1、Designation: D7817 12Standard 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 case of revision, the
2、 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 raceway brine, br
3、ine-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 use. It is theresp
4、onsibility 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 Salt Pre-served (Cured) Hides and Skins for
5、Chemical and Physi-cal 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 brineor pre-charge raceway
6、 liquor are serially diluted and plated onagar containing 7 % NaCl and an antibiotic solution. The platesare incubated at 2025C 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 areequated as halophilic o
7、rganisms. Salt tolerant yeast and moldhave been known to cause damage to hides and skins inraceway brine.5. Apparatus5.1 Incubator, 2025C.5.2 Colony counter(not mandatory, but highly recom-mended).5.3 Sterile pipets.5.4 Stomacher, for mixing initial dilution. (If stomacher isunavailable, hand-mix.)5
8、.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 manufacturersdirections for steril
9、ization 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 mixer, for mixing dilutio
10、n 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 15 min at 121C.NOTE 1Typic
11、al autoclave setting is 120124C. (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 bottles and 9 mLdilution tubes.
12、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, 2012. Published October 2012. DOI: 10.1520/D7817-122For referenced ASTM standard
13、s, 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 onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19
14、428-2959. United States16.3 Potato Dextrose Agar (PDA).6.4 Antibiotic solution(Chloramphenicol3) (needed toinhibit bacterial growth on agar).6.5 Distilled or deionized water.6.6 Salt (NaCl), Sodium chloride reagent grade.6.7 1N 6N NaOH.6.8 Bacillus stearothermophilus spore suspensions or strips(comm
15、ercially 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 specimen shall be sampled in a
16、ccordance withPractice D6715, and placed in sterile containers.9. Preparation of Potato Dextrose Agar and AntibioticSolution9.1 Prepare the Antibiotic stock (10000 ppm) solution bydissolving1gofchloramphenicol in 100 mL sterile deionizedor distilled water. Store this stock solution in a dark locatio
17、n at#5C 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. Add 10 mL ofchloramphenicol stock solution per litre of agar to give aconcentration of 100 ppm. Sterilize in t
18、he 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 23 h before use,provided the water level in the waterbath is 23 cm above thesurface of the agar. Final pH of the agar: 5.6 6 0.2.10. Procedure10.1 Using a sterile scalpel, a
19、septically weigh a 20 6 0.1 gspecimen in a sterile bag. For brine-cured hides and skinsinclude both flesh and hair side.10.2 Add 180 g of BPD w/salt (6.2) diluent into the samesterile bag (10.1). Stomach or hand-massage for 1 min. Thisprovides a 1:10 dilution.10.3 Prepare the following sample diluti
20、ons: 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 the sample plates.Example: To obtain a 10-2dilution, mix the 10-1dilution andpipet 1 mL of that 10-1dilution into
21、 a 9mL 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 the appropriate,separate petri dishes.10.5 Pour prepared agar (9.3) that has been previouslytempered to 45 6 1C into
22、 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 to ASTM International Headquarters. Your comments willreceive careful consideration at a meeting of the responsibl
23、e technical committee,1which you may attend.FIG. 1 PlatingD7817 122NOTE 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.10.6 Swirl the plate gently in a figure-eight motion to evenlydistribute the
24、sample.10.7 Allow agar to solidify.10.8 Incubate at 2025C for 5 days (a cabinet at roomtemperature is acceptable for use).NOTE 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
25、extraneous growths that are not part ofthe original colony.10.9 Following incubation, count only those plates that have10150 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 ha
26、ve 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 cm2.Therefore, the estimated count for that plate is556=280, and theestimated count for that dilution is 280 10,000 = 2,80
27、0,000. 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 count as A, and record the mold as B.NOTE 6Yeast colonies will appear as 23mm in diameter with asatin-like or matte finish. Mo
28、st 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 surface)in shape.NOTE 7Mold colonies will have a whiskery or cotton tuft-likeappearance and may tend to spread over the surface
29、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 has occurred. If mold colonies are present, do notopen the plates. Tape them shut before proper disposal.NOTE 8If mainly yea
30、sts 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 be loweredat the discretion of the analyst.11. Calculation of Results for Yeast and Mold11.1 Calculate by using the followi
31、ng 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 step 10.10, andC = dilution factor (see Table 1).12. Report12.1 Report the results from 11.1 as Yeast (salt tolerant) cfupergof
32、sample, 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 details aregiven in an ASTM Research Report.413.1.1 Repeatability limit (r)Two test results obtainedwithin one laboratory shall be
33、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 operatorusing the same equipment on the same day in the samelaboratory.13.1.1.1 Repeatability li
34、mits 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 the interval representing the criticaldifference between two test results for the same material,obtained by different operators
35、 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 (repeatability limit and reproduc-ibility limit) are used as specified in Practice E177.13.1.4 Any judgment in accordance with 1
36、3.1.1 wouldnormally have an approximate 95 % probability of beingcorrect, however the precision statistics obtained in this ILSmust not be treated as exact mathematical quantities which areapplicable to all circumstances and uses. The limited numberof locations tested and laboratories reporting resu
37、lts guaranteesthat there will be times when differences greater than predictedby the ILS results will arise, sometimes with considerablygreater or smaller frequency than the 95 % probability limitwould imply. Consider the repeatability limit as a general4Supporting data have been filed at ASTM Inter
38、national Headquarters and maybe obtained by requesting Research Report RR:D31-1016.TABLE 1 Dilution FactorTest Tube Plated (10.4) Dilution Factor10-210010-31,00010-410,00010-5100,00010-61,000,00010-710,000,000TABLE 2 Halophilic Bacteria (log count)Material AverageRepeatabilityStandardDeviationRepeat
39、abilityLimitxsrrHGS 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 123guide, and the associated probability of 95 % as only a roughindicator of what can be expected.13.2 BiasAt the time of the study, no
40、 accepted referencematerial suitable for determining the bias for this test methodwas utilized, therefore no statement on bias is being made.13.3 The precision statement was determined through sta-tistical examination of 30 results, from one laboratory, on sixmaterials. These materials were describe
41、d 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 hoursMaterial 4: IPHS Hair Hair side in process 6 hoursMaterial 5: CHS Flesh Flesh side cured 16 hoursMaterial 6: CHS Hair Hair side cured 1
42、6 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 test material.14. Keywords14.1 bacteria; brine; halophilic; hides; mold; raceway; salttolerant; skins; yeastASTM International takes no
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45、 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 Committee on Standards, at the addr
46、ess 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).D7817 124
