Requirements for Bacterial growth and development

A brief lecture about bacterial nutrition

1. Microbiology
BIOL 333
1
Lecture 4
Microbial Nutrition
Dr. Dalia El-Badan

2. Microbial Nutrition
•Nutritionisaprocessbywhichchemicalsubstancescalled
nutrientsareacquiredfromtheenvironmentandusedin
cellularactivitiessuchasmetabolismandgrowth.
•Alllivingorganismsrequirenutrientsformaintenance,growth
andreproduction.Theconcentrationofnutrientsinaculture
mediumcanaffecteitherthemicrobialpopulationgrowthrate
orthetotalcellyieldoftheculture.
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3. •Whentheconcentrationofnutrientsincreases,moreofthem
canbeconvertedtocellularmaterial,andasaresultincrease
inthenumberofcellsintheculture.
•Innature,nutrientsareoftenlimitedandcompetitionfor
themisgreat.Toovercomethesedifficulties,many
microorganismshavedevelopedgreatflexibilityinthe
utilizationofvarioussubstratesavailableintheirenvironment
andhaveevolvedformsthataidtheminassimilatingnutrients.
•Cellsaccumulatenutrientsbymeansofproteinscalled
permeases,whicharepartoftheplasmamembranes.
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4. •Thesourceofnutrientsisextremelyvaried:Somemicrobes
obtaintheirnutrientsentirelyfrominorganicsources,and
othersrequireacombinationoforganicandinorganic
sources.
•Parasitescapableofinvadingandlivingonthehumanbody
deriveallessentialnutrientsfromhosttissues,tissuefluids,
secretions,andwastes.
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5. •Aninorganicnutrientisanatomorsimplemoleculethat
containsacombinationofatomsotherthancarbonand
hydrogen.
•Incontrast,themoleculesoforganicnutrientscontaincarbon
andhydrogenatomsandareusuallytheproductsofliving
things.Theyrangefromthesimplestorganicmolecule,
methane(CH
4),tolargepolymers(carbohydrates,lipids,
proteins,andnucleicacids).
•Ingeneral,alllivingthingsrequireasourceofelementssuchas
carbon,hydrogen,oxygen,phosphorus,potassium,nitrogen,
sulfur,calcium,iron,sodium,chlorine,magnesium,andcertain
otherelements.
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6. •Microorganismsdifferinthewaystheytakeupnutrientsfrom
theirenvironmentalthoughtheyhavethesamebasic
nutritionalrequirements.Bacteriaandfungiarecalled
osmotrophsbecausetheyobtaintheirnutrientsinsolutions.
•Osmotrophicmicroorganismsareunabletotransportlarge
molecules(suchasproteinsorpolysaccharides)throughtheir
membranes,andthereforemustdigestthemoutsidethecell.
•Largemoleculesarebrokendownbyexoenzymesthatare
secretedbythecelltotheexternalenvironment
•Othermicroorganismssuchasprotozoa(phagotrophs)ingest
foodparticlesbyphagocytosis.
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7. •Twocategoriesofessentialnutrientsaremacronutrientsand
micronutrients.Macronutrientsarerequiredinrelativelylarge
quantitiesandplayprincipalrolesincellstructureand
metabolism.
•Micronutrients,ortraceelements,arepresentinmuchsmaller
amountsandareinvolvedinenzymefunctionand
maintenanceofproteinstructure.
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8. Chemical analysis of microbial cytoplasm
•Examiningthechemicalcompositionofabacterialcellcan
indicateitsnutritionalrequirements.
•Severalimportantfeaturesofcellcompositioncanbe
summarizedasfollows:
•Watercontentisthehighestofallthecomponents(70%).
•Proteinsarethenextmostprevalentchemical.
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9. •About96%ofthecelliscomposedofsixelements
(representedbyCHNOPS).
•Acellas“simple”asE.colicontainsontheorderof5,000
differentcompounds,yetitneedstoabsorbonlyafewtypes
ofnutrientstosynthesizethisgreatdiversity.Theseinclude
(NH4)
2SO
4,FeCl
2,NaCl,traceelements,glucose,KH
2PO
4,
MgSO
4,CaHPO
4,andwater.
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10. 10

11. Culture methods
Direct isolation:
•Somemicroorganismsarerecognizedbytheircolonial
characteristics,accumulationofsubstances,orchangesin
theirenvironment.
•Byproducingvisibleevidenceoftheirpresencemanyofthese
microorganismsaresuitablefor directisolation.
Appropriategrowthmediaandconditionsforsuchorganisms
couldbereadilydetermined.Manydifferenttypes
ofmicroorganisms,couldbeinvestigatedonlyafter
thetechniquesofenrichmentculturehaddeveloped.
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12. Enrichment culture:
•Enrichmentconditionsforagivenorganismarethosethat
enableittocompetesuccessfullyandoutgrowotherorganisms
thatmaybepresentintheinoculum.
•Thus,onecanimposeenvironmentconditionsbychoiceofa
numberoffactors(natureofcarbon,energyandnitrogen
sources,hydrogenacceptors,gaseousatmosphere,
temperature,light,pH,etc.).
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13. •Oninoculationofsuchamediumwithamixedpopulationof
microorganismssuchasasampleofsoil,theorganismthatare
mosthighlyadaptedtotheparticulargrowthconditionsprovided
willoutgrowtheothersandgaindominance(innumber).
•Byrepeatedsubculturinginthesamemediumandunderthe
sameconditionsandfinallyplatingoutonsolidmediumofthe
samecomposition,theenrichedstrainorspeciescanthenbe
isolated.
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14. Culture media
•Theculturemediummaybeinaliquidorsolidform:
•Liquidculturemediaarereferredtoasbroths.
•Solidmediaaresolidifiedwithagar(ahighlybranched
polysaccharideobtainedfromalgae).
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15. •Agarisidealsolidifyingagentforseveralreasons:
(1)agarisrarelyusedasnutrientbymicroorganismsand
thereforeitisnotreadilydegradedwhenmicrobesgrowon
culturemediasolidifiedwithagar(veryfewbacteriaareableto
digestit).
(2)agarmeltsatonly100°Cbutremainsliquiddownto45°C
(orless).Agarisaddedtoaqueousnutrientsolutionsat
concentrationof15-20g/L.
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16. •Ifsolidmediawithoutanyorganiccomponentarerequired,
silicagelisusedasthesolidifyingagent.
•Gelatinisrarelyusedassolidifyingagent,becauseitmeltsat
26-30°Candmanymicroorganismscanliquefyit.
•Semisolidmedium(gel)ismadewithhalftheamountofagar
neededinsolidmedia.
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17. Microorganismsrequireabouttenelementsinlargequantities,
becausetheyareusedtoconstructcarbohydrates,lipids,
proteins,andnucleicacids.Severalotherelementsareneeded
inverysmallamountsandarepartsofenzymesandcofactors.
1.Macronutrients
2.Micronutrients
3.Growthfactors
4.Environmentalfactors:temperature;pH;Oxygen
Dr. Dalia El-Badan
Microbial growth requirements

18. 1.Macronutrients:nutrientsthatarerequiredinlargequantities
Microorganismsrequiresabout10elementsinlargequantities
carbon,oxygen,hydrogen,nitrogen,sulfur,phosphorus,
potassium,calcium,magnesiumandiron.
Firstsix(C,O,H,N,SandP)arecomponentsofcarbohydrates,
lipids,proteinsandnucleicacidswhiletheremainingfour
macroelementsexistinthecellascationsandplayavarietyof
roles;
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19. Oxygen
•Becauseoxygenisamajorcomponentoforganiccompounds
suchascarbohydrates,lipids,andproteins,itplaysan
importantroleinthestructuralandenzymaticfunctionsofthe
cell.
•Oxygenislikewiseacommoncomponentofinorganicsalts
suchassulfates,phosphates,nitrates,andwater.Freegaseous
oxygen(O
2)isabsolutelyessentialtothemetabolismofmany
organisms.
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20. Hydrogen
•Hydrogenisamajorelementinallorganiccompoundsand
severalinorganicones,includingwater(H
2O),salts(Ca[OH]
2),
andcertainnaturallyoccurringgases(H
2S,CH
4,andH
2).
•Thesegasesarebothusedandproducedbymicrobes.
•Hydrogenperformsthefollowingoverlappingrolesinthe
biochemistryofcells:(1)maintainingpH,(2)forminghydrogen
bondsbetweenmolecules,and(3)servingasthesourceoffree
energyinoxidation-reductionreactionsofrespiration.
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21. Carbon:Itisneededforalltheorganiccompoundsthatmake
upthelivingcell.
Nitrogen:ThesynthesisofDNAandRNA,requirenitrogenand
phosphorous
•Nitrogenisneededtoformaminogroupinaminoacidsof
protein
Sulfur:Usedtosynthesizesulfur-containingaminoacidsand
enzymes
Phosphorus:Usedtosynthesizenucleicacidsand
phospholipidsofthecellmembrane
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22. potassium (K
+
) is required for activity by number of enzymes,
including some of those involved in protein synthesis.
Calcium (Ca
+2
)contributes to the heat resistance of bacterial
endospores. Magnesium (Mg
+2
)serves as a cofactor for many
enzymes, complexes with ATP and stabilizes ribosomes and cell
membranes. Iron (Fe
+2
and Fe
+3
)is a part of cytochromes and a
cofactor for enzymes and electron–carrying proteins.
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23. 2-Micronutrients or trace elements which are nutrients that
are required in relatively small amounts, such as: manganese,
zinc, cobalt, molybdenum, nickel and copper. They are a part
of enzymes and cofactors and they aids in the catalysis of
reactions and maintenance of protein structure.
Zinc (Zn)is present at the active site of some enzymes.
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24. 24

25. 3.GrowthFactors:areessentialorganiccompoundsthatthe
organismisunabletosynthesizeitsuchas:aminoacids,purines
andpyrimidines,vitamins
•Aminoacidsareneededforproteinsynthesis,
•purinesandpyrimidinesfornucleicacidsynthesis.
•Vitaminsaresmallorganicmoleculesthatusuallymakeup
allorpartenzymecofactors,andonlyverysmallamounts
arerequiredforgrowth.
MostcommonvitaminsareBiotin,FolicacidandRiboflavin
(B2).Organismsthathavesuchaccessoryrequirementsare
knownasauxotrophsindistinctiontoprototrophs,whichare
independentofaccessoryfoodfactors.
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26. 26

27. Nutritional types of microorganisms
•Autotrophs: is an organism that uses CO
2, an inorganic gas, as
its carbon source. Because autotrophs have the special
capacity to convert CO
2into organic compounds, they are not
nutritionally dependent on other living things
•Heterotrophs: is an organism that must obtain its carbon in an
organic form. Because organic carbon originates from the
bodies of other organisms, heterotrophs are dependent on
other life forms.
•Oligotrophs:are organisms adapted to live in environments
where nutrients are scarce
–For example, rivers, other clean water systems
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28. Trophic categories
•Defined by:
–Source of carbon for cell synthesis
–mechanisms used to generate energy
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29. Energy Source
Light
(Phototrophs)
PS I: anaerobic, H2S
PS I+II: aerobic, H2O
Chemical
(Chemotrophs)
Inorganic
(Chemolithotrophs)
Aerobic (majority)
Anaerobic (few)
Organic
(Chemoorganotrophs)
Aerobic respiration
Anaerobic respiration
Fermentation
Carbon Source
CO2
(Autotrophs)
Organic
(Heterotrophs)
Photoautotrophs
Photoheterotrophs
Chemolithoautotrophs
Chemoorganoheterotrphs
Chemolithoheterotrophs
(or Mixotrophs)
Chemoorganoautotrophs
Classification 29

30. Categories of culture media
•Thecompositionofculturemediadetermineswhich
typesofmicroorganismscangrowinthem.
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31. •Thereare5differentcategoriesofculturemedia:
1-Chemicallydefinedmedia(orsyntheticmedia):
Theirexactchemicalcompositionisknown.
Theingredientsusedareusuallyhighlypurifiedinorganicsalts
andsimpleorganiccompoundssuchasglucoseorpurified
aminoacids.
Byknowingtheexactcompositionoftheculturemedium,itis
possibletomaintainahighdegreeofconsistencyfrombatchto
batch.Oneofthedrawbacksofadefinedmediumisthatitcan
beveryexpensivetoprepareanditmaysupportthegrowthof
onlyafewspecies.
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32. •Example of a simple synthetic medium:
K
2HPO
4 0.5 g
NH
4CI 1.0 g
MgSO
4.7H
2O 0.2 g
FeSO
4.7H
2O 0.01 g
CaCl
2.2H
2 0.01 g
Glucose 10.0 g
Water 1000 ml
•Trace element stock solution:
ZnCl
2 70 mg
MnCl
2.4H
2O 100 mg
CoCl
2.6H
2O 200 mg
NiCl
2.6H
2O 100 mg
CuCl
2.2H
2O 20 mg
NaMoO
4.2H
2O50 mg
Na
2SeO
3.5H
2O26 mg
Distilled water100 ml
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33. 2-Livingmedia:Somemicroorganismswillproliferate
onlywhengrowinginalivinghost.Forex.Chlamydias,
Rickettsiasandvirusesrequirelivingcellstoreproduce.
Theyaregrowninthelaboratoryinlivingcellsgrownin
tissuecultures.Tissueculturesconsistofanutrient
solutionthatcansupportthegrowthofthehostcells
derivedfromthetissueofvariousanimalsorplants.
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34. •3-Complexmediaarepreparedusingnaturalproductssuchas
meatextractsorvegetableinfusions.Althoughnatural
productscontainmanyoftheessentialnutrientsnecessaryfor
microbialreproduction,theexactconcentrationofeach
nutrientisunknown.Complexculturemediaareused
extensivelyinthemicrobiologylaboratorybecausetheyare
easytoprepare,inexpensive,andabletosupportthegrowth
ofmanydifferenttypesofmicroorganisms.
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35. •Themajorshortcomingofcomplexmediaisthevariability
oftheircompositionfrombatchtobatch,thevariabilityin
thechemicalcompositionofthemediummayalterthe
growthcharacteristicsofcultivatedmicroorganisms.
Complexmediamaycontainyeastextract,beefextract,
peptone,maltextractorcaseinhydrolysate.
•Nutrientagarmediumusedforcultivationofaerobic
heterotrophs is
composedof(g/L);peptone,5;beefextract,3;agar,15-20;
water(L).Sometimes5g/lNaClisadded.
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36. •Beefextractisacomplexmixtureofprotein,carbohydrates,lipids
andotherbiochemicalconstituentsobtainedbyextractingthe
watersolublecomponentsfrombeeftissue.
•Peptoneisanenzymaticdigestofproteincontainingaminoacids
andotherN
2-containingcompounds,aswellasvitaminsand
others.
•Yeastextractisanaqueousextractofyeastcellscontaining
vitaminsandothergrowthfactors.
•Caseinhydrolysateisanorganiccompoundusedasasourceof
carbonandnitrogenobtainedfromtheenzymaticoracid
hydrolysisofmilkcasein,itisamixtureofaminoacidsand
peptides.
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37. 4-Selectivemedia:areculturemediacontainingatleastone
ingredientthatinhibitsthereproductionofunwanted
organismsbutpermitthereproductionofaspecificgroupof
microorganisms.
Theingredientsincludeoneormoreselectiveagents,suchas
dyesascrystalvioletormalachitegreen;antibioticssuchas
penicillinorstreptomycin,andchemicalssuchassodiumazide
orNaCl.
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38. •Ex1:Mannitolsaltagar(MSA)isaselectivemediumusedforthe
isolationofpathogenicstaphylococcifromhumanskin.Themedium
containsmannitol,aphenolredindicator,and7.5%sodiumchloride.
Thehighsaltconcentrationinhibitsthegrowthofmostbacteriaother
thanstaphylococci.OnMSA,pathogenicStaphylococcusaureus
producessmallcoloniessurroundedbyyellowzones.
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ThereasonforthischangeincoloristhatS.
aureusfermentsthemannitol,producingan
acid,which,inturn,changestheindicator
fromredtoyellow.Thegrowthofothertypes
ofbacteriaisgenerallyinhibited.

39. •Ex.2:Theadditionofcrystalviolettothemediumallowsthegrowthof
Gram-vebacteriaandpreventthegrowthofGram+ve.
•Ex.3:ThetwogeneraEscherichiaandEnterobacterareentericbacteriagrow
onthesamemedium.Membersoftheformergenusarenormallyinhabitants
oftheintestinaltactofwarm-bloodedanimals,whilemembersofthelatter
genusarecommonsoilinhabitants.Escherichiastrainsareadaptedtogrow
athighertemperature(44.5˚C)thanEnterobacterstrains.
•Ex.4:Isolationofsporeformingbacteria(generaBacillusandClostridium)
fromsoilbytheexposureoftheinoculumtoahightemperature(5-10minat
80°C)whichkillsallvegetativecells.
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40. 5-Differentialmediaarespecialformulationsdesignedtoreveal
differencesbetweenmicroorganisms.
Differentialmediaarewidelyusedinmicrobiologybecausetheyaidin
theisolationandidentificationofmicroorganisms.
Differentialmediausuallycontainachemicalthatisutilizedoraltered
bysomemicroorganismsbutnotbyothers.
Ex.Bloodagarmediumdifferentiatesbetween3groupsofbacteria:l.
Non-haemolyticbacteriawhichgrowwithouthaemolysisofredblood
cells.
2.-haemolyticbacteriawhichgrowandpartiallyhaemolyzeredblood
cellsformingadarkgreenzonearoundthebacterialcolonies.
3.β-haemolyticbacteriawhichperformcompletehaemolysisofred
bloodcellsformingaclearzonearoundbacterialcolonies.
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41. A. β-haemolytic
B.-haemolytic
C. Non-haemolytic
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42. •Somemediamaypossessbothselectiveanddifferentialproperties,allowingthem
togrowcertaingroupsoforganismsofinterestandgiveawaytodifferentiatethem
basedonavisiblereactionwiththemedia.Thesemediacanbepowerfuldiagnostic
toolsinbothmedicalandenvironmentalsettings.
•MacConkeyagarisselectiveforGram-negativespecies(itcontainscrystalvioletdye
toinhibitGram-positive)anddifferentialwithrespecttolactosefermentation.
MacConkeyagarisusedforthedetectionofcoliformsandentericpathogensbased
ontheirabilitytofermentlactose(acidproduceddecreasespHofmediumand
neutralredindicatorinmediumgivescolonies
apinkcolor).
•Lactose-fermentingbacteriaappear
redtopink.
•Non-lactosefermentingbacteriaappear
ascolorlessortransparentcolonies.
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43. Thank you
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