 mL of each dilution
20、 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 12 min after adding dilution to avoidadherence of sample to bottom of dish. Do not pour agar directly on thesample. Replace the cover.10.5 Swirl t
21、he plate gently in a figure-eight motion to evenlydistribute the sample.10.6 Allow agar to solidify.10.7 Incubate at 2025C for 5 days (a cabinet at roomtemperature is acceptable for use).3The sole source of supply known to the committee at this time is Sigma-Aldrich, Cat. # C0378 (25 g). If you are
22、aware of alternative suppliers, pleaseprovide this information to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.FIG. 1 PlatingD7819 122NOTE 4Do not stack plates higher than 3, and do not inve
23、rt the plates.Let plates remain undisturbed until time for counting. Moving the platescould dislodge spores thus creating extraneous growths that are not part ofthe original colony.10.8 Following incubation, count only those plates that have10150 colonies.NOTE 5Estimated counts can be made on plates
24、 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 of 56 when estimating the count. Example: 1 mLof a 10-4dilutionwas plated and the plate has an average count of 5 colonies per
25、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 with 10 colonies: report as estimatedcounts.10.9 Record each plates dilution and count on the work-sheet. Record the yeast count
26、 as A, and record the mold as B.NOTE 6Yeast colonies will appear as 23 mm in diameter with asatin-like or matte finish. Most are opaque white but they may bepigmented (orange or pink), sometimes yellow. Most produce a fermentedfruity or bakery aroma. They are convex or conical (raised off the surfac
27、e)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-green and green in color, and sometimesbecome dark gray or even black. Count from the underside of the platewhen mold overgrowth ha
28、s 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 areusually countable. However, if substantial amounts of mold are present,depending on the type of mold, the upper countable limit may b
29、e 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 colonies 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 1
30、0.9, 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, the counts for yeast and mold may becombined as one count, and reported as “Yeast the details aregiven in an ASTM Research Report
31、.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; “r”istheinterval representing the critical difference between two testresults for the same material, obtained by the same operator
32、using 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 shall bejudged not equivalent if they differ by more than the “R” valuefor that material; “R” is the interval representing the cr
33、iticaldifference between two test results for the same material,obtained by different operators using different equipment indifferent laboratories.13.1.2.1 Reproducibility limits are listed in Tables 2 and 3below.13.1.3 The above terms (repeatability limit and reproduc-ibility limit) are used as spe
34、cified 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 the precision statistics ob-tained in this ILS must not be treated as exact mathematicalquantities which are applicable to all cir
35、cumstances and uses.The limited number of materials tested and laboratories report-ing results guarantees that there will be times when differencesgreater than predicted by the ILS results will arise, sometimeswith considerably greater or smaller frequency than the 95 %probability limit would imply.
36、 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 expected.13.2 BiasAt the time of the study, there was no acceptedreference material suitable for determining the bias for this t
37、estmethod, 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 laboratories, ona single uncured hide material.4Supporting data have been filed at ASTM International Headquarters and maybe obtained by r
38、equesting 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 Colony Forming Units per gramAvgARepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibility
39、LimitxsrsRrRFresh(uncured)hide68643 14807 15379 41461 43062AThe average of the laboratories calculated averages.D7819 12314. Keywords14.1 hides; mold; skins; yeastASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this s
40、tandard. 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 technical committee and must be re
41、viewed 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 consideration at a meeting of therespons
42、ible 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 Harbor Drive, PO Box C700, West
43、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
44、 to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).TABLE 3 Log Yeast and MoldAvgARepeatabilityStandardDeviationReproducibilityStandardDeviationRepeatabilityLimitReproducibilityLimitxsrsRrRFresh(uncured)hide4.826 0.100 0.103 0.280 0.290AThe average of the laboratories calculated averages.D7819 124
