TestsToDeterminePhysiological

Characteristics

StreakPlateMethod
Inordertoidentifybacteria,itisnecessaryto
obtainapureculture.Thisis
donebyusingthestreakplatemethod.
Bacterialcellsarespreadoverthe
surfaceofanagarplateinacontinuous
dilution,thisallowsthecellstobeseparated
fromeachother.Whentheplateisincubated,
thoseindividualcellswillgrowintocolonies
thatoriginatedfromasinglecell.

Trypic SoyAgarPlates
Trypticase soyagarisabacterialgrowth
medium.
TSAisageneralpurposemediaproducedvia
enzymaticdigestionofsoybeanmealand
casein.
Itisfrequentlythebasemediaofothertypes
ofagaranditsupportsgrowthofmanysemi
fastidiousbacteria

GrowthCharacteristicsofEMB
EMBagarisbothaselectiveanddifferentialagar.
EMBagarisinhibitorytogrampositivebacteria,
andisindicativeoflactosefermentation.
Itisselectivebecauseitcontainsbilesaltsand
eosinandmethylene bluedyes,whichinhibit
grampositivebacteriafromgrowing.
Itisalsodifferential,inthatlactosefermenting
bacteria,willproducedarkcolonieswithagreen
metalic sheen,whilebacteriathatcannot
fermentlactoseproducecolorlesscolonies.

EMBAgar

Hektoenentericagar(HEA)
HEAcontainsdyesandbilesaltswhichinhibitgrowthof
Grampositivesandhighconcentrationsofthreesugars:
lactose,sucrose,andtheplantglycosidesalicin. dyesand
bilesaltspreventgrowthofGrampositivebacteria.
Acidproductionfromanyofthesugarsresultsincolor
changeofthedyes. Acidproducersformorangeorpink
colonies,nonfermenters ofallthreesugarsformgreenish
colonies.Ifanorganismfermentslactose,fermentationof
theothertwosugarscannotbedetected.
HEAcontainssodiumthiosulfate andferriccitrate. Some
bacteriausethiosulfate anionasanelectronacceptor,
reducingittosulfide.Inthiscaseablackprecipitateofiron
sulfideformsinthecentersofcolonies.

GrowthCharacteristics
ofMAC
MacConkey agarisbothaselectiveanddifferential
agaritisinhibitorytogrampositivebacteria,and
isindicativeoflactosefermentation.
Itisselectivebecauseitcontainsbilesaltsand
crystalvioletdye,whichinhibitgrampositive
bacteriafromgrowing.
Itisalsodifferential,inthatlactosefermenting
bacteria,produceredcolonies,whilebacteria
thatcannotfermentlactoseproducecolorless
colonies.

MotilityTest
Themotilitytestisusedtodeterminethe
presenceofflagella
Bacteriathataremotilehaveflagella.
Nonmotilebacteriadonothaveflagella.

MotilityTest

Procedure:

Inoculateanisolatedcolonyintotryptic soybrothorbrainheart
infusionbroth.

Incubate theculturefor24hours

Placeadropofthebroth ontoacleanmicroscopeslide,andcover
withacoverslip.

Observemicroscopicallyforthepresenceofmotility.

Apositivetestresultisindicatedbyindividualbacterialcellsmoving
inrandomdirections.

OxidativeandFermentativeTests
FermentationTests:determineiftheunknown
iscapableofcarryingoutvarious
fermentationreactions

OxidativeTests:determinesiftheunknown
carriesoutrespiratoryfunction
.

FermentationTests
O/FGlucoseTest
DurhamTubesugarfermentations
Mixed AcidFermentation(methyl
red[MR]test)
Butanediol Fermentation(Voges
Proskauer [VP]Test)
CitrateTest

O/FGlucoseTest
determinesiftheunknownorganismis
oxidative,fermentativeorcapableofboth
kindsofmetabolism
Toperformthistestanunknownorganismis
inoculatedintoatubeofO/FGlucoseby
stabbingwithaninoculatingneedle.Mineral
oilisaddedandthetubesareincubatedat37
degreesfor24hours.

O/FGlucoseTest

Interpretation
Result

Interpretation

AnaerobicAerobic
YellowYellow

Oxidativeandfermentatative

GreenYellow

OxidativeMetabolism

GreenGreen

Sugarnotmetabolized
(nonsaccharolytic)

 IfitisdeterminedbytheO/FGlucoseTest
thattheunknownbacteriaisoxidativeand
notcapableoffermentingsugarsthenthe
bacteriumcannotbeidentifiedby
fermentationtests
Ifitisdeterminedthattheorganismis
fermentativeinitsmetabolismitisimportant
towhichspecificsugarsarefermentedor
whichfermentationpathwaysareusedfor
growth

DurhamTubeSugarFermentations
PhenolRedFermentation
Ifanorganismfermentsasugaracidisusually
producedandgasmaybeanendproduct.
Toperformthistestanunknownorganismis
inoculatedintoaDurhamsugartube.The
carbohydratetubeisthenincubatedfor24
hoursat37degrees.

DurhamFermentationTube
Thepresenceofanacidisindicatedbyacolor
changeinthepHindicator(phenolred)from
red(alkalinevalues)toyellow(acidicvalues)
Theproductionofgassuchashydrogenand
carbondioxideisrevealedbythe
displacementofmediumfromtheDurham
Tube.

DurhamFermentationTube

MixedAcidFermentation
MethylRedTest
Importantindifferentiatingsomegramnegative
intestinalbacteriaisthatofmixedacid
fermentation.
Somebacteriafermentsglucosetoproducea
numberoforganicacidssuchaslactic,acetic,
succinic andformicacids.
CO2,H2andethanolarealsoproducedinthis
fermentation
Apositivetest,whichturnsredafterMethylred
isadded,indicatestheorganismcarriedout
mixedacidfermentation.

MethylRedTest
Toperformthistestaglucosebrothisusedthat
hasbeenbufferedwithpeptoneanddipotassium
phosphateisinoculatedwithanorganism.The
tubeisthenincubatedat37degreesfor35days.
34dropsofMethylRedisthenaddedtothe
tube
Ifthetubebecomesredimmediatelythisisa
positiveresult.Ifthereisnochangethisisa
negativeresult.

MethylRedTest

2,3 Butanediol Fermentation

VoguesProskauer Test
TheMethylRedTestandVPtestare
importanttestsindifferentiatinggram
bacteria
Somegramnegativeintestinalbacteriadonot
carryoutmixedacidfermentationbutrather
theyfermentglucosetoproducelimited
amountsofsomeorganicacidsandprimarilya
moreneutralendproduct,2,3butanediol.

VoguesProskauer Test
Toperformthistestaglucosebrothisusedthat
hasbeenbufferedwithpeptoneanddipotassium
phosphateisinoculatedwithanorganism.The
tubeisthenincubatedat37degreesfor35days.
1mlofcultureisremovedwithapipetteand
mixedwith18dropsofBaritts ReagentA(alpha
naphthol)and18DropsofBarrits ReagentB
(KOH).
Thetubeisthenvigorouslyshaken(tooxidizethe
2,3butanedioltoacetoin)and islefttostandfor
30minutes.

VoguesProskauer Test
VoguesProskauer testafteradditionof
Barritts A&Bandwaiting2025minutes.
Theredcolorontherighttubeisa(+)test.

VoguesProskauer TestResults
ApositiveVPtestwillturnredtopinkincolor.
AnegativeVPtestwillremainunchanged
Ifbutanediol isproduceditisVPpositiveand
isusuallyMethylRednegative

CitrateTest
Organismsthatdegradecitratemustuse
ammoniumsalts,thatserveasasolenitrogen
sourceforgrowth,andintheprocessthey
produceammoniathatcausesthemediumto
becomealkaline.
UnderalkalineconditionsthepHindicator
turnsthemediumfromdarkgreentoa
Persianblue

CitrateTest
Toperformthistestanunknownorganismis
inoculatedintoatubeofSimmons Citrateby
streakingtheslantandthenstabbingthe
mediumwiththeinoculatingneedle.The
tubesareincubatedat37degreesfor2448
hours.

CitrateTest

OxidativeTests
Oxidase Test
Catalase Test
Nitratereduction

Oxidase Test
Theoxidase testassaysforthepresenceof
cytochrome oxidase.
Cytochrome oxidase occursinbacteriathat
carryoutrespirationandwhereoxygenisthe
terminalelectronacceptor
Thetestdifferentiatebetweenthosebacteriathat
havecytochrom oxidase anduseoxygenasa
terminalelectronacceptorandthosethatuse
oxygenasaterminalelectronacceptorbuthave
othertypesofterminaloxidases.

Oxidase Test
Theenzymeisdetectedbytheuseofan
artificialelectronacceptor,whichchanges
fromyellowtopurplewhenelectronsare
transferredfromreducedcytochrome ctothe
artificialacceptor.

Oxidase Test
AswabofabacteriafromaTSAplateistaken
byasterileswab,alternativelyasampleisalso
transferredfromtheplatetoapieceoffilter
paper.Severaldropsofoxidase reagentare
addedtothecellsonthepaper.

Oxidase Test
Positiveculturewillcausethereagenttoturn
fromyellowtopurplein1030seconds(a)
Nochangeorachangeafter30secondsis
consideredanegativeresult(b)

Catlase Test
Thistestisperformedtodetectforthepresence
oftheenzymecatalase whichdegradeshydrogen
peroxideintooxygenandwater.
Strictanaerobesandaerotolerant bacterialack
thisenzyme.
Thepresenceofcatalayse isonewayto
determineaerobesandfacultive areobes which
havethisenzyme.
Organismscontainingthecatalase enzymewill
formoxygenbubbleswhenexposedtohydrogen
peroxide.

Catalase Test
Toperformthistestawoodenstickisusedto
transferasmallamountofgrowthtoaslide.
Hydrogenperoxideisthenadded.
Ifcatalase isproducedtherewillbevigorous
bubblingduetothebreakdownofhydrogen
peroxideandtheproductionofoxygengas.
Asamplecannotbetakenfrombloodagar
Awireloopcannotbeused

Catalase Test
Positive

Negative

NitrateReduction
SomeFacultativeanaerobesusenitrateasa
terminalelectronacceptorinatypeof
anaerobicrespirationcallednitrate
respiration.
Enzymesinvolvedinnitratereductionare
inducibleandareonlyproducedifnitrateis
presentaanaerobicconditionsexistfor
growth.

NitrateReductionProcedure
23dropsofReagentAand23dropsofReagentBis
addedtoAnitratebrothculturewithDurhamtubesof
theunknownorganism.
Ifadeepredcolorfailstoappeartheorganismdidnot
reducenitrateoritmayhaveproducesanongaseuos
nitratereduction.
IfnegativeZincisthenaddedtothecultureandthen
shaken.Ifaredcolordevelopsinthetubethenirate
wasreducedbythezincindicatinganegativetestfor
Nitratereduction.Ifnotcolordevelopsanongaseous
endproductmayhavebeenformed,whichmeansthe
unknownreducednitrate.

HydrolyticandDegradative Reactions
StarchHydrolysis
Bacteriathathydrolyzesstarchproduceamalses
thatdegradethestarchmoleculeintomolecules
ofmaltose,glucose,sucroseanddextrins.
Starchhydrolysisisdetectedbyaddingiodineto
starchmedium.
Iodinecomplexeswiththestarchmacromoleculeand
causesthemediumtoturnblue
Ifthestarchhasbeendegradedthemediumadjacent
tothebacterialgrowthwillbeclearafteriodineis
added

StarchHydrolysis

HydrolyticandDegradative Reactions

CaseinHydrolysisTest
Casease isanexoenzyme thatisproducedby
somebacteriainordertodegradecasein.
Caseinisalargeproteinthatisresponsiblefor
thewhitecolorofmilk.
Thistestisconductedonmilkagarwhichisa
complexmediacontainingcasien,peptone
andbeefextract.

CaseinHydrolysisTest
Ifanorganismcanproducecasein,thenthere
willbeazoneofclearingaroundthebacterial
growth.

HydrolyticandDegradativeReactions
FatHydrolysis/SpiritBlueAgar
Fathydrolysisisachievedwhentheglycerol
moleculeinatriglycerideiscleavedfromthe
adjoiningfattyacids.Thisreactioniscatalyzed
bytheexoenzymelipase.
Spiritblueagarcontainstributyrinasa
substrateforlipaseandapHindicatorthat
becomescolorlessasthepHofthemediumis
loweredinresponsetothebuildupoffatty
acidsduetothefathydrolyzingactionof
lipase

 Youmustconfirmthenegativeresultby
confirmingtheshinyappearanceoftheagar
Adull,matteappearanceoraclearingofthe
bluecolorinthemediaisconsidereda
positiveresultforfathydrolysis.

HydrolyticandDegradativeReactions
TryptophanDegradation
Tryptophanisdegradedby tryptophanase
intoindole,ammonia,pyruvicacid.
Pyruvicacidistheninvolvedinmetabolic
pathwaysothatATPenergyforbacterialcell
isgenerated. WhileotherproductslikeNH3
andPyruvateismetabolized,indoleisnot.
Anditstaysinthemedium.

TryptophanDegradation
UponadditionofKovacsreagent,deepred
ring atthetopoftheagar/broth isformed
whenKovacsreagentreactswithindole.This
isthe(+)result.

HydrolyticandDegradativeReactions
UreaHydrolysis
Themediumusedtoperformthistestisa
Ureabroththatcontainsaphenolred
indicator
Urea,istheendproductofaminoacid
metabolism. Someorganismscontainurease,
whichallowsthemtobreakdownureato
formCO2andammonia.Ammoniareactswith
watertoformammoniumhydroxide.

UreaTest
Brothbecomesredpurplecoloriftestis
positive duetoproductionofammonium
hydroxide.
Ifnegative,brothremainsorange.

HydrolyticandDegradativeReactions
Phenylalaninedeaminase
Phenylalaninedeaminasemediumteststhe
abilityofanorganismtoproducetheenzyme
deaminase.Thisenzymeremovestheamine
groupfromtheaminoacidphenylalanineand
releasestheaminegroupasfree
ammonia. Asaresultofthisreaction,
phenylpyruvicacidisalsoproduced.
Itisusedtodifferentiatemembersofthe
generaProteus,Morganella,andProvidencia
fromotherEnterobacteriaceae.

PhenylalanineDeaminaseTest
Afterincubation,10%ferricchlorideisadded
tothemedia;
ifphenylpyruvicacidwasproduced,itwill
reactwiththeferricchlorideandturndark
green.
Ifthemediumremainsastrawcolor,the
organismisnegativeforphenylalanine
deaminaseproduction.

PhenylalanineDeaminaseTest
PositiveResultNegativeResult

MultipleTestMedia
IMViCTests
IMViCisanacronymthatstandsforindole,
methylred,VogesProskauer,andcitrate
EnterobacteriaeaeareGramnegative
bacteriathatgrowintheintestinaltractof
humansandotheranimals.TheIMViCtests
arefrequentlyemployedforidentificationof
thisgroupofmicrobes.

MultipleTestMedia
IndoleTest
Thetestorganismisinoculatedintotryptone
broth,arichsourceoftheaminoacid
tryptophan.Indolepositivebacteriasuchas
producetryptophanase,anenzymethat
cleavestryptophan,producingindoleand
otherproducts.
Theindoletestmustbereadby48hoursof
incubationbecausetheindolecanbefurther
degradedifprolongedincubationoccurs.

IndoleTest
WhenKovac'sreagent(isaddedtoabroth
withindoleinit,adarkpinkcolordevelops.

MultipleTestMedia
KligersIronAgar(KIA)Test
Thisisadifferentialmedium.Ittestsfor
organisms abilitiestofermentglucoseand
lactosetoacidandacidplusgasendproducts.
Itallowsforidentificationofsulfurreducers
whichbindswithirontoformablack
precipitate.
Glucosefermentationwillcreateacidic
byproductsthatwillturnthephenolred
indicatorinthemediayelllow.

KIATest
Withyourneedlepickmaterialfromacolonyand
stabthroughthecenteroftheagartothebottomof
thetube.
Thenstreakthesurfaceoftheslant.Thisdouble
inoculationallowsgrowthofyourunknownorganism
ontheaerobicsurfaceoftheslantandinthelargely
anaerobicbuttofthetube.
IncubateyourKliglerslants,whichmustbe
interpretedafterincubationfor24hours.

KIAresults
Lactose(+)or().Thuslactose(+)organismsyieldayellow
slantandlactose()organismsyieldaredslant.
Glucose(+)or()glucose(+)organismsyieldayellowbutt.
Fermentationoflactose.alllactosepositiveorganismsare
alsoglucosepositive.Ifablackcolorfromironsulfide
obscuresthecolorofthebuttyoucanpresumeitisyellow.
H2S(+)or().IfanorganismformsH2S,thelowerportionof
thetubewillturnblack,duetoformationofironsulfide.
Gasformation(+)or().Ifanorganismformsgasfromglucose
orlactosetheagarinthebuttwillshowbubblesorcracks.
Positive
Negative
Lactoseyellowslant
redslant
Glucoseyellowbutt redbutt

KIAresults

MultipleTestMedia
SulfurIndoleMotility(SIM)
Thisisadifferentialmedium.Itteststhe
abilityofanorganismto:reducesulfur,
produceindoleandbemotile.
SulfurcanbereducedtoH2Seitherby
catabolismoftheaminoacidcysteinebythe
enzymecysteinedesulfuraseorbyreduction
ofthiosulfateinanaerobicrespiration.
Ifhydrogensulfideisproduced,ablackcolor
formsinthemedium.

SulfurIndoleMotility(SIM)
SIMtubesareinoculatedwithasinglestabtothe
bottomofthetube.Ifanorganismismotilethanthe
growthwillradiatefromthestabmarkandmakethe
entiretubeappearturbid.
Bacteriathathavetheenzymetryptophanase,can
converttheaminoacid,tryptophanetoindole.
IndolereactswithaddedKovacsreagenttoform
rosindoledyewhichisredincolor(indole+).
Ifhydrogensulfideisproduced,ablackcolorforms
inthemedium.

SIMresults

Negative
for indole
production
.

Positive for
indole
production

Positive for
motility and
sulfur
reduction

Positive
for motility
and
negative
for sulfur
reduction

MultipleTestMedia
LitmusMilk
Todifferentiatebacteriabasedonvarious
reactionsthatoccurinskimmilk
supplementedwithalitmuspHindicator.
Bacterialenzymesalterthemediaandmay
bringaboutvariouschanges. Litmusisadded
tothemediumtodetectpHchangesthatmay
occurasaresultoftheseenzymatic
reactions. AboveapHof8.3litmusisblue,
whilebelowapHof4.5litmusisred.

PossibleResults:
A. Acid/Reduction/Curd
B. Reduction/Curd(arrowdenotesgaspocket)
C. UninoculatedControl
D. AcidFormation
E. Proteolysisofcasein
F. AlkalineReaction

Decarboxylase,
Lysine,Aginine,Ornithine
Decarboxylase

Decarboxylasebrothtestsfortheproduction
oftheenzymedecarboxylase,whichremoves
thecarboxylgroupfromanaminoacid.
Decarboxylasebrothcontainsnutrients,
dextrose,pyridoxal,andthepHindicators
bromcresolpurpleandcresolred.
Bromcresolpurpleturnspurpleatanalkaline
pHandturnsyellowatanacidicpH.

Decarboxylase,
Lysine,Aginine,Ornithine
Decarboxylase

Iftheinoculatedmediumisyellow,orifthereisnocolor
change,theorganismisdecarboxylasenegativeforthat
aminoacid. Ifthemediumturnspurple,theorganismis
decarboxylasepositiveforthataminoacid.
Mineraloilisusedtopromotefermentationbylockingout
oxygen,anditalsopreventsfalsealkalinizationatthesurface
ofthemedium.
Ornithinedecarboxylationyieldsputrescine.
Lysinedecarboxylationresultsincadaverine.
ThesebyproductsaresufficienttoraisethepHofthemedia
sothatthebrothturnspurple

Lysine
positive
reaction
asthe
medium
has
turned
purple

displays a
negative result

GelatinHydrolysis(Liqefication)Test
Nutrientgelatinisadifferentialmediumthat
teststheabilityofanorganismtoproducean
exoenzyme,calledgelatinase,thathydrolyzes
gelatin.
Gelatinaseallowstheorganismsthatproduce
ittobreakdowngelatinintosmaller
polypeptides,peptides,andaminoacidsthat
cancrossthecellmembraneandbeutilized
bytheorganism.

GelatinaseTest
Whengelatinisbrokendown,itcannolonger
solidify. Ifanorganismcanbreakdown
gelatin,theareaswheretheorganismhas
grownwillremainliquidevenifthegelatinis
refrigerated.

Gelatinhydrolysis

thetubeonthetheleft
ispositivefor
gelatinase
production,as
evidencedbythe
liquidationofthe
media.
Whereasthetubeonthe
rightisnegative