1、Designation: D7817 12 (Reapproved 2016)Standard Test Method forEnumeration of Yeast and Mold in Raceway Brine, Brine-Cured Hides and Skins1This standard is issued under the fixed designation D7817; the number immediately following the designation indicates the year oforiginal adoption or, in the cas
2、e of revision, 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. This test method is applicable to
3、 raceway brine, brine-cured hides and skins, and pre-charge raceway liquor.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
4、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. Referenced Documents2.1 ASTM Standards:2D6715 Practice for Sampling and Preparation of Fresh orSalt-Preserved (Cu
5、red) Hides and Skins for Chemical 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 brine-cured hides and skins, raceway bri
6、ne,or pre-charge raceway liquor are serially diluted and plated onagar containing 7 % NaCl and an antibiotic solution. The platesare incubated at 20 25C for 5 days.4. Significance and Use4.1 This test method enumerates salt tolerant yeast andmold, and under the conditions of this test method those a
7、reequated as halophilic organisms. Salt tolerant yeast and moldhave been known to cause damage to hides and skins inraceway brine.5. Apparatus5.1 Incubator, 20 25C.5.2 Colony counter(not mandatory, but highly recom-mended).5.3 Sterile pipets.5.4 Stomacher, for mixing initial dilution. (If stomacher
8、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) insideglassware for a full autoclave cycle. Follow manufact
9、urersdirections 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, asneeded for cutting cured hides and skins).5.12 Vortex
10、 mixer, for mixing dilution tubes (optional).5.13 Autoclave thermometer.6. Reagents and Materials6.1 Butterfields Phosphate Stock SolutionDissolve 34 gKH2PO4(Potassium 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
11、15 min at 121C.NOTE 1Typical autoclave setting is 120 124C. (See 5.7.)6.2 Butterfields Phosphate Diluent with salt (BPD w/salt)Take 1.25 mL of Butterfields Phosphate Stock solution (6.1)and bring to 1 Lwith DI water, then add 77 g of salt (NaCl) perlitre prior to autoclaving. Dispense into 1-L bottl
12、es and 9-mLdilution tubes. Sterilize for 15 min at 121C. (See Note 1.)1This test method is under the jurisdiction ofASTM Committee D31 on Leatherand is the direct responsibility of Subcommittee D31.02 on Wet Blue.Current edition approved Sept. 1, 2016. Published October 2016. Originallyapproved in 2
13、012. Last previous edition approved in 2012 as D7817 12. DOI:10.1520/D7817-12R16.2For 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
14、 onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.3 Potato Dextrose Agar (PDA).6.4 Antibiotic solution(Chloramphenicol)3 (needed toinhibit bacterial growth on agar).6.5 Distilled or deionized water.6.6 Salt (NaCl),
15、 Sodium chloride reagent grade.6.7 1N 6N NaOH.6.8 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 precau-tions and instructions on
16、 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 (10 000 ppm) solution bydissolvin
17、g1gofchloramphenicol 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 L ofdeionized or distilled water and heat to boiling to dissolvecompletely.9.3 Add 77 g of NaCl per litre of agar.
18、Add 10 mL ofchloramphenicol stock solution per litre of agar to give aconcentration of 100 ppm. Sterilize in the autoclave for 15 minat 121C. (See Note 1.) Cool to 45 6 1C in a waterbath. Oncemedium has been tempered, it can be held for 2 3 h beforeuse, provided the water level in the waterbath is 2
19、 3 cm abovethe surface of the agar. Final pH of the agar: 5.6 6 0.2.10. Procedure10.1 Using a sterile scalpel, aseptically weigh a 20 6 0.1 gspecimen in a sterile bag. For brine-cured hides and skins,include both flesh and hair side.10.2 Add 180 g of BPD w/salt (6.2) diluent into the samesterile bag
20、 (10.1). Stomach or hand-massage for 1 min. Thisprovides a 1:10 dilution.10.3 Prepare the following sample dilutions: 10-2,10-3,10-4,10-5,10-6, and 10-7(see Fig. 1).10.3.1 Control BlankIn 10.5, pour melted media that hasbeen previously tempered to 45 6 1C into a dish, thencontinue with 10.6 as with
21、the sample plates.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 between the tubes, the analystmust use a different pipet or pipet tip for each transfer.10.4 Pipet 1 mL of each dilution into th
22、e appropriate,separate petri dishes.10.5 Pour prepared agar (9.3) that has been previouslytempered to 45 6 1C into the dish.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
23、to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.FIG. 1 PlatingD7817 12 (2016)2NOTE 3Add agar within 1 2 min after adding dilution to avoidadherence of sample to bottom of dish. Do not pour a
24、gar directly on thesample. Replace the cover.10.6 Swirl the plate gently in a figure-eight motion to evenlydistribute the sample.10.7 Allow agar to solidify.10.8 Incubate at 20 25C for 5 days (a cabinet at roomtemperature is acceptable for use).NOTE 4Do not stack plates higher than 3, and do not inv
25、ert 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 colony.10.9 Following incubation, count only those plates that have10 150 colonies.NOTE 5Estimated counts can be made on pla
26、tes 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 p
27、er 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.10.10 Record each plates dilution and count on the work-sheet. Record the yeast c
28、ount 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 (orange or pink), sometimes yellow. Most produce a fermentedfruity or bakery aroma. They are convex or conical (raised off the
29、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-green and green in color, and sometimesbecome dark gray or even black. Count from the underside of the platewhen mold overgro
30、wth 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 areusually countable. However, if substantial amounts of mold are present,depending on the type of mold, the upper countable limit
31、 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 colonies counted in step 10.10, andC = dilution factor (see Table 1).Mold 5 B 3 C (2)where:B = number of colonies counted in
32、 step 10.10, andC = dilution factor (see Table 1).12. Report12.1 Report the results from 11.1 as Yeast (salt tolerant) cfupergofsample, Mold (salt tolerant) cfu per g of samplerespectively.NOTE 9When requested, the counts for yeast and mold may becombined as one count, and reported as “Yeast the det
33、ails 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; “r”istheinterval representing the critical difference between two testresults for the same
34、 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 Table 2.13.1.2 Reproducibility limit (R)Two test results shall bejudged not equivalent if they differ by more than the “R” valuefor that material; “R” is
35、 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 limits cannot be determined withdata from only one reporting laboratory.13.1.3 The above terms
36、(repeatability limit and reproduc-ibility limit) are used as specified in Practice E177.13.1.4 Any judgment in accordance with 13.1.1 wouldnormally have an approximate 95 % probability of beingcorrect, however the precision statistics obtained in this ILSmust not be treated as exact mathematical qua
37、ntities which areapplicable to all circumstances and uses. The limited numberof locations tested and laboratories reporting results guaranteesthat there will be times when differences greater than predictedby the ILS results will arise, sometimes with considerablygreater or smaller frequency than th
38、e 95 % probability limitwould imply. Consider the repeatability limit as a general4Supporting data have been filed at ASTM International Headquarters and maybe obtained by requesting Research Report RR:D31-1016.TABLE 1 Dilution FactorTest Tube Plated (10.4) Dilution Factor10-210010-31,00010-410,0001
39、0-5100,00010-61,000,00010-710,000,000TABLE 2 Halophilic Bacteria (log count)Material AverageRepeatabilityStandardDeviationRepeatabilityLimitxsrrHGS Flesh 4.71 0.44 1.25HGS Hair 4.79 0.35 0.98IPHS Flesh 4.31 0.24 0.66IPHS Hair 3.62 0.31 0.87CHS Flesh 3.55 0.17 0.48CHS Hair 2.97 0.17 0.48D7817 12 (201
40、6)3guide, and the associated probability of 95 % as only a roughindicator of what can be expected.13.2 BiasAt the time of the study, no accepted referencematerial suitable for determining the bias for this test methodwas utilized, therefore no statement on bias is being made.13.3 The precision state
41、ment was determined through sta-tistical examination of 30 results, from one laboratory, on sixmaterials. These materials were described as the following:Material 1: HGS Flesh Flesh side uncured (green)Material 2: HGS Hair Hair side uncured (green)Material 3: IPHS Flesh Flesh side in process 6 hours
42、Material 4: IPHS Hair Hair side in process 6 hoursMaterial 5: CHS Flesh Flesh side cured 16 hoursMaterial 6: CHS Hair Hair side cured 16 hours13.3.1 To judge the equivalency of two test results, it isrecommended to choose the material described as closest incharacteristics and concentration to the t
43、est material.14. Keywords14.1 bacteria; brine; halophilic; hides; mold; raceway; salttolerant; skins; yeastASTM 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 th
44、at 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 reviewed every five years andif not revised, either reappr
45、oved 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 theresponsible technical committee, which you may attend. If you f
46、eel 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 Conshohocken, PA 19428-2959,United States. Individual re
47、prints (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 to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 12 (2016)4
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