ASTM_D_7818_-_12.pdf

Designation:D781812Standard Test Method forEnumeration of Proteolytic Bacteria in Fresh(Uncured)Hides and Skins1This standard is issued under the fixed designation D7818;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 bacteria thatcan hydrolyze protein/collagen in fresh(uncured)hides andskins.This test method is applicable to uncured hides andskins.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 uncured hides and skins are serially dilutedand plated on agar containing casein from skim milk.Theplates are incubated under aerobic conditions at 35C for 48 h.After incubation,to determine bacteria that can hydrolyzeprotein(proteolytic),the plates are flooded with dilute acid andthe colonies showing a halo are counted.4.Significance and Use4.1 This test method enumerates proteolytic bacteria.Pro-teolytic bacteria have been known to cause damage to hidesand skins.5.Apparatus5.1 Incubator,35 6 1C.5.2 Colony counter(not mandatory,but highly recom-mended).5.3 Sterile pipets.5.4 Bent glass rods,sterile.5.5 Stomacher,for mixing initial dilution.(If stomacher isunavailable,hand-mix.)5.6 Balance.5.7 Sterile petri dishes.5.8 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.)5.9 Stomacher bags,or sterile,sealable quart plastic bag(e.g.food storage type,sterile bag).5.10 Cutting tool,sterile(e.g.scalpel blade and forcep,asneeded for cutting fresh hides and skins).5.11 Vortex mixer,for mixing dilution tubes(optional).5.12 pH meter.5.13 Waterbath,45 6 1C.5.14 Autoclave thermometer.6.Reagents and Materials6.1 5%acetic acid.6.2 Butterfields Phosphate Stock Solution:Dissolve 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 5.8.)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/D7818-122For 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 Butterfields Phosphate Diluent(BPD):Take 1.25 mLofButterfields Phosphate Stock solution(6.2)and bring to 1 Lwith DI water.Dispense into 1 L bottles and 9 mL dilutiontubes.Sterilize for 15 min at 121C.(See Note 1.)6.4 Standard plate count agar containing 100 mL of 10%powdered skim milk solution per litre of agar.6.5 Alcohol(for flame sterilizing),e.g.70%Isopropylalcohol.6.6 Bent glass rod(“hockey-stick”).6.7 Powdered skim milk.6.8 Distilled or deionized water.6.9 Bacillus stearothermophilus spore suspensions or strips(commercially available),or equivalent.6.10 1N 6N NaOH.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 Standard Plate Count Agar9.1 Prepare the standard plate count agar per manufacturerlabel directions.9.2 Autoclave the prepared agar for 15 min at 121C.(SeeNote 1.)9.3 Prepare a 10%powdered skim milk mixture by adding10 g powdered skim milk to 100 mL DI water,then stirring themixture to dissolve it.Autoclave the mixture for 15 min at121C.(See Note 1.)9.4 Cool the agar(9.2)to 45 6 1C,then add 100 mL of thesterile 10%powdered skim milk mixture(9.3)per li