8.Isolation of pure cultures and preservation of cultures.pdf

Isolation of pure culture, its various method.

1. Isolation of pure cultures and
preservation of cultures
Agricultural Microbiology (ABB 156)
College of Horticulture and Forestry
Rani Lakshmi BaiCentral Agricultural University,
Jhansi-284003

2. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
What is Pure cultures ???????
Cultures that contain only one type of cell, ideally with
the culture derived from an initial single cell.
Apurecultureisapopulationofcellsormulticellular
organismsgrowingintheabsenceofotherspeciesor
types.
OR
Pure culture
Mixed culture

3. Isolation of Pure cultures
1.POURPLATETECHNIQUE
2.SERIALDILUTIONTECHNIQUE
3.SPREADPLATETECHNIQUE
4.ENRICHMENT METHOD
5.STREAKPLATETECHNIQUE
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi

4. 1.POURPLATETECHNIQUE
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
Solidsamplese.g.Soil,
Foodmaterial,rootcrush
etc.
Liquidsamplese.g.water,
milk,blood,urine,leaf
sapetc.
Serial Dilution method
1 ml

5. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
➢Innature,microbialpopulationsdonotsegregate
themselvesbyspeciesbutexistwithamixtureofmany
othercelltypes.
➢Thepour-platetechniquerequiresaserialdilutionofthe
mixedculturebymeansofalooporpipette.
➢Moltenagarcooledto45°C,ispouredintoaPetridish
containingaspecifiedamountofthedilutedsample.
➢Followingtheadditionofthemolten-agarmedia,theplates
gentlyrotatedinacircularmotiontoachieveuniform
distributionofmicroorganisms.
➢Incubatedovernighttogrowandmultiplythemicrobialcell.
➢Countthecolonies
Purpose of pour plate method
➢Tocheckthemicrobialloadinasamples(water,soil,and
plantsample).

6. Themostcommonmethodfordeterminingthetotal
viablecountisthepour-platemethod.
Thepourplatetechniquecanbeusedtodeterminethe
numberofmicrobes/mLinaspecimen.
Ithastheadvantageofnotrequiringpreviously
preparedplatesandisoftenusedtoassaybacterial
contaminationoffoodstuffs.
The use of relatively hot agar carries the risk of killing
some sensitive contaminants, so giving a low result.
Small colonies may be overlooked.
Advantages of pour plate technique
Disadvantages of pour plate technique
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi

7. 2.SERIALDILUTIONTECHNIQUE
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
✓Aserialdilutionisthestepwisedilutionofa
substanceinsolution.
✓Usuallythedilutionfactorateachstepisconstant,
resultinginageometricprogressionofthe
concentrationinalogarithmicfashion.
✓Theheavypopulationofmicrobesisdilutedfor
furtherisolationpurposes.

8. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
➢Prepare a series of at least 6 test tubes containing
9 ml of sterile distilled water.
➢Using a sterile pipette, add 1ml of sample in the
first tube of the set. Label it as 10
-1
.
➢Mix the contents well by swirling the tube upside
down few times.
➢From the first tube, take 1ml of the sample and
transfer to second tube. Label it as 10
-2
.
➢Repeat the procedure with all the remaining tubes
labelling them until 10
-6
.

9. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
Advantages
1.Ithelpstoreduceadensecultureofcellstoamore
usableconcentration.
2.Aspecificamountofbacteriaarereducedwith
everydilution.
3.Thenumberofcoloniesculturedfromserial
dilutionsofthesamplearecountedtoestimatethe
concentrationofanunknownsample.
Disadvantages
1.Itdoesnotseparatebacterialikeastreakplate.

10. 3.SPREADPLATETECHNIQUE
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
✓Spreadplatetechniqueisamethodemployedto
platealiquidsampleforthepurposeofisolatingor
countingthebacteriapresentinthatsample.
✓Aperfectspreadplatetechniquewillresultsvisible
andisolatedcoloniesofbacteriathatareevenly
distributedintheplateandarecountable.
✓Thetechniqueismostcommonly appliedfor
microbialtestingoffoods,plantsamplesorto
isolateandidentifyvarietyofmicrobialflora
presentintheenvironmentalsamplese.g.soil.

11. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi

12. Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
➢Pipetteout0.1mlfromtheappropriatedesireddilution
seriesontothecenterofthesurfaceofanagarplate.
➢DiptheL-shapedglassspreader(hockeystick)into
alcohol.
➢FlametheglassspreaderoveraBunsenburner.
➢Spreadthesampleevenlyoverthesurfaceofagarusing
thesterileglassspreader,carefullyrotatingthePetri
dishunderneathatanangleof45
o
atthesametime.
➢Incubatetheplateat30-37°Cfor24hours.
➢Calculatetheconlonyformingunits(CFU)valueofthe
sample.
Method of Spread Plate

13. Tomakeaccuratedilutionsusingpipettes(master
serialdilutiontechnique).
Toapplyabalancedspreadtechniqueusingaglass
spreadertospreadtheinoculumevenlyontheagar
surface.
Torespectthenecessary“short”timeintervalbetween
agarinoculationandspreading.
➢Onceyoucountthecolonies,multiplybytheappropriate
dilutionfactortodeterminethenumberofCFU/mLinthe
originalsample.
Rani
Lakshmi
Bai
Central Agricultural University, Jhansi
Check Points

14. Forexample:Supposetheplateofthe10
-6
dilution
yieldedacountof130colonies.Then,thenumberof
bacteriain1mloftheoriginalsamplecanbe
calculatedasfollows:
CFU(Bacteria)/ml=130/0.1mlx10
6
=130×10
7
=1.3x10
9
or1300,000,000Cells/ml
Total No. of colonies
Volume of culture Spreadedon plate
xDilution factorCFU/ml =
Calculationofresult:

15. 4.ENRICHMENT METHOD
➢Enrichmentcultureisbasicallyanisolationtechniquedesigned
tomakeconditionsofgrowthveryfavorableforanorganismof
interestwhilehavinganunfavorableenvironmentforany
competition.
Or
“Enrichmentcultureisthetechniquethatisusedtoenhancethe
populationdensityofaparticulargroupofmicroorganismswithin
thetotalmicrobialpopulationofasample”.
➢Enrichmentisgenerallydonebyintroducingnutrientsintothe
certaingrowthmediatofavourthegrowthofaparticular
microorganism overothers,enrichingasampleforthe
microorganismofinterest.
➢Enrichmentculturetechniquesareusedtoincreaseasmall
numberofthedesiredorganismstodetectablelevels.Thisallows
forthedetectionandidentificationofmicroorganismswitha
varietyofnutritionalneeds.

16. Forexample:1.Methanogenicenrichmentsareusually
performedinmediathathasanutrientcomposition,
environmentalpHvalue,temperatureandoxygen-free
conditions,similartothoseofnaturalmethanogenic
environments.
2.Fortheisolationofphosphatesolubilizingmicrobes
generallycalciumtriphosphateissupplementedinthe
media,Sothattargetedmicrobescanbeisolated.
3.Similarlyfortheisolationofsalttolerantmicrobes,the
mediaissupplementedwithNaClwithdifferent
concentration.

17. Observable Characteristics for the isolation of
Desired Bacteria
1.OdorProduction:Somebacteriaproduceauniquearoma(gas)as
aresultoftheirmetabolicprocesses.Microbiologistwillalways
makenoteofthespecificaromaoftheorganismunderstudy.
Examplesofaromaareammonia(NH
3),rottenegg(H
2S),alcoholic(-
OH),fecal,etc.
2.PigmentProduction:Pigmentsproducedby
bacteriaareeithercellbound(limitedtocells
andthecolony),extracellular(pigmentis
releasedintothemedium)orboth.Thecolorof
thepigmentshouldbenoted,asthis
characteristiccouldbeveryhelpfulinthe
identificationoftheorganism.

18. 3.ColonialMorphology:Thiscategoryincludesthecolonydensity,
texture,elevation,cohesiveness,shapeandmargin.
a.Colonydensity:Holdtheplateinfrontofalightsourceto
determineifthecoloniesareclear,opaque(nolightpassesthrough
thecolony)ortranslucent.
b.Colonytexture:This
isbestobservedwhen
directlightisreflected
offthecolonies.The
textureisusually
eithersmooth(even
surface)orrough
(irregular,non-smooth
surface).

19. 5.STREAKPLATETECHNIQUE
✓Itisusedforisolatingindividualcoloniesofbacteria.During
streaking,youuseasterilelooptopickasinglecolonyfromthe
cultureplate.
✓Thisinvolvesjustlightlytouchingthelooptothecolonyof
interest.
✓Then,yousmear,or“streak”theloopwiththebacteriaontoa
freshplatetoisolatethedifferentbacteriacolonies.
✓Themodernstreakplatemethodhasprogressedfromtheefforts
ofRobertKoch.

20. Quadrant streaking
Zig-ZagstreakingSingle Line streaking

21. ✓Toproduceisolatedcoloniesofanorganism(mostly
bacteria)onanagarplate.
✓Thisisusefulwhenweneedtoseparateorganismsin
amixedculture(topurify/isolateparticularstrain
fromcontaminants)orwhenweneedtostudythe
colonymorphologyofanorganism.
✓Toidentifytheorganism:biochemicalteststoidentify
bacteriaareonlyvalidwhenperformedonpure
cultures.
Purpose of streaking

22. Preservation of Cultures
✓Theprimaryaimofculturepreservationistomaintainthe
organismalive,uncontaminatedandwithoutvariationor
mutation.

23. ➢Refrigeratororcoldroomstorageisofuseonlyforshorttime
preservationofcultures.
✓Liveculturesonaculturemediumcanbesuccessfullystoredin
refrigeratorsorcoldrooms,whenthetemperatureis
maintainedat4ºC.
✓Atthistemperaturerangethemetabolicactivitiesofmicrobes
slowsdowngreatlybutdonotaltogetherstop.
✓Asaresult,bacterialmetabolismwillbeveryslowandonly
lessquantityofnutrientswillbeutilized.
✓Thismethodcannotbeusedforaverylongtimebecausetoxic
productsgetaccumulatedwhichcankillthemicrobes.
1. Storage at Refrigerator Or Cold Room Storage

24. Cultures are storage at Refrigerator

25. ➢Freezingisacommonprocessforstorageofbacteria.Thus,thick
bacterialsuspensionscanbefrozenatatemperatureof-30ºC.
➢Metabolicratesarereducedbyloweringthetemperature.
➢Freezingandthawingisawellknowntechniqueforactually
disruptingcells.
➢Culturescanbepreservedveryeffectivelyiffrozeninthe
presenceofacryoprotectant(Glycerolordimethylsulphoxide
(DMSO),whichreducesdamagefromicecrystals.
➢Thesimplestwaytopreserveacultureistoadd15%(v/v)glycerol
tothecultureandthentostoreitat-20ºCor-80ºCinafreezer.
2. Storage By Freezing
➢Culturescanbepreservedforanumberofyearsinglycerol,ata
temperatureof-40ºCinafreezer.

26. Freeze-drying(lyophilization)isoneofthemosteconomicaland
effectivemethodsforlong-termpreservationofbacteriaandother
microorganisms.
➢Inthismethod,about2mlofglycerolsolutionisaddedonto
theagarslantculture.Shakingcanemulsifytheculture.
Emulsionisthentransferredtoampoules,witheachampoule
having5mloftheculture.
Inthismethodbacterialculturesorvirussuspensionsaredried
andkeptinthedrystateundersuitableconditions.
3. Freeze-drying (lyophilization)
Freeze-dryingisamultistageprocess:
➢Itbeginswithfreezingbelow0ºC(e.g.-20ºC),atemporarystop
tometabolicactivity.
➢Thencontinueswiththeremovalofwaterwithoutthawing
(sublimation).

27. ➢Endswithadriedproduct.Thedriedproductis
sealedeitherundervacuumorunderaninertgas,
canbestoredatroomtemperaturewithnofurther
metabolicactivityuntilwaterandnutrientsare
restored.
Ampoule containing lyophilized
microorganisms

28. 4. Storage In Silica Gel
➢Bothbacteriaandyeastcanbestoredinsilicagel
powderatlowtemperatureforaperiodof1-2years.
➢Inthismethod,finelypowdered,heatsterilizedand
cooledsilicapowderismixedwithathick
suspensionofcellsandstoredatlowtemperature.
➢Thebasicprincipleinthistechniqueisquick
desiccationatlowtemperature,whichallowsthecell
toremainviableforalongperiod.

29. 5. Preservation using Paraffin and mineral oil

30. ➢VariousfungisuchasFusarium,Penicillium,Alternaria,
Rhizopus,Aspergillusetc.provedsuccessfulforstoragein
sterilesoil.
➢Soilstorageinvolvesinoculationof1mlofsporesuspension
intosoil(thathasbeenautoclavedtwice)andincubatingat
roomtemperaturefor5-10days.
➢Thisinitialgrowthperiodallowsthefungustousetheavailable
moistureandgraduallytobecomedormant.Thebottlesare
thenstoredatrefrigerator.
➢Sprayingfewsoilparticlesonasuitablemediumretrieves
culture.
6. Storage in Sterile Soil