ASTM_D_7817_-_12.pdf

Designation:D781712Standard 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 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,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 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(Cured)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 brineor pre-charge raceway 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 organisms.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.)5.5 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 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 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 15 min at 121C.NOTE1Typical 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 L with DI water,then add 77 g of salt(NaCl)perlitre prior to autoclaving.Dispense into 1 L bottles and 9 mLdilution tubes.Sterilize for 15 min at 121C.(See Note 1.)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 Sept.1,2012.Published October 2012.DOI:10.1520/D7817-122For 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 onthe ASTM website.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 6.3 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(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 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(10000 ppm)solution bydissolving 1 g of chloramphenicol 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.Add 10 mL ofchloramphenicol stock solution per litre of agar to give aconcentration of 100 ppm.