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AnaerobicSystemsAntibioticSensitivityTestingCultureMedia,SupplementsandRawMaterialsArethereanyguidelinesforpreparationandreconstitutionofculturemedia?

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BLOODPRODUCTSHorseblood,defibrinatedSR0050Horseblood,oxalatedSR0049Horseblood,haemolysedSR0048Sheepblood,defibrinatedSR0051HorseserumSR0035Horseandsheepbloodarethemostwidelyusedanimalbloodproductsinculturemedia.Thechoiceofanimalislargelytraditional,withtheUSAandmuchofcontinentalEuropepreferringsheepblood,whilsttheUKandCommonwealthpartnerspreferhorseblood.

Thehaemolyticreactionsofhorseandsheepbloodarenotidenticalandbloodagarmediadesignedforhorsebloodmaynotbesatisfactorywithsheepbloodandviceversa.SeeBloodAgarBase(Sheep)CM0854.

Itisnotpossibletosterilisewholebloodproductsandthereforetheymustbecollectedaseptically.Freshlydrawnbloodfromhealthyanimalscandestroysmallnumbersofbacteriawhichmaytransientlyinfectitduringcollectionbutconsiderableexpertiseandskillarerequiredtomaintainhighquality,sterileblood.Itisdifficulttoobtainsterilebloodfromabattoirsthereforebleedingiscarriedoutinsuitablepremisesfromanimalswhicharespeciallymaintainedonhighproteindiets.

Defibrinationisnowacceptedasthebestmethodofpreventingbloodclotting.Itmustbecarriedoutimmediatelyafterthedrawingthebloodandtheagitationmustbesufficienttodenaturethefibrinogenbutnottocauseruptureoftheerythrocytesandhaemolysis.SomelysisisinevitableinthisprocessbutthereleaseofNADinhorsebloodfromtherupturederythrocytesstimulatesthegrowthofHaemophilusinfluenzae.Thiseffectisnotseenwithsheepblood.

Theuseofpotassiumoxalateasananticoagulantincreasestheyieldofwholebloodbutitremainsasapotentialchelateofdivalentmetals.Thischelateeffect,whichalsoappliestosodiumcitratetreatedblood,canbeseenintheoverallsmallercolonieswhichgrowonanticoagulantcontainingbloodagar.

Toprolongtheshelflifeofbloodproductsintheuser'shands,bloodiscollected,bottledanddispatchedonthesameday.Thephysicalcharacteristicsofappearance,haemoglobincontentandpackedcellvolumearecheckedbeforereleasebutsterilitytestsarecarriedoutbeyondthetimeofdispatch.Shouldsamplesofindividualanimalbloodcollectionsshowinfectionthenallrecipientsofthesuspectbloodarenotifiedbytelephone,faxortelex.

Haemolysedorlakedhorsebloodisusedforspecialpurposesinculturemedia.IthasbeenusedformanyyearsinCorynebacteriumdiphtheriaemedia,wherebettergrowthwasobservedafterlysisofthehorsebloodbythetelluriteinthemedium.

LysedbloodisstimulatoryformanyClostridiaandHaemophilusspeciesbutFildesPepticDigestofBloodSR0046canalsobeusedforthispurpose.

Inantibioticsusceptibilitytesting,lysedhorsebloodisaddedtothemediumtoimprovethereactionswithtrimethoprimandsulphonamides.Mostculturemedia,unlessspeciallyprocessedforsusceptibilitytesting,containamountsofthymidinewhichcanantagonisetheinhibitoryeffectsoftheseantimicrobials.Whenhorsebloodislysedtheerythrocytesreleaseanenzymethymidinephosphorylasewhichconvertsthymidineintothemuchlessantagonisticcompoundthymine.Thisenzymedoesnotexistinsheeperythrocytes.Thelyticagentusedforlysedhorsebloodiswhitesaponinwhichappearsnottoaffectthegrowthofbacteriaatthelevelrequiredforlysis.

SerumforCultureMediaHorseserumSR0035

Totallyasepticprocessingisnotrequiredforbloodserumproduction.Bloodiscollectedincleanvesselsinacleanenvironment.Afterclottinghastakenplacethebloodisstoredat28Ctoallowtheclottoretractandtheserumseparate.Theseparatedserumisthenfiltersterilisedandfilledoutintosterilebottles.Allserashouldbestoredat28Cbutforlongertermstorageitisbestfrozenat20C.

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DipslidesGeneralToxinDetection

Serumcanbecomecloudyonstoragecausedbylipaseenzymesandlipidchanges.Heatingtheserumto56Cfor30minutes(inactivation)willusuallyovercomethisproblem.

SheepErythrocytesforImmunologicalReactionsSheepblood(inAlseversolution)SR0053

Afterwashingthesheeperythrocytes,bycentrifugingwithphysiologicalsalineuntilthesupernatantsalineisfreefromhaemolysis,thewashedcellsmaybeusedforanyimmunological/serologicaltestwheresheepcellantigensarerequirede.g.RoseWaaler,PaulBunnellandvariousvirologicalcomplementfixationtests.

Thesheepbloodshouldbestoredat28Candusedbeforetheexpirydateonthelabel.

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