Microbiological techniques: Sterilization

Students can learn the basics of the sterilization techniques

1. Unit 4 Microbiological
techniques
Dr. Deepak P
Microbiology
and
Microbiological
techniques

2. Components of Microbial media
•Microbialmedia,alsoknownasgrowthmediaorculturemedia,arenutrient-rich
substancesusedtocultivatemicroorganismslikebacteria,fungi,andprotistsin
laboratorysettings.
•Thesemediatypicallycontainamixtureoforganicandinorganiccompoundsthat
providethenecessarynutrientsformicrobialgrowth.
•Thecomponentsofmicrobialmediacanvarydependingonthetypeof
microorganismbeingculturedandthespecificrequirementsoftheexperimentor
study.

3. Components of Microbial media
•Water
•Carbon sources
•Nitrogen sources
•Minerals
•Vitamins
•Agar
•pH indicators
•Selective agents
•Indicator organisms
•Buffering agents

4. •Courtesy: https://images.app.goo.gl/kseoBMyoMehnj2TP6

5. Natural microbiological media
•Naturalmicrobiologicalmediaarethosethatutilizecomponents
deriveddirectlyfromnaturalsourcesratherthansyntheticor
chemicallydefinedingredients.
•Thesemediamimicthenaturalenvironmentofmicroorganisms
morecloselyandareoftenusedforspecificpurposessuchas
environmentalmonitoring,isolationofindigenous
microorganisms,orstudyingmicrobialinteractionsinnatural
habitats.
•Someexamplesofnaturalmicrobiologicalmediainclude:

6. Natural microbiological media
•Soil extract agar: Soil components
•Water agar: organic and inorganic content
•Leaf infusion agar:
•Milk agar:
•Blood agar:
•Shellfish extract agar:
•Plant tissue agar:
•Manure agar:

7. Synthetic Microbial medium
•Syntheticmicrobialmediaareculturemediacomposedof
preciselydefinedchemicalcomponents,oftenwithknown
concentrations.
•Unlikenaturalmedia,whichcontaincomplexmixturesof
organicandinorganiccompoundsderivedfromnatural
sources,syntheticmediaarepreparedusingpurified
chemicals.
•Thesemediaareadvantageousinmicrobiologicalresearch
becausetheyoffergreatercontrolovernutrientcomposition
andcanbetailoredtospecificexperimentalneeds.
•Somecommonexamplesofsyntheticmicrobialmediainclude

8. Minimal media
•Minimalmediacontainonlyessentialnutrients
requiredformicrobialgrowth,typicallyincludinga
carbonsource,nitrogensource,salts,andwater.
•Theyareoftenusedforstudyingmicrobial
metabolism,nutrientrequirements,andgenetic
manipulation.

9. Defined media
•Definedmediaaresimilartominimalmediabutmaycontain
additionalspecificcomponentssuchasvitamins,aminoacids,
ortracemineralsatdefinedconcentrations.
•Thesemediaareusefulforstudyingthenutritional
requirementsofmicroorganismsandforcontrolled
experimentswhereprecisenutrientcompositionsare
necessary.

10. Synthetic complete media
•Syntheticcompletemediaareenrichedformulationsthat
containallnecessarynutrientsrequiredtosupportthe
growthofaspecificmicroorganism.
•Theytypicallyincludeacarbonsource,nitrogensource,
vitamins,minerals,andotheressentialnutrients.
•Syntheticcompletemediaarecommonlyusedforroutine
laboratoryculturingofmicroorganismsandforgenetic
manipulationexperiments.

11. Synthetic selective media
•Syntheticselectivemediaaredesignedtosupportthe
growthofspecificmicroorganismswhileinhibitingthe
growthofothers.
•Theycontainselectiveagentssuchasantibiotics,dyes,
orinhibitorsthattargetparticularmetabolicpathwaysor
physiologicalcharacteristicsofcertainmicroorganisms.
•Thesemediaareoftenusedforisolatingandidentifying
specificbacterialstrainsfrommixedcultures.

12. Synthetic differential media:
•Syntheticdifferentialmediaaresimilartoselective
mediabutalsocontainindicatorsthatallowforthe
differentiationofmicrobialcoloniesbasedonspecific
metabolicactivitiesorbiochemicalreactions.
•Examplesincludemediafortestingcarbohydrate
fermentation,ureaseactivity,orhydrogensulfide
production.

13. Chemically defined media
•Chemicallydefinedmediaaresyntheticmedia
composedentirelyofchemicallydefinedcomponents,
witheachcomponentpreciselycharacterizedand
known.
•Thesemediaofferthehighestlevelofcontrolover
nutrientcompositionandarecommonlyusedin
researchapplicationssuchasbioprocessoptimization,
whereconsistencyandreproducibilityarecritical.

15. Chemically defined Microbial Media
•Chemicallydefinedmicrobialmediaareprecisely
formulatedculturemediacomposedofpurechemical
compounds with known structuresand
concentrations.
•Thesemediaprovidecompletecontroloverthe
nutritionalcompositionandareoftenusedin
researchsettingswherereproducibilityand
consistencyareessential.

16. •M9MinimalMedium:
•M9minimalmediumisaclassicexampleofachemicallydefinedmedium
usedforculturingbacteriasuchasEscherichiacoli.Itcontainssaltssuchas
ammoniumsulfate,potassiumphosphate,sodiumchloride,andmagnesium
sulfate,alongwithacarbonsourcesuchasglucoseorglycerol.Additional
supplementssuchasaminoacidsorvitaminscanbeaddedasneeded.
•E-MEM(EssentialMinimalEagle'sMedium):
•E-MEMisachemicallydefinedmediumusedforculturingmammaliancells
intissueculture.Itcontainsessentialaminoacids,vitamins,inorganicsalts,
glucose,andothernutrientsrequiredforcellgrowth.E-MEMprovidesa
consistentandwell-definedenvironmentforcellcultureexperiments.

17. •YNB(YeastNitrogenBase):
•YNBisachemicallydefinedmediumusedforculturingyeast
cellssuchasSaccharomycescerevisiae.Itcontainssalts,
vitamins,andtracemineralsbutlacksacarbonsource.YNB
canbesupplementedwithdifferentcarbonsourcessuchas
glucoseorgalactoseandadditionalnutrientsasneeded.

18. Complex microbial medium with examples
•Complexmicrobialmediacontainavarietyofnatural
ingredientsderivedfrombiologicalsourcessuchas
animaltissues,plantextracts,ormicrobialproducts.
•Thesemediaprovidearichandundefinedmixtureof
nutrients,growthfactors,andothercompoundsthat
supportthegrowthofawiderangeofmicroorganisms.

19. •NutrientAgar:
•Nutrientagarisawidelyusedcomplexmediumforculturingbacteria
andfungi.Ittypicallycontainsbeefextract,peptone(partially
digestedprotein),agar,andwater.Nutrientagarprovidesageneral-
purposemediumsuitableforthecultivationofmanymicroorganisms.
•TrypticSoyAgar(TSA):
•Trypticsoyagarisacomplexmediumcomposedofenzymaticdigest
ofcasein(soybeanmeal)andsoybeanpeptone,alongwithagarand
water.TSAiscommonlyusedfortheisolationandenumerationof
bacteriafromvarioussourcesduetoitsnutritivepropertiesand
abilitytosupportthegrowthofawiderangeofmicroorganisms.

20. •BloodAgar:
•Bloodagarisacomplexmediumconsistingofagarsupplementedwith
sheeporhorseblood.Itisoftenusedfortheisolationand
differentiationofpathogenicbacteriabasedontheirhemolytic
properties.Bloodagarsupportsthegrowthoffastidiousbacteriaand
providesnutrientssuchashemoglobinfortheirgrowth.
•ChocolateAgar:
•Chocolateagarisacomplexmediummadebyheatingbloodagarto
lyseredbloodcells,releasingnutrientssuchashemoglobin.Itis
particularlyusefulforthecultivationoffastidiousbacteriasuchas
HaemophilusinfluenzaeandNeisseriagonorrhoeae.

21. •MacConkeyAgar:
•MacConkeyagarisaselectiveanddifferentialmediumusedforthe
isolationanddifferentiationofgram-negativebacteria,particularly
membersofthefamilyEnterobacteriaceae.Itcontainsbilesaltsand
crystalviolettoinhibitthegrowthofgram-positivebacteria,aswellas
lactoseandneutralredtodifferentiatelactose-fermentingandnon-
fermentingorganisms.
•SabouraudDextroseAgar(SDA):
•Sabourauddextroseagarisacomplexmediumusedfortheisolationand
cultivationoffungiandyeasts.Itcontainsdextrose(glucose)asacarbon
source,peptone,agar,andmaybesupplementedwithantibioticssuchas
chloramphenicolorgentamicintoinhibitbacterialgrowth.

22. •R2AAgar:
•R2Aagarisacomplexmediumspecificallydesignedforthe
enumerationandcultivationofbacteriafromwatersamples.It
containsareducednutrientconcentrationcomparedtoothermedia,
makingitsuitablefortherecoveryofbacteriathatmaybestressed
orslow-growinginenvironmentalsamples.
•BrainHeartInfusion(BHI)Agar:
•BHIagarisacomplexmediumcontainingbrainandheartextracts,
peptone,andagar.Itiscommonlyusedforthecultivationof
fastidiousmicroorganisms,includingpathogenssuchasStreptococcus
andStaphylococcusspecies.

23. •Tryptic Soy Agar (TSA):
ChocolateAgar:
TrypticSoyAgar(TSA): BloodAgar
•Courtesy: https://images.app.goo.gl/3kEQ16kAFQQXdUSq5

24. Selective microbial Medium
•Selectivemicrobialmediaareformulatedtoencouragethe
growthofcertaintypesofmicroorganismswhileinhibitingthe
growthofothers.
•Thesemediatypicallycontainadditivesthatselectively
suppressthegrowthofunwantedorganisms,allowingthe
targetmicroorganismstothriveandbeeasilyidentified.

25. •MacConkeyAgar:
•Composition:MacConkeyagarcontainsbilesalts,crystal
violet,neutralreddye,lactose,andpeptone,alongwithagar
asasolidifyingagent.
•Application:MacConkeyagarisselectiveforgram-negative
bacteria,particularlymembers ofthe family
Enterobacteriaceae,whileinhibitingthegrowthofmostgram-
positivebacteria.Itisdifferentialbasedonlactose
fermentation,allowingforthedifferentiationoflactose-
fermentingandnon-fermentingbacteria.

26. •MannitolSaltAgar(MSA):
•Composition:MSAcontainsmannitol,salt(typically
sodiumchloride),phenolreddye,peptone,andagar.
•Application:MSAisselectiveforhalophilic(salt-
tolerant)bacteria,particularlyStaphylococcusspecies,
whileinhibitingthegrowthofotherbacteria.Itis
differentialbasedonmannitolfermentation,allowing
fortheidentificationofmannitol-fermentingandnon-
fermentingbacteria.

27. •XyloseLysineDeoxycholate(XLD)Agar:
•Composition:XLDagarcontainsxylose,lysine,
deoxycholate,phenolreddye,sodiumthiosulfate,and
agar.
•Application:XLDagarisselectiveforgram-negative
entericbacteriasuchasSalmonellaandShigella
specieswhileinhibitingthegrowthofotherbacteria.It
isdifferentialbasedonthefermentationofxyloseand
lysine,aswellastheproductionofhydrogensulfide
(H
2S)gas.

28. •HektoenEntericAgar(HEA):
•Composition:HEAcontainsbilesalts,lactose,sucrose,
salicin,ferricammoniumcitrate,bromothymolblue,acid
fuchsin,andagar.
•Application:HEAisselectiveforgram-negativeenteric
bacteria,particularlySalmonellaandShigellaspecies,while
inhibitingthegrowthofmostotherbacteria.Itisdifferential
basedonthefermentationoflactose,sucrose,andsalicin,as
wellastheproductionofhydrogensulfide(H
2S)gas.

29. https://images.app.goo.gl/k6bnYBdXFBBpgjFr8

30. Differential microbial media
•Differentialmicrobialmediaaredesignedtodistinguish
betweendifferenttypesofmicroorganismsbasedon
theirmetabolicorbiochemicalcharacteristics.
•Thesemediatypicallycontainindicatorsorsubstrates
thatundergospecificchangesinresponsetomicrobial
activity,allowingforthevisualdifferentiationof
bacterialcolonies.

31. •EMBAgar(EosinMethyleneBlueAgar):
•Composition:EMBagarcontainseosinYandmethylenebluedyes,
lactose,peptone,agar,andwater.
•Application:EMBagarisselectiveforgram-negativebacteria,
particularlyEnterobacteriaceae,whileinhibitingmostgram-positive
bacteria.
•Itisdifferentialbasedonlactosefermentationandtheabilityof
certainorganismstoreducemethyleneblue.
•Lactose-fermentingbacteriaproducedarkcolonieswithagreen
metallicsheenduetotheprecipitationofeosinandmethyleneblue
dyes.Non-fermentingbacteriaappearcolorlessorpale.

32. •XyloseLysineDeoxycholate(XLD)Agar:
•Composition:XLDagarcontainsxylose,lysine,deoxycholate,phenolred
dye,sodiumthiosulfate,andagar.
•Application:XLDagarisselectiveforgram-negativeentericbacteria
suchasSalmonellaandShigellaspecies,whileinhibitingmostother
bacteria.
•Itisdifferentialbasedonthefermentationofxyloseandlysine,aswell
astheproductionofhydrogensulfide(H
2S)gas.
•SalmonellaandsomeShigellaspeciesproduceblackcoloniesduetoH2S
production,whilenon-H2S-producingbacteriaappearcolorlessorred.

33. https://images.app.goo.gl/7KZjDRn21PrqHzo9A

34. Indicator medium
•Indicatormediaarespecializedmicrobialgrowth
mediathatcontainindicators,substancesthat
undergoobservablechangeswhenspecific
metabolicactivitiesoccur.
•Thesechangescanbevisualizedandusedto
identifyordifferentiatebetweendifferenttypes
ofmicroorganismsorbiochemicalreactions.

35. •PhenolRedBroth:
•Composition:Phenolredbrothtypicallycontainspeptone,
phenolreddye,acarbohydratesuchasglucose,lactose,or
sucrose,andsometimesapHindicator.
•Application:Phenolredbrothisusedtodifferentiate
betweenbacteriabasedontheirabilitytoferment
carbohydratesandproduceacid.Whencarbohydratesare
fermented,acidisproduced,whichlowersthepHofthe
broth,causingthephenolreddyetochangecolor.For
example,inphenolredglucosebroth,themediumturns
yellowwhenglucoseisfermented,whereasinphenolred
lactosebroth,themediumturnsyellowwhenlactoseis
fermented.

36. •MethylRed-Voges-Proskauer(MR-VP)Broth:
•Composition:MR-VPbrothcontainspeptone,glucose,anda
phosphatebuffer.Itisdividedintotwoparts:onefortheMethylRed
testandtheotherfortheVoges-Proskauertest.
•Application:MR-VPbrothisusedtodifferentiatebetweenbacteria
basedontheirmixed-acidfermentationpathway(MethylRedtest)
andacetoinproduction(Voges-Proskauertest).IntheMethylRed
test,theadditionofMethylReddyeturnsthemediumredwhenthe
pHisbelow4.4,indicatingmixed-acidfermentation.IntheVoges-
Proskauertest,theadditionofalpha-naphtholandpotassium
hydroxidefollowedbyincubationwithoxygenresultsinaredcolorif
acetoinispresent.

37. https://images.app.goo.gl/sC74tX4WTdUcYajLA

38. •TripleSugarIron(TSI)Agar:
•Composition:TSIagarcontainspeptone,sugars(glucose,lactose,and
sucrose),ferroussulfate,andapHindicator(phenolred).
•Application:TSIagarisusedtodifferentiatebetweenbacteriabased
ontheirabilitytofermentsugarsandproducehydrogensulfide(H2S)
gas.Fermentationofsugarsleadstotheproductionofacid,which
turnsthemediumyellow.ProductionofH2Sresultsintheformationof
blackprecipitates.Thepatternofcolorchangesandgasproduction
canhelpidentifybacteriaanddeterminetheirmetabolicpathways.

39. https://images.app.goo.gl/XQAUkyWXkocv4KC9A

40. •UreaBroth:
•Composition:Ureabrothcontainspeptone,urea,andapH
indicator(phenolred).
•Application:Ureabrothisusedtodifferentiatebetween
bacteriabasedontheirabilitytohydrolyzeureausingthe
enzymeurease.Urease-positivebacteriahydrolyzeurea,
producingammonia,whichraisesthepHofthebroth,
turningitpink.Urease-negativebacteriadonothydrolyze
urea,andthemediumremainsyellow.

41. https://images.app.goo.gl/jPbbALHwQWZcNm2a7

42. •SimmonsCitrateAgar:
•Composition:Simmonscitrateagarcontainssodium
citrate,ammoniumdihydrogenphosphate,bromothymol
blue,andagar.
•Application:Simmonscitrateagarisusedtodifferentiate
betweenbacteriabasedontheirabilitytoutilizecitrateas
thesolecarbonsource.Citrate-positivebacteriaproduce
alkalinebyproducts,whichraisethepHofthemedium,
turningitblue.

43. https://images.app.goo.gl/y5X3RNB7VTeNiTGy7

44. Enriched medium
•Enrichedmicrobialmediaaredesignedtosupportthe
growthoffastidiousmicroorganisms,whichhave
complexnutritionalrequirementsandmaynotgrowwell
onstandardmedia.
•Thesemediacontainadditionalnutrientsorgrowth
factorsthatprovideoptimalconditionsforthe
cultivationofspecificmicroorganisms.

45. •BloodAgar:
•Composition:Bloodagarisacomplexmediumcontainingagar
supplementedwithsheeporhorseblood.
•Application:Bloodagarisenrichedwithnutrientssuchas
hemoglobinandotherfactorspresentinblood,makingitsuitable
forthecultivationoffastidiousmicroorganisms,particularlythose
thatrequireadditionalnutrientsforgrowth.
•Itiscommonlyusedinclinicalmicrobiologyfortheisolationand
identificationofpathogenicbacteria,includingstreptococci,
staphylococci,andotherhemolyticbacteria.

46. •ChocolateAgar:
•Composition:Chocolateagarispreparedbyheatingblood
agartolyseredbloodcells,releasingnutrientssuchas
hemoglobin.
•Application:Chocolateagarishighlyenrichedwith
hemoglobin,aminoacids,andothergrowthfactors,makingit
particularlysuitableforthecultivationoffastidiousbacteria
suchasHaemophilusinfluenzaeandNeisseriameningitidis.It
iscommonlyusedinclinicalmicrobiologyfortheisolation
andidentificationofthesepathogensfromclinical
specimens.

47. •BrainHeartInfusion(BHI)Agar:
•Composition:BHIagarcontainsbrainandheartextracts,peptone,
andagar.
•Application:BHIagarisenrichedwithnutrientsderivedfromanimal
tissues,makingitsuitableforthecultivationofawiderangeof
fastidiousmicroorganisms,includinganaerobicbacteria,
streptococci,staphylococci,andotherfastidiousorganisms.Itis
commonlyusedinclinicalmicrobiologyandresearchlaboratoriesfor
theisolationandcultivationofvariousmicroorganisms.

48. •TrypticSoyAgar(TSA)with5%SheepBlood:
•Composition:TSAwith5%sheepbloodcontainstrypticsoyagar
supplementedwith5%sheepblood.
•Application:TSAwith5%sheepbloodprovidesadditionalnutrients
andgrowthfactorsfromblood,makingitsuitableforthecultivation
ofawiderangeoffastidiousbacteria,includingstreptococci,
staphylococci,andotherhemolyticbacteria.Itiscommonlyusedin
clinicalmicrobiologyfortheisolationandidentificationof
pathogenicbacteriafromclinicalspecimens.

49. Enrichment medium
•Enrichmentmicrobialmediaaredesignedto
promotethegrowthofspecificgroupsof
microorganismsbyprovidingselectiveconditions
thatfavortheirproliferationwhileinhibitingthe
growthofothers.
•Thesemediatypicallycontainnutrientsthat
supportthegrowthoftargetorganismswhile
minimizingcompetitionfromcontaminants.

50. •SeleniteBroth:
•Composition:Selenitebrothtypicallycontainspeptone,
sodiumselenite,andothersaltsinabufferedsolution.
•Application:Selenitebrothisusedfortheenrichmentand
isolationofSalmonellaandShigellaspeciesfromclinicaland
environmentalsamples.Sodiumseleniteinhibitsthegrowthof
mostotherbacteria,whileSalmonellaandShigellaspeciesare
abletogrowandmultiplyundertheseconditions.After
enrichment,thebrothisplatedontoselectiveagarmediafor
furtherisolationandidentification.

51. https://images.app.goo.gl/fVE5PXZCCAhB7E9f8

52. •Rappaport-Vassiliadis(RV)Broth:
•Composition:RVbrothcontainspeptone,magnesiumchloride,
malachitegreen,andpotassiumtelluriteinabuffered
solution.
•Application:RVbrothisusedfortheenrichmentofSalmonella
speciesfromfoodandenvironmentalsamples.Itcontains
selectiveagentssuchasmalachitegreenandpotassium
tellurite,whichinhibitthegrowthofmostotherbacteriawhile
allowingSalmonellatogrow.RVbrothisoftenusedin
combinationwithotherselectivemediafortheisolationand
identificationofSalmonella.

53. https://images.app.goo.gl/kfsKb5dExtQSmxdf9

54. •AlkalinePeptoneWater:
•Composition:Alkalinepeptonewatercontainspeptoneand
sodiumhydroxideinabufferedsolution.
•Application:Alkalinepeptonewaterisusedfortheenrichment
ofVibriospecies,particularlyVibriocholerae,fromclinicaland
environmentalsamples.ThealkalinepHofthemediuminhibits
thegrowthofmostotherbacteria,whileVibriospeciesare
abletogrowandmultiplyundertheseconditions.After
enrichment,thebrothisplatedontoselectiveagarmediafor
furtherisolationandidentification.

55. https://images.app.goo.gl/6KV2wB8fRVfZtpTy6

56. •TetrathionateBroth:
•Composition:Tetrathionatebrothcontainspeptone,potassium
tetrathionate,andothersaltsinabufferedsolution.
•Application:Tetrathionatebrothisusedfortheenrichment
andisolationofSalmonellaspeciesfromclinicaland
environmentalsamples.Potassiumtetrathionateinhibitsthe
growthofmostotherbacteria,whileSalmonellaspeciesare
abletogrowandmultiplyundertheseconditions.After
enrichment,thebrothisplatedontoselectiveagarmediafor
furtherisolationandidentification.

57. •Muller-Kauffmann Tetrathionate-Novobiocin Broth
(MKTTn):
•Composition:MKTTnbrothcontainspeptone,potassium
tetrathionate,novobiocin,andothersaltsinabufferedsolution.
•Application:MKTTnbrothisusedfortheenrichmentandisolation
ofSalmonellaspecies,particularlySalmonellatyphiand
Salmonellaparatyphi,fromclinicalspecimens.Novobiocininhibits
thegrowthofmostotherbacteria,whileSalmonellaspeciesare
abletogrowandmultiplyundertheseconditions.After
enrichment,thebrothisplatedontoselectiveagarmediafor
furtherisolationandidentification.

58. Pure culture methods: Serial dilution and plating
methods (pour plate, spread plate and streak
plate methods
•SerialDilution:
•Process:Serialdilutioninvolvesdilutingasamplecontainingamixture
ofmicroorganismsinaseriesofstepstoreducethenumberoforganisms
ineachsuccessivedilution.Thisistypicallydonebytransferringasmall
volumeoftheoriginalsampletoatubecontainingaknownvolumeof
sterilediluent(e.g.,salineorbroth)andmixingthoroughly.Subsequent
dilutionsaremadebytransferringaportionofthepreviousdilutiontoa
newtubeofdiluentandmixingagain.
•Purpose:Serialdilutionisusedtoreducethemicrobialpopulationtoa
levelwhereindividualcoloniescanbeisolatedandcountedonagar
plates.Italsohelpsensurethatisolatedcoloniesarisefromsinglecells
orclumpsofcells,facilitatingtheformationofpurecultures.

59. https://images.app.goo.gl/rigi2ejbW8dJNCm1A

60. Pure culture methods: Serial dilution and plating
methods (pour plate, spread plate and streak
plate methods
•PlatingMethods:
•PourPlateMethod:
•Process:Inthepourplatemethod,aseriesofdilutedsamplesare
addedtoseparatePetridishescontainingmoltenagarmedium.The
mixtureisthengentlyagitatedtoensureevendistributionofthe
samplewithinthemedium.Aftersolidification,coloniesgrowboth
onthesurfaceandwithintheagar.
•Purpose:Thepourplatemethodallowsfortheisolationof
microorganismsbothonthesurfaceandwithintheagar,makingit
usefulfororganismsthatmaynotgrowwellonthesurfacealone.It
alsoprovidesahigherchanceofisolatingsinglecoloniescompared
toothermethods.

61. https://images.app.goo.gl/EhUvPX5b5PLtZAD86

62. Pure culture methods: Serial dilution and plating
methods (pour plate, spread plate and streak
plate methods
•SpreadPlateMethod:
•Process:Inthespreadplatemethod,asmallvolumeofthe
dilutedsampleisspreadevenlyoverthesurfaceofanagar
plateusingasterilespreadingtoolsuchasaspreaderor
glassrod.Afterallowingtheplatetodry,coloniesgrowon
thesurfaceoftheagar.
•Purpose:Thespreadplatemethodiscommonlyusedwhen
theobjectiveistoobtainisolatedcoloniesonthesurface
oftheagar.Itisrelativelyquickandstraightforward
comparedtothepourplatemethod.

63. https://images.app.goo.gl/QoXspAxT568otyMz8

64. Pure culture methods: Serial dilution and plating
methods (pour plate, spread plate and streak
plate methods
•StreakPlateMethod:
•Process:Inthestreakplatemethod,asmallvolumeoftheoriginal
sampleisstreakedontothesurfaceofanagarplateusingan
inoculatingloop.Theloopisthensterilizedandusedtostreaktheagar
surfaceinapatternthatgraduallydilutesthesampleacrosstheplate.
Afterincubation,isolatedcoloniesformindistinctregionsoftheplate.
•Purpose:Thestreakplatemethodisparticularlyusefulforobtaining
isolatedcoloniesfromamixedculture.Itreliesonthedilutionofthe
samplethroughsequentialstreaking,allowingfortheisolationof
individualcoloniesontheplatesurface.

65. https://images.app.goo.gl/dcZVk8NLEy3UmhgT7

66. Cultivation of microbes
•Cultivationofmicrobesreferstotheprocessof
growingandmaintainingmicroorganismsin
laboratorysettingsforvariouspurposes,including
research,diagnostics,andindustrialapplications.
Microbialcultivationinvolvesprovidingsuitable
growthconditions,suchasnutrient-richmedia,
appropriatetemperature,pH,andoxygenlevels,to
supportthegrowthandreproductionof
microorganisms.

67. https://images.app.goo.gl/8t1KF4nSem6BmmtU7

68. •Selection of Microorganisms:
•Preparation of Growth Media:
•Inoculation:
•Incubation:
•Monitoring Growth:
•Subculturing:
•Characterization and Identification:
•Storage and Preservation:

69. Maintenance and preservation/ stocking of
pure culture
•RegularSubculturing:
•Topreventgeneticdrift,maintainvitality,andensurethepurityof
cultures,it'sessentialtoregularlysubculturemicroorganismsontofresh
growthmedia.Subculturinginvolvestransferringasmallportionofthe
originalculturetonewmedia,typicallyatregularintervals,suchas
weeklyormonthly,dependingonthegrowthrateofthemicroorganism.
•StorageatRefrigerationTemperature:
•Manymicroorganismscanbestoredshort-term(weekstomonths)at
refrigeratortemperatures(2-8°C).Refrigerationslowsdownmetabolic
processesandreducestherateofmicrobialgrowth,helpingtomaintain
cultureviability.However,culturesshouldstillbesubcultured
periodicallytopreventagingandmaintainviability.

70. https://images.app.goo.gl/a9NpENCzEgyZHexK7

71. •Cryopreservation:
•Cryopreservationinvolvesfreezingmicrobialculturesatultra-lowtemperatures,
typicallyinliquidnitrogen(-196°C)ordeepfreezers(-80°C).Cryopreservation
ensureslong-termviabilityandstabilityofculturesforyearstodecades.Before
freezing,culturesareusuallymixedwithacryoprotectant,suchasglycerolor
dimethylsulfoxide(DMSO),toprotectcellsfromdamageduringfreezingand
thawing.
•Lyophilization(Freeze-Drying):
•Lyophilizationisamethodofpreservationthatinvolvesremovingwaterfrom
microbialculturesundervacuumconditions,followedbysealingthedried
culturesinairtightcontainers.Freeze-dryingpreservesculturesinadehydrated
state,whichsignificantlyextendstheirshelflife.Lyophilizedculturescanbe
storedatroomtemperatureforyearsandareeasilyreconstitutedbyaddinga
suitablerehydrationmedium.

72. •Long-TermMaintenanceinCultureCollections:
•Culturecollections,suchastheAmericanTypeCultureCollection(ATCC)
andothermicrobialrepositories,specializeinthelong-termstorageand
distributionofdiversemicrobialstrains.Thesecollectionsuse
cryopreservation,lyophilization,orotherspecializedtechniquesto
maintainculturesandprovidethemtoresearchersworldwide.
•MaintainingProperRecords:
•It'simportanttomaintainaccurateanddetailedrecordsofeachpure
culture,includingstrainidentification,dateofisolation,sourceofthe
culture,growthcharacteristics,storageconditions,andanysubculturing
orpreservationmethodsused.Properdocumentationensurestraceability
andreproducibilityofresearchresults.

73. Cultivation of anaerobic bacteria
•AnaerobicChambers:
•Anaerobicchambers,alsoknownasgloveboxesoranaerobic
workstations,areessentialforcultivatinganaerobicbacteria.
Thesechambersprovideacontrolledenvironmentwithlow
oxygenlevels(typicallylessthan1%)byreplacingthe
atmospherewithinertgasessuchasnitrogenandhydrogen.The
atmosphereinsidethechamberismaintainedbyagascontrol
systemandmonitoredwithoxygensensors.

74. Cultivation of anaerobic bacteria
•AnaerobicCultureMedia:
•Specializedanaerobicculturemediaareusedtosupportthegrowthof
anaerobicbacteria.Thesemediacontainreducingagentsthathelpcreate
anaerobicconditionsandprovidenutrientsrequiredforbacterialgrowth.
Commonlyusedanaerobicculturemediainclude:
•FluidThioglycollateMedium:Thioglycollatereducesoxygentowater,creatingan
anaerobicenvironment.Italsocontainsnutrientssuchaspeptones,yeastextract,
anddextrosetosupportbacterialgrowth.
•AnaerobicAgar:Anaerobicagarisasolidmediumthatcontainsreducingagentssuch
ascysteineorthioglycolate,alongwithnutrientsandindicatorstovisualizebacterial
growth.
•Brewer'sAnaerobicAgar:Brewer'sanaerobicagarcontainsheminandvitaminK1,
whicharenecessaryforthegrowthofcertainanaerobicbacteria,particularly
anaerobicGram-positivecocci.

75. Staining Techniques: Principles of staining, Types
of Stains-simple, structural and differential
•Stainingtechniquesareessentialtoolsinmicrobiology
forvisualizingandstudyingmicroorganisms.
•Thesetechniquesinvolvetheapplicationofspecificdyes
orstainstohighlightvariousstructuralcomponentsof
cells,allowingfortheirobservationunderamicroscope.

76. Principles of Staining:
•AffinityforCellularComponents:Stainshaveanaffinityfor
specificcellularcomponentsbasedontheirchemicalproperties.
Forexample,somestainsbindtonucleicacids,whileothersbind
toproteinsorcarbohydrates.
•ContrastEnhancement:Stainsincreasethecontrastbetween
differentpartsofthecellandthebackground,makingcellular
structuresmorevisibleunderamicroscope.

77. Principles of Staining:
•Fixation:Priortostaining,cellsareoftenfixedtopreservetheirstructure
andpreventthemfrombeingwashedawayduringthestainingprocess.
Fixativessuchasheat,alcohol,orchemicalagentsmaybeused.
•Differentiation:Afterstaining,excessdyeisremovedthroughaprocess
calleddifferentiation.Thisstephelpsenhancethecontrastbetweenstained
cellsandthebackground.
•Visualization:Stainedcellsareobservedunderamicroscope,allowingfor
theidentificationandcharacterizationofcellularstructuresand
morphologies.

78. Types of Stains:
•SimpleStains:
•Principle:Simplestainsinvolvetheuseofasingle
dyethatbindstoallcellsorstructuresinauniform
manner,providingcontrastbetweenthecellsand
thebackground.
•Examples:Crystalviolet,methyleneblue,safranin,
andbasicfuchsinarecommonlyusedsimplestains.

79. https://images.app.goo.gl/fUPswUxDyEBdf5eF7

80. Types of Stains:
•StructuralStains:
•Principle:Structuralstainstargetspecificcellularstructuresor
components,allowingfortheirvisualizationandidentification.These
stainshighlightparticularstructureswithinthecell.
•Examples:
•GramStain:Distinguishesbetweengram-positiveandgram-negativebacteriabased
ondifferencesincellwallcomposition.
•Acid-FastStain:Detectsacid-fastbacteria,whichhaveawaxycellwall,suchas
Mycobacteriumtuberculosis.
•EndosporeStain:Highlightsendospores,whichareresistantstructuresproducedby
certainbacteriaunderunfavorableconditions.
•CapsuleStain:Visualizesbacterialcapsules,whichareprotectivelayerssurrounding
somebacteria.

81. https://images.app.goo.gl/8N9m26YGsj7im8oo6

82. Types of Stains:
•DifferentialStains:
•Principle:Differentialstainsdifferentiatebetweendifferenttypesof
microorganismsorcellularstructuresbasedontheirstainingproperties.
Thesestainsproducecontrastingcolorsfordifferentcelltypesor
structures.
•Examples:
•GramStain:Gram-positivebacteriastainpurple,whilegram-negativebacteriastain
pinkaftercounterstaining.
•Ziehl-NeelsenStain:Acid-fastbacteriaretaintheprimarystain(carbolfuchsin)and
appearred,whilenon-acid-fastbacteriaarecounterstainedblue.
•SporeStain:Endosporesstaingreen,whilevegetativecellsstainredorpink.
•HematoxylinandEosin(H&E)Stain:Usedinhistology,hematoxylinstainscellnuclei
blue-purple,whileeosinstainscytoplasmandextracellularstructurespink.

83. https://images.app.goo.gl/AXD5EMNeTqTjrYAP7