ASTM_D_7816_-_12.pdf

Designation:D781612Standard Test Method forEnumeration of Halophilic and Proteolytic Bacteria inRaceway Brine,Brine-Cured Hides and Skins1This standard is issued under the fixed designation D7816;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 tolerate high salt concentrations or can hydrolyze protein/collagen,or both.This test method is applicable to racewaybrine,brine-cured hides and skins,and pre-charge racewayliquor.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 TestsE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3.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 casein from skim milk.Theplates are incubated under aerobic conditions at 35C for 48 h.After incubation,to determine salt tolerant(halophilic)bacteria,all colonies are counted.To determine bacteria thatcan hydrolyze protein(proteolytic),the plates are flooded withdilute acid and the colonies showing a“halo”(semi-clearzones)are counted.4.Significance and Use4.1 This test method enumerates salt tolerant(halophilic)bacteria,and proteolytic bacteria that are also salt tolerant.Under the conditions of this test method those bacteria areequated as halophilic organisms.Salt tolerant proteolyticbacteria have been known to cause damage to hides and skinsin raceway brine.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(“hockey-stick”),sterile.(If non-sterile,will need to flame sterilize.)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 cured 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,or equivalent for monitoringautoclave temperature.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/D7816-122For 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.Reagents and Materials6.1 5%acetic acid.6.2 1N 6N NaOH.6.3 Standard plate count agar containing 100 mL of 10%powdered skim milk solution and 77 g of salt per litre of agar.6.4 Ready-To-Use Plate,(optional)3:Plate CountAgar+10%skim milk+7.7%NaCl.6.5 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 at 15 psi.(See 5.8.)6.6 Butterfields Phosphate Diluent with salt(BPD w/salt)Take 1.25 mL of Butterfields Phosphate Stock solution(6.5)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.)6.7 Alcohol(for flame sterilizing),e.g.70%Isopropylalcohol.6.8 Powdered skim milk.6.9 Distilled or deionized water.6.10 Salt(NaCl),Sodium chloride reagent grade.6.11 Bacillus stearothermophilus spore suspensions orstrips(commercially available),or equivalent.7.Hazards7.1 All reagents and chemicals should be handled with care.Before us