Bacterial isolation

Details about different steps in the bacterial isolation process.

1. BACTERIAISOLATION
Introduction:
Thereareapproximately10,000namedspeciesofmicrobes.Itisestimatedthat
therearebetween10,000and100,000moreunidentifiedspeciesforevery
identifiedone.Notonlyaretheremanytypesofbacteria,therearealotof
individualbacteria.Asinglespoonfulofsoilcanhave100millionindividual
bacteria.Ascrapingofyourgumscanyield1millionbacteriapercm2(acm2is
aboutthesizeofyourlittlefingernail).Thebacteriainandonourbodiesmakes
upabout10%ofourdrybodyweight.
Mostofthecurrentlyknownspeciesofbacteriahavebeenidentifiedusing
traditionalmicrobiologicaltechniquessuchasthegramstainreaction,
morphology,andmetabolicreactions.Bacteriararelylivealonebutin
communitieswithotherbacteria.Thisistruebothintheenvironmentandinand
onourbodies.Thisclassfocusesontheroleofbacteriaindisease.Isolatinga
singlebacteriumspeciesisthefirststepinidentifyingthebacteriapossibly
responsibleforadiseaseprocess.
Thefirstrequirementforphysicallyisolatingabacteriumisthatitcanbe
culturedinthelaboratory.Thisrequiresknowledgeofoptimaltemperaturefor
growth,optimaloxygenrequirements,andoptimalnutritionalneeds.Wework
withaverylimitednumberofbacteriainthiscourse.Thebacteriaweworkwith
arealsoveryeasytocultureinthelab.
IsolationofMicroorganisms:
Microorganismsoccurinnaturalenvironmentlikesoil.Theyaremixedwith
severalotherformsoflife.Manymicrobesarepathogenic.Theycauseanumber
ofdiseaseswithavarietyofsymptoms,dependingonhowtheyinteractwiththe
patient.Theisolationandgrowthofsuspectedmicrobeinpurecultureisessential
fortheidentificationandcontroltheinfectiousagent.
Theprimaryculturefromnaturalsourcewillnormallybeamixedculture
containingmicrobesofdifferentkinds.Butinlaboratory,thevariousspeciesmay
beisolatedfromoneanother.Aculturewhichcontainsjustonespeciesof
microorganismiscalledapureculture.Theprocessofobtainingapureculture
byseparatingonespeciesofmicrobefromamixtureofotherspecies,isknown
asisolationoftheorganisms.
MethodsofIsolation:

2. Therearespecialtechniquesemployedtoobtainpureculturesofmicroorganisms.
Infewcasesitispossibletosecurepureculturebydirectisolationordirect
transfer.Thiscanbedoneonlyinthosesituationsinwhichpurecultureoccurs
naturally.Kindsofspecimenstakenforculturingwilldependonthenatureand
habitatofmicrobes.
Differentpathogenscanbeisolatedfrombodytissuesandfluidssuchasblood,
urine,sputum,pus,faces,spinalfluid,bile,pleuralfluids,stomachfluidsetc.In
thebloodstreamofapatientsufferingwithtyphoidfever,thebacteria
Salmonellatyphosamaybepresent.
Apurecultureofthisbacteriummaybeobtainedbydrawingbloodsampleusing
asterilizedhypodermicsyringeandtreatingthebloodwithanticoagulantsuchas
heparinandpotassiumoxalate.Thepresenceoftheanticoagulantpreventsthe
pathogenicmicrobefromentrappinginfibrinclot.Thesampleofthebloodmay
beinoculatedintoasuitablemedium.
Followingisolationmethodsareemployedtoisolatemicrobesfrommixed
cultures:
1.Streaking
2.Plating
3.Dilution
4.Enrichedprocedure,and
5.Singlecelltechnique.
1.Streaking:
Thisismostwidelyusedmethodofisolation.Thetechniqueconsistsofpouring
asuitablesterilemediumintosterilepetriplateandallowingthemediumto
solidify.Bymeansofasterileloopeorstraightneedleorasterilebentglass-rod
asmallamountofgrowthpreferablyfromabrothcultureorbacterialsuspension
isstreakedbackandforthacrossthesurfaceofagaruntilaboutonethirdofthe
diameteroftheplatehasbeencovered.
Theneedleisthenflamedandstreakingindoneatrightanglestoandacrossthe
firststreak.Thisservestodragbacteriaoutinalonglinefromtheinitialstreak.
Whenthisstreakingiscompletedtheneedleisagainflamedandstreakingis
doneatrightanglestothesecondstreakandparalleltothefirst.

3. 2.Plating:
Itincludesdilutingofamixtureofmicroorganismsuntilonlyafewhundred
bacteriaareleftineachmillilitreofthesuspension.Averysmallamountofthe
dilutionisthenplacedinasterilepetriplatebymeansofasterilelooporpipette.
Themeltedagarmediumiscooledtoabout45°Candispouredintoplate.The
microorganismandagararewellmixed.Whentheagarissolidifiedthe
individualbacteriumwillbeheldinplaceandwillgrowtoavisiblecolony.
3.Dilution:
Thismethodisusedforthemicroorganismswhichcannotbeeasilyisolatedby
streakingorplatingmethod.Sometimeswhenseveralorganismsarepresentina
mixture,withoneorganismpredominating,thepredominatingformmaybe
isolatedbythismethod.Forexample,whenrawmilkisallowedtosouratroom
temperatureitwill,atthetimeofcurding,haveamixtureofmicroorganisms
withhighpercentageofStreptococcuslactis.
If1mlofthesourmilkistakenintoatubecontaining9ml.ofsterilemilk(in
whichnoorganismsarepresent)then1ml.ofthismixtureistransferredwitha
sterilepipetteintoasecondtubeofsterilemilkandtheprocedureisrepeatedi.e.
fromsecondtothirdtube,thirdtofourthtubeuntilaseriesofabout10tubesare
inoculated.Bythisserialdilution,thechancesarethatapurecultureofS.lactis
willbeobtained.

4. 4.EnrichmentProcedure:
Thisprocedureinvolvestheuseofmediaandconditionsofcultivationwhich
favourthegrowthofthedesiredspecies.Forexample,whenamansufferswith
typhoid,theintestinaldischargepossessmallnumberofSalmonellatyphosa
whencomparedwithE.coliandotherforms.
Itisalmostimpossibletoisolatethetyphoidorganismsbecausetheyrepresent
onlyafractionofapercentofthetotalmicroorganismspresent.Themediaare
thereforederived,whichallowtherapidgrowthofthedesiredorganisms,atthe
sametimeinhibitingthegrowthofothermicroorganisms.
5.SingleTechnique:
Thisisoneofthemostidealanddifficultmethodofsecuringpureculture.Inthis
methodasuspensionofthepurecultureisplacedontheunder-sideofasterile
cover-glassmountedoveramoistchamberonthestageofthemicroscope.
Whilelookingthroughthemicroscope,asinglecellisremovedwiththehelpof
sterilemicropipetteandtransferredtoasmalldropofsterilemediumonasterile
cover-glassandismountedonasterilehangingdropslide,whichisthen
incubatedatsuitabletemperature.Ifthesinglecellgerminatesinthisdrop,few
cellsaretransferredintoatubecontainingsterileculturemediumwhichisplaced
intheincubatortoobtainpurecultureoriginatedfromsinglecell.

5. Othermethods:
Theisolationofanaerobicmicroorganismsisverydifficult.Therearecertain
specialtechniquesbywhichtheseorganismsareisolated.
CultivationofMicroorganisms:
Forcultivatingmicrobesinlaboratory,werequireculturemedia.Thevarious
mixturesofnutritivesubstancesusedforthelaboratorycultivationof
microorganismsarecollectivelyknownasculturemedia.Theculturemedia
serveassoilinwhichbacteriaareplantedforthepurposeofstudy.
CultureMedia:
Culturemediamustcontainalltheessentialnutrientsrequiredbytheorganism
foritsgrowthandreproduction.Asuitablesourceofenergy,buildingmaterials
andgrowthfactorsmustbesuppliedinadequateamounts.
Soaculturemediummustcontain:

6. Sincemicroorganismsshowaconsiderablevariationintheirnutritional
requirements,nosinglemediumissuitableforgrowthofallofthem.
Thedifferenttypesofculturemediaemployedfallintothreegroups:
1.Definedorsyntheticmedia:
Thesearethemediapreparedfromchemicalcompounds.Theyarehighly
purifiedandspecific,aninvestigatorworkinginanotherlaboratorycanduplicate
them.
2.Complexornon-syntheticmedia:
Mediathatarepreparedfromingredientsthathavenotbeenpreciselydefined.It
containshydrolysedproteinsandvitaminextracts.Thistypeofmediumcannot
beduplicatedbyanotherworkerinanotherlaboratory.Peptoneisusually
producedbyboilingbeef,bythehydrolysisofitsprotein.Caseinpeptoneand
milkpeptonearealsousedincomplexmediaasthesourceofaminoacidsand
nitrogen.
Allliquidmedia,whethercomplexorsyntheticmaybeconvertedtosolidmedia
byaddingeithergelatin(aprotein)oragar-agar,(acomplexpolysaccharide)
extractedfromredmarinealgae.Theuseofagarhasanadvantage.Themost
bacteriaareunabletohydrolyzethismoleculeintomoresimplecomponents.
Sincegelatinisaliquidatroomtemperature,theuseagarallowsthemediumto
remaininasolidformwhilemicrobesaregrowingonitssurface.
3.Livingcells:
Theseareusedforthecultivationofviruses.Forexample,fertilizedeggs
incubatedfor8to12daysareabletosupportthegrowthofmanyviruses.
Inanotherclassificationculturemediaaregroupedintofollowingfour
types:
1.Naturalmedia:
Includessubstancesoccurringinnature,as1)Milk2)Eggs3)Blood4)Extract
ofplantandanimaltissues.
2.Derivedmedia:
Includesknownsubstancesbutthechemicalcompositionofeachisunknown.
Examplesare1.Nutrientbroth(preparedbyboilingbeeftoextractnutrientsand
addinganaminoacid-nitrogensource.)2.Nutrientagar3.Nutrientgelatin.

7. 3.Chemicallydefinedmedia:
Exactchemicalcompositionknown.
4.Specialmedia:
Includecombinationsoftheotherthreetypesofmedia.
Therearefourcategoriesofmediausedinlaboratory:
Theyare:
1.Enrichment
2.Selective
3.Differentialand
4.Propagation.
1.Enrichmentmedia:
Theyarepreparedwithingredientsthatwillenhancethegrowthofcertain
microbes.Enrichmentmediaencouragethegrowthofthesuspectedpathogenso
thatitwillbecomethemostpre-dominanttypeofmicrobeintheculture.Two
typesofenrichmentmediaarebloodagarandchocolateagar.
2.Selectivemedia:
Theyarepreparedwithingredientsthatinhibitthegrowthofunwantedmicrobes
whichmightbeinthespecimen.Theinhibitormaybeanantibiotic,saltorother
chemical.Mixedcultureofmicrobesoriginallygrowninenrichmentmediamay
beinoculatedintoselectivemediatoisolatethedesiredmicrobe.
3.Differentialmedia:
Theyaredesignedtodifferentiateamongmicrobes.Differentbacterialspecies
mayproducedissimilarcolonycolourswhengrownondifferentialagar.Whilein
differentialbrothcultures,themediachangecolour.Differentialmediaareused
toconfirmtheidentityofamicrobethathasalreadybeenisolatedbyculturingin
enrichmentandselectivemedia.
4.Propagationmedia:
Theyareusedtopropagate,orkeepmicrobesgrowingforalonglime.Samples
grownonthesemediamaybetakenforanalysis.Themostcommonpropagation
mediaarenutrientbrothandagar.

8. PreparationofMedia:
Therearethreemainstepsinthepreparationofmedia:
(a)PreparationassolutionsofchemicalsandadjustingthepH.
(b)Dispensingthemedia,and
(c)Sterilization.
Abrothispreparedbydissolvingtheappropriateamountofthecomponentsin
distilledwaterandpHisadjustedbytheadditionofeitherdiluteNaOHorHcl.
Thebrothisdispensedintocleanrimless‘Pyrex’testtubeswhichareplugged
withnon-absorbantcottonwoolplugs.Thetesttubesareplacedinwirebaskets
whicharecoveredwithgreaseproofpaper.
Themediaaresterilizedbyautoclavingatatemperatureof121°Candapressure
of151b/in2for15minutes.Butmediumcontainingheat-sensitivesubstances
aresterilizedeitherbyfilteringthesolutionatroomtemperature,using
bacteria-prooffilterorbyaprocesscalledTyndallization.
Inthismethod,theliquidsaresteamedforonehouradayonthreeconsecutive
daysandtheliquidsareincubatedat25-30°C.Duringthefirststeaming,allthe
heatsensitivevegetativecellsarekilled,leavingonlythespores.Duringthefirst
incubationperiod,mostofthesporesgerminateintovegetativecells.These
vegetativecellsarekilledbythesecondsteamperiod.
Inthesecondincubationperiod,therestofthesporesgerminateintovegetative
cellswhicharekilledbythethirdsteamingperiod.Inthisway,theliquidsare
sterilizedwithouttemperaturerisingabove100°C.
MaintenanceofPureCulture:
Afterobtainingthepurecultureofaparticularmicrobe,itmaybegrown
andmaintainedasapurecultureindifferentways:
1.Themostcommonpracticeistogrowthecultureonsuitablemediumuntilit
reachesthestationaryphaseofgrowth,andthenstoreinarefrigerator.Iftheyare
tobekeptaliveforalongperiodallculturemustbetransferredtoafreshsterile
medium.Thusbysuccessivetransfer,aculturemaybekeptforanindefinite
period.
2.Asecondmethodinvolvesfreezingofyoungcultureanddesiccatingitunder
vaccum.Thecellsofapureculturewillremainviableforalongperiodoftimeif
theyaremixedwithsterilebloodserum,sterileskimmedmilk,beforefreezing

9. anddrying.Theydriedculturesarekeptinthesealed,evacuatedtubesandare
storedincoolplaces.
3.Thismethodofmaintainingpurecultureismostsuitableforsporeforming
species.Themicroorganismsaregrowninpurecultureinsuitablemedia.A
suspensionofmicroorganismsisthentransferredtocottonstopperedtubesof
sterilizeddrysoil.Thesporesremainviable,thoughdormant,forlongperiodsof
time,indrysoil.Theorganismcanbegrownafteradesiredperiod,by
transferringthesoilintoasuitablemediumandincubatingitundersuitable
temperature.
PRINCIPLE:
Bacterialisolation,purificationandidentificationarethefirststepsto
bacteriologicalstudies.Isolationisdonetoobtainpurebacterialcultures.
Bacteriaareusuallyisolatedfromfishkidneyandspleen;andfromthe
hepatopancreas,lymphoidorganandmusclesofshrimp.Thesetissuesare
monitororgansthatusuallyharborthedisease-causingbacteriaduringinfection.
Toobtainapurebacterialcultureisthefirststeptobacterialidentification.Pure
cultureisessentialinthestudyofthemorphology,physiology,biochemical
characteristics,andsusceptibilitytoantimicrobialagentsofaparticularbacterial
strain.Pureculturesarebestobtainedbyusingsolidmedia,bystreakplateor
pourplatemethod.Streakplate,ifproperlydone,isthemostpracticalmethod.In
thestreakplatemethod,aloopfuloftheinoculumisplacedneartheperipheryof
theplatewithagarmediumandspreadorstreakedontheupperportionofthe
platewithparalleloverlappingstrokes.Theinoculumisstreakedoverother
portionsoftheplatesothatisolatedcoloniescouldbeobservedinthelast
streakedarea.Theidentificationofabacterialpathogenisimportantinfish
diagnosis.Treatmentcouldbeimplementedonlyafterthecausativeagentorthe
bacteriumhasbeenidentified.Bacterialspeciesdifferinmorphological,
physiologicalandbiochemicalcharacteristicsandthosecanbeusedwhencoding
orlabellingthem(Appendix1.1).Therefore,identificationisaccomplishedby
performingseveralmorphological,physiologicalandbiochemicaltests.Results
ofthesetestsarecomparedtoestablishedtaxaoridentificationschemes
(Appendix1.2).Bacterialculturesshouldbepreservedforfuturestudy.Storing
inappropriatemediumpreservesbacterialcultures.Thesimplestmethodisby
sub-culturingorbytransferringtheorganismtofreshsolidmediumthathasa
minimalnutrientcontenttopreventbacterialovergrowth.Thebacteriaare
allowedtogrowbeforestoringintherefrigeratororarecoveredwithparaffinoil
andstoredatroomtemperatureinthedark.Anothersimplemethodisby
deep-freezingofthebacterialculture,stockedinabrothmediumwithglycerol.
Glycerolisaddedtopreventthedessicationofbacterialcells.Bacterialcultures

10. mayalsobepreservedbyfreeze-dryingorlyophilization.Inthismethod,wateris
removedfromthefrozenbacterialsuspensionbysublimationundervacuum.
Bacterialculturesshouldbeproperlylabeledorcodedbeforestorage.Itis
importanttolabelthetubeorvialforstoringbacterialcultureswithanindelible
ink.Thelabelorcodeshouldincludethereferencenumberandotherpertinent
informationsuchassourceofsample(hostanimal,location),dateofisolation,
specialproperties,identification,nameofthepersonwhoisolatedtheorganism
andthedateofpreparationofthestockculture.
OBJECTIVES:
Afterreadingthischapter,youwillbeableto:
Expalinthestepsinvolvedintheisolationofbacteria.
describethesignificanceofSpecimencollection.
describethesignificanceofPreservationandtransportationofspecimen.
explaintheroleofmicroscopyinisolationofbacteria.
explainvariousmethodsforisolationofbacteria.

ISOLATIONOFBACTERIA:
Isolationofbacteriaformsaverysignificantstepinthediagnosisand
managementoftheillness.Isolationofbacteriainvolvesvarioussteps
Specimencollection
Preservationandtransportationofspecimen
Microscopicexaminationofsample
Variousmethodsusedforisolationofbacteria
SpecimencollectionManydifferentspecimensaresentformicrobiological
examinationfrompatientswithsuspectedbacterialinfection.Common
specimensincludeurine,faeces,woundswabs,throatswabs,vaginalswabs,
sputum,andblood.Lesscommon,butimportantspecimensincludecerebrospinal
fluid,pleuralfluid,jointaspirates,tissue,boneandprostheticmaterial(e.g.line
tips).
Sometypesofspecimenarenormallysterilee.g.blood,CSF.Thesesamplesare
usuallyobtainedviaapercutaneousroutewithneedleandsyringe,using
appropriateskindisinfectionandanaseptictechnique.Thecultureofbacteria
fromsuchspecimensisusuallyindicativeofdefiniteinfectionexceptiftheyare
skincontaminants(bacteriainhabitantsofnormalskin).

11. Incontrast,manymicrobiologicalspecimensareobtainedfromnon-sterilesites
e.g.vaginalorthroatswabs,urinesample,stoolsample.Suchsamplesoften
containbacteriaofnoclinicalrelevanceinadditiontopossiblepathogens,
makingtheinterpretationofcultureresultsmoredifficult.Ingeneralitis
preferabletosendsamplesfromsterilesitesifavailable.
Itispreferredtoobtainthesamplesforbacteriologicalculturebeforeantibiotic
therapyisstarted.Thismaximizesthesensitivityoftheinvestigationsand
reducesfalse-negativeresults.Similarly,samplesoftissueorpusarepreferred
overswabs,tomaximizetherecoveryofbacteriainthelaboratory.
Specimensmustbeaccuratelylabelledandaccompaniedbyaproperly
completedrequisitionform,indicatingthenatureofthespecimen,thedateof
samplecollection,relevantclinicalinformation,theinvestigationsrequired,and
detailsofantibiotictherapy,ifany.
Thisallowsthelaboratorytoperformthecorrectrangeoftests,andhelpsinthe
interpretationofresultsandreporting.Alongwithclinicalspecimens,medical
microbiologylaboratoriesalsoprocesssamplesoffood,waterandother
environmentalsamples(e.g.airsamplingfromoperatingtheatres)aspartof
infectioncontrolprocedures.
High-risksamples:
Certainbacterialinfectionsareaparticularhazardtolaboratorystaff,and
specimensthatmightcontainthesepathogensshouldbelabelledas‘highrisk’to
allowforadditionalsafetymeasuresifnecessary.Forexample-bloodcultures
fromsuspectedtyphoid(Salmonellatyphi)orbrucellosis(Brucellaspecies),and
samplesfromsuspectedMycobacteriumtuberculosis.
PreservationandTransportofspecimen:
Mostspecimensaresenttothelaboratoryinsterileuniversalcontainers.Swabs
areplacedinasuitabletransportmedium(eg.charcoalmedium)otherwiseit
leadstofalsenegativereporting.

12. Specimensshouldbetransportedassoonaspossibletothelaboratory.Incasea
delayisanticipatedthespecimenshouldbestoredat4°C.
Immediatetransportisnecessaryinorderto:
(i)Preservetheviabilityofthe‘delicate’bacteria,suchasStreptococcus
pneumoniaeorHaemophilusinfluenzae(delaysinprocessingcancause
false-negativecultureresults);
(ii)(ii)Minimizethemultiplicationofbacteria(e.g.coliforms)within
specimensbeforetheyreachthelaboratory.Inparticularurineandother
specimensthatutilizeasemiquantitativeculturetechniqueforthierdetection,as
delaysintransportcangiverisetofalselyhighbacterialcountswhenthe
specimenisprocessed.
Microscopy:
AGramstainhelpswiththevisualizationofbacteria,andgivesanindicationof
thetypeofbacteriapresent,basedontheshapeofthebacteriaandthestaining
properties(Grampositive:purple;Gramnegative:pink/red).
AGramstainalsohelpstoidentifymixturesofbacteria,helpstodeterminethe
appropriaterangeofagarplatestobeusedforsubsequentculture,andhelpswith
theinterpretationofcultureresults.

13. Forliquidspecimense.g.CSF,thesampleisfirstcentrifugedtoconcentrateany
bacterialcellsinthedeposit,andGramstainandcultureisperformedfromthe
depositafterthesupernatantisdecanted.Thishelpsincreasethesensitivityof
bothmicroscopyandculture.
Ziehl-Neelsen(ZN)stainisusedtodemonstratethepresenceofMycobacteria.
Mycobacteriacanalsobevisualizedusingthefluorescentdyeauramineanda
fluorescencemicroscope.Directimmunofluorescenceisemployedtodetect
certainpathogens(e.g.Legionella,Pneumocystis)usingspecificantibodies
conjugatedtoafluorescentdye.
Anothermicroscopictechniqueisdarkgroundmicroscopy.Thisismainlyused
todetectthethinspirochaetalcellsofTreponemapallidum(syphilisbacteria).
METHODSOFISOLATIONOFBACTERIA:
Methodsofisolationofbacteriacanbebroadlyclassifiedintotwo
Culturemethods
OnSolidmedia
OnLiquidmedia

14. Automatedsystems
Non-culturemethods
Culturemethods:
Thespecimensreceivedinthelaboratoryareplatedontheculturemedia.The
appropriateculturemediaisselecteddependinguponthebacteriasuspected.The
followingprecautionsneedtobetakenintoconsiderationwhentheculture
methodsareprocessed
Optimalatmosphericconditions
Optimaltemperature
Growthrequirementofthebacteria
Atmosphericconditions:Coloniesofbacteriaareusuallylargeenoughto
identifyafter18–24hoursofincubation(usuallyat37°C),butforsomebacteria
longerincubationtimesarerequired(from2daystoseveralweeks).Culture
platesareincubated(1)inair,(2)inairwithaddedcarbondioxide(5%),(3)
anaerobically(withoutoxygen)or(4)micro-aerophilically(atraceofoxygen)
accordingtotherequirementsofthedifferenttypesofbacteriathatmaybe
presentinspecimens.
IncaseofMycobacteriaespeciallythescotochromogentheculturebottlesare
placedindarkorthebottlesarecoveredwithblackpaperandkeptforincubation
at37°C.Temperature:Mostofthebacteriarequiresatemperatureof37°Cfor
optimalgrowth.Thistemperatureisprovidedplacingtheinoculatedculture
platesintheincubatorsetat37°Ctemperature.

15. Growthrequirementofthebacteria:
Differentbacteriahavedifferentgrowthrequirements.ForegStreptococcus
pneumoniaerequiresfactorVandfactorXforitsgrowth,whicharefoundin
chocolateagar.ThusforsamplesuspectedofS.pneumoniaethesamplesare
platedonchocolateagar.Similarlydependinguponthegrowthrequirementsthe
appropriateculturemediaareused.
CULTUREONSOLIDMEDIA:
Theprincipalmethodforthedetectionofbacteriafromclinicalspecimensisby
cultureonsolidculturemedia.Bacteriagrowonthesurfaceofculturemediato
producedistinctcolonies.
Differentbacteriaproducedifferentbutcharacteristiccolonies,allowingforearly
presumptiveidentificationandeasyidentificationofmixedcultures.Thereare
manydifferenttypesofculturemedia.Agarisusedasthegellingagenttowhich
isaddedavarietyofnutrients(e.g.blood,peptoneandsugars)andotherfactors
(e.g.buffers,saltsandindicators).
Someculturemediaarenonselective(e.g.bloodagar,nutrientagar)andthese
willgrowawidevarietyofbacteria.Whilesomee.g.MacConkeyagararemore
selective(inthiscasethroughtheadditionofbilesaltsselectingforthe
‘biletolerant’bacteriafoundinthelargeintestinesuchasEscherichiacoliand
Enterococcusfaecalis).MacConkeyagaralsocontainslactoseandanindicator
systemthatidentifieslactose-fermentingcoliforms(e.g.Escherichiacoli,
Klebsiella)fromlactose-nonfermentingcoliforms(e.g.MorganellaSalmonella).
Mediacanbemadeevenmoreselectivebytheadditionofantibioticsorother
inhibitorysubstances,andsophisticatedindicatorsystemscanallowfortheeasy
detectionofdefinedbacteriafrommixedpopulations.
Methodofinoculatingthesolidculturemedia:
Methodusedforinoculatingthesolidmediadependsuponthepurposeof
inoculation-whethertohaveisolatedcoloniesortoknowthebacterialloadofthe
sample(quantitativeanalysis).
Forobtainingtheisolatedcoloniesstreakingmethodisused,themostcommon
methodofinoculatinganagarplateisstreaking.

16. Media:
Bacterialisolationcanbedoneusingageneralmedium,whereinvariousbacteria
cangrow,andselectivemediathatallowsgrowthofspecificgenera.Examplesof
generalmediaarenutrientagar(NA),trypticsoyagar(TSA),andbrainheart
infusionagar(BHIA).Examplesofselectivemediaarethiosulfatecitratebile
sucroseagar(TCBS)forvibrios,andglutamatestarchphenolredagar(GSP)for
aeromonadsandpeudomonads.Mediaaresupplementedwith1-2%sodium
chloride(NaCl)iftobeusedformarinespecies.AdjustthepHoftheculture
mediato7.2-7.4byadding0.1NNaOH.
Streaking:
1.Usinginoculatingloop,getsamplesofshrimp(suchasthehepatopancreas
andmuscle)andfish(kidney,spleen)tissuesandstreakontotheupperonefourth
portionofanagarplatewithparalleloverlappingstrokes.Useoneagarplatefor
eachanimalsample.Theplatecanbedividedintohalfandstreakedwithtwo
differenttissuesfromthesamesample.Besuretolabeltheplate.
2.Flametheloopandallowittocool.Turntheplateatrightangle.Overlapthe
previousstreakonceortwiceandrepeatthestreakingprocessonone-halfofthe
remainingarea.
3.Repeatprocedure2.

17. 4.Incubateplatesovernightat30°C.Photoatrightshowsastreakedplateafter
incubation.
5.Afterincubationfor16-20hours,checkforbacterialgrowth.Checkfor
luminescenceunderdarkconditions,markingtheluminouscoloniesontheplate
withapentelpen.Isolatedcoloniesshouldbeobservedinthelaststreakedarea.
6.Selectrepresentativebacterialcoloniesbasedonthedifferenceinshape,size
andcolor.Markselectedcoloniesfromeachplate.Subcultureontotrypticasesoy
agar(TSA)plateandincubateovernight.

18. Storage:
1.Observethecoloniesontheagarplatetodeterminethepurityoftheculture.
Pureculturesshouldshowthesamecolonycharacteristicsandnotoverlapping.
2.Selectapurewellisolatedcolony.Stabeachstraininto2tubesof1.2%TSA,
labelandincubate.Thesewillserveasstockcultures.

19. 3.Keepthestockculturesinthelowestcompartmentoftherefrigerator(8-12°C)
oratroomtemperatureuntiluse.Donotstockculturesintheseconditionsfor
over6months.
4.Purifiedbacterialculturesmayalsobestockedinnutrientbrothwith20%
glycerolandstoredat–80°Cuntiluse.Bacterialculturesmaybestockedinthis
conditionfor2years.
IDENTIFICATION:
Bacterialisolatesmaybeidentifiedusingconventionalmethodsbasedontheir
morphological,biochemicalandphysiologicalcharacteristics.Thefollowingare
importantbiochemicaltestsfortheidentificationofbacterialgenerathatare
importantinaquaculture:
1.Gramreaction
2.Oxidasetest
3.Motility
4.Oxidationandfermentationtest
5.O/129sensitivitytest6.Sensitivitytonovobiocin
Furtherbiochemicalcharacterizationmustbecarriedoutifthereisaneedto
identifyuptothespecieslevel.

20. Cultureinliquidmedia:
Bacteriacanalsobegrowninliquidmedia(broth).Likeagarplates,broth
culturesmaybenonselectiveorselective.Bacterialgrowthiseasytodetectas
theclearliquidturnsturbid,usuallywithin24–48hr,butincubationmayneedto
beextendedto14daysormore.
Theadvantageofbrothcultureisthatitissignificantlymoresensitivethandirect
cultureonagar.Thedisadvantageisthat,byitself,itisnoteasytodeterminethe
typeofbacteriapresentorwhetheramixedgrowthhasoccurred,andinmost
casesthebrothmustbesubculturedontosolidagarplates.Thiscausesan
additionaldelayincultureresults.Brothculturesarealsopronetocontamination
Brothenrichmentmediaareusedwhenhighsensitivityisrequirede.g.for
detectionofbacteriafromCSF,ortodetectsmallnumbersofSalmonellaina
stoolsamplecontainingmanymillionsofotherbacteria.

21. Automatedsystem:
Automatedbloodculturesystemseg.BACTEC,BacteAlertutilizeliquidculture.
Bacterialgrowthmaybedetectedbyavarietyofmethods(e.g.detectionof
bacterialCO2production).
Automatedliquidculturesystemsarealsoavailableforthecultureof
Mycobacteria,andsimilartechnologycanbeusedtoautomatesensitivity.
TheadvantageofautomatedsystemareRapidity:theyaidinfastergrowthof
bacteria.Thuslesstimeconsuming.
Theincidenceofcontaminationduringtheprocessingofsampleareminimised.
Realtimemonitoringofthegrowth.
Oneofthemainlimitationsisthecommercialviability.

22. Nonculturemethods
Isolationofbacteriacanalsobecarriedoutbynon-culturemethods.Inparticular
themoreadvancedAmplificationtechniqueslikePolymerasechainreaction
(PCR),ligasechainreaction(LCR),stranddisplacementamplification(SDA),
andnucleicacidsequencebasedamplification(NASBA)arebeingusedin
clinicallaboratoriesforisolationandidentificationofbacteria.
Thefollowingaresomeofthefactorsthatareconsideredininterpreting
bacteriologicalcultureresults:
typeofspecimenzanydelaysinprocessing
typesofbacteriarecovered
knowledgeofthenormalhumanfloraatdifferentsites
clinicalinformationprovidedontherequestform
detailsofrecentantibiotictherapy
Theremustbegoodliaisonbetweenhealthcareworkersandthemicrobiology
laboratory,inordertoensurethatthemostappropriateinvestigationsare
performed,resultsareinterpretedcorrectly,andclinicallyrelevant
bacteriologicalreportsareproduced.
REFERENCES:
1.Bailey,W.R.andE.G.Scott.1966.DiagnosticMicrobiology,SecondEdition.
ToppanCompanyLtd.,Japan,342pp.
2.Tonguthai,K.,S.Chinabut,T.Somsiri,P.ChanratchakolandS.
Kanchanakhan.1999.DiagnosticProceduresforFinfishDiseases.Aquatic
AnimalHealthResearchInstitute,Bangkok,Thailand.
3.IsolationandCultivationofMicroorganisms,ArticleSharedbyAkshay
Sisodiya