R.KAVITHA, M.

PHARM
LECTURER
DEPARTMENT OF PHARMACEUTIC
SRM COLLEGE OF PHARMACY
KATTANKULATUR

IDENTIFICATIONMETHOD
Themostimportanttaskofabacteriologyisto
identifythepathogensfromtheclinicalsampleso
thatappropriatetreatmentcanbeinstituted.

Thereareseveralmethodstoidentifiedthedifferent
typeofbacteria.
1.
2.
3.
4.
5.
6.

Isolationinpureform
Stainingreaction
Morphologyofbacterialcolony
Culturalcharacteristics
Metabolism
Biochemicalproperties

1.Isolationinpureform
y Studiesonthebiochemical,antigenicandothercharacters

ofbacteriacanbedoneonlyiftheorganismavailableinthe
pureform.
Technique:
a. Platingonsolidculturemedia clinicalsampleisstreaked
ontoasolidmedium(like:MacConkeyagar,nutrientagar
orbloodagar)insuchawaysoastoensureisolated
discretecolonies.
b. Useofselectivegrowthconditionmostimportant
exampleofthisisthegrowthofanaerobicbacteriawhich
willnottakeplaceinanenvironmenthavingoxygen.

2.Stainingreaction
a.

Theageofthecultureisimportant.Inoldercultures,
stainingcharacteristicseithervaryorarenotbroughtout
well.Simplestainsbringoutthebestmorphology.
Differentialandspecialstainsarenecessarytobringout
characteristicslike:gramnegativeandgrampositive
bacteria,Acidfastandnonacidfast,spirochetes,capsule
andFlagella,etc.

a.Gramstain
a.

GramstaindividesthebacteriaintoGrampositive&Gram
negative.

Thebasicproceduregoeslikethis:
i.
Takeaheatfixedbacterialsmear.
ii. FloodthesmearwithCRYSTALVIOLET for1minute,thenwash
withwater.[PRIMARYSTAIN]
iii. FloodthesmearwithIODINEfor1minute,thenwashwith
water.
iv. FloodthesmearwithETHANOLACETONE,quickly,thenwash
withwater.[DECOLORI
v.
FloodthesmearwithSAFRANINfor1minute,thenwash
withwater.[COUNTERSTAIN]
vi. Blotthesmear,airdryandobserve.

contd
y Examineundermicroscope

i.Grampositivebacteria violet
ii.Gramnegativebacteria pink

ShapeofBacteria
y Bacteriadisplaythreebasicshapes:

round cocci,(fromtheGreek kokkos aberry),

ii. rodshaped bacilli(fromtheLatin bacillus astick
orrod),
iii. spiral(quelled).
i.

i.Coccus

Staphylococcus species

Streptococcusspecies

ii.Bacillus

Clostridium spp.

Listeria spp.

ZiehlNeelsen Staining
y AcidFastbacilli red

Mycobacteriumtuberculosis

c.Indiaink(capsulestain)
y Thecapsulestainemploysanacidicstainandabasic

staintodetectcapsuleproduction.
y Capsulesareformedbyorganismssuchas Klebsiella
pneumoniae .Mostcapsulesarecomposedof
polysaccharides,butsomearecomposedof
polypeptides.

Staining procedure
y Place aloopfulof India ink onthesideofaclean slide
y A small portion ofthe solid culture issuspended

in saline onthe slide near the ink andthenemulsifiedin

the drop of ink,orelse,mixaloopful
of liquid culture of specimens likeCSF withthe ink.
y Place aclean cover slip over
the preparation avoiding air bubbles.
y Press down,or blot gentlywitha filter paper strip togeta
thin,even film
y Examineunder dry objectives followedby oil immersion

Examineundermicroscope

Klebsiellapneomonae

y Spirochetes brownishblack

Spirochetes

Treponemapallidum

Leptospira

3.Morphologyofthebacterial
colony
Shape:circular,irregular,radiateorrhizoid.
ii. Size:diameterinmm
iii. Elevation:flat,raised,lowconvex,domeshaped
iv. Margin:Entire,wavy,lobate,filiform
v. Surface:smooth,wavy,rough,granular,papillate,
glisteningetc.
i.

Shapeofthecolony

Elevationofthecolony

Marginsofthecolony

4.Culturalcharacteristics
Theseprovideadditionalinformationforthe
identificationofabacterium.

A.
i.
ii.

iii.
iv.
v.
vi.
vii.
viii.

Onsolidmediumthefollowingcharacters
areobserved
Shape:circular,irregular,radiateorrhizoid.
Size: Thesizeofthecolonycanbeausefulcharacteristic
foridentification.Thediameterofarepresentative
colonymaybemeasured.
Elevation:
Margin:Entire,wavy,lobate,filiform
Surface:smooth,wavy,rough,granular,papillate,
glisteningetc.
Sizeinmm
Texture :dry,moist,mucoid,brittle,viscous,butyrous
(buttery).
Color :colorless,pink,black,red,bluishgreen.

MacConkeyAgar

Enterobactercloacaeon
MacConkeyAgar:
growthwithpinkcolonies

EschericiacolionMacConkeyAgar:
growth,withpinkcolonies

contd.

Staphylococcusaureus

Streptococcuspyogens

contd

Bacillussubtilis

Proteusspp.

B.INAFLUIDMEDIUMFOLLOWING
CHARACTERSAREOBSERVED

Degreeofgrowth Absence,scanty,moderate,
abundantetc.
ii. Presentofturbidityanditsnature.
iii. Presenceofdepositanditscharacter.
iv. Natureofsurfacegrowth.
v. Easeanddisintegrationandodor.
i.

5.METABOLISM
Toclassifythedifferentiatespeciesfollowingaspectsare
studied
i. Requirementofoxygen
ii. Theneedofco2
iii. Capacitytoformpigments
iv. Powerofhemolysis

LaboratoryObjectives

TestsToKnow
y CaseStudyTests
y Indole
y MethylRed/VogesProskauer
y Citrate
y H2SproductioninSIM
y Motility
y Lactosefermentation
y Sucrosefermentation
y Glucosefermentation&gasproduction
y TripleSugarIronAgar(TSI)test

y Staphylococcusidentificationtests
y MSA

IndoleTest
Principle:
Indoletestisperformedtodeterminetheabilityoftheorganismtosplit
tryptophanmoleculeintoIndole.Indoleisoneofthemetabolicdegradation
productoftheaminoacidtryptophan
Bacteriathatpossesstheenzymetryptophanasearecapableofhydrolyzing
anddeaminatingtryptophanwiththeproductionofIndole,Pyruvicacidand
ammonia.

Propertyittestsfor:
y Thistestisperformedtohelpdifferentiatespeciesofthefamily

Enterobacteriaceae.

MediaandReagentsUsed:
y Tryptonebrothcontainstryptophan.
y Kovacsreagentcontainshydrochloricacid,dimethylaminobenzaldehyde,

andamylalcoholyellowincolor.

Indole test
Procedure:
InoculateTryptonebrothwith
thetestorganismandincubate
for18to24hrsat37 c
Add15dropsofKovacsreagent
downtheinnerwallofthetube
Interpretation:
Developmentofbrightredcolor
attheinterfaceofthereagent
andthebrothwithinseconds
afteraddingthereagentis
indicativeofthepresenceof
Indoleandisapositivetest

IndolePositive:
E.coli
Proteusvulgaris
IndoleNegative:
Salmonellaspp.
Klebsiellaspp.
Enterobacteraerogens

MethylRed/VogesProskauer(MR/VP)
y Propertiesthesetestfor:Bothtestsareusedtodifferentiate

speciesofthefamilyEnterobacteriaceae.
y MediaandReagentsUsed:
y GlucoseBroth
y MethylRedindicatorforMRtest
y VogesProskauerreagents A:5%AlphaNaphthol&ethanol,
B:PotassiumHydroxide;(3:1ratio)&DeionizedWater.
PrincipleofMRtest:
Totesttheabilityoftheorganismtoproduceandmaintain
stableacidendproductsfromglucosefermentationandto
overcomethebufferingcapacityofthesystem
Thisisaqualitativetestforacidproduction.

MRtest(contd)
Procedure:

InoculatetheMR/VPbrothwithapurecultureofthetestorganismand
incubateat35 for48to72hrs.
Add5dropsofMRreagenttothebroth

Resultinterpretation:
Positiveresultisred(indicatingpHbelow6)
Negativeresultisyellow(indicatingnoacidproduction)
MRPositive:
E.coli
MRNegative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.
Left:negative/Right:positive

VogesProskauerTest(acetoinproduction)
Principle:
Todeterminetheabilityoftheorganismsto
produceneutralendproductacetylmethyl
carbinol (acetoin)fromglucosefermentation

Procedure:
Innoculate purecultureofthetestorganism
intoMR/VPbrothandincubatefor24hrsat
37c
Aliquot1mlofthebrothtoasteriletesttube
andadd0.6mlofVP(A)followedby0.2mlof
VP:left+andright
VP(B)
Shakethetubegentlytoexposethemediumto Positive
atmosphericoxygenandallowthetubeto
Klebseilla pneumoniae
remainundisturbedfor10to15mins
Enterobacter

Intrepretation:
Positive:Pinkishredcoloratthesurfaceofthe Negative
medium
E.coli
Negative:Yellowcoloratthesurfaceofthe
medium

CitrateUtilizationtest:
y Thistestisoneofseveraltechniqueusedtoassistintheidentificationof

enterobacteria.Thetestisbasedontheabilityofanorganismtousecitrate
asitsonlysolesourceofcarbonandammoniaasitsonlysourceof
nitrogen.
y Principle:
Thetestorganismisculturedinamediumwhichcontainssodiumcitrate,an
ammoniumsaltandtheindicatorbromothymolblue.Growthinthemediumis
shownbyturbidityandachangeincolouroftheindicatorfromlightgreentoblue,
duetoalkalinereactionfollowingcitrateutilization.

y Procedure:
InoculumisstreakedovertheslantofSimmonscitrate
agarinatubeandincubatedfor2448hrs.

y Resultinterpretation:
Growthontheslantandchangeincolour
toblueofthemediumindicatespositiveresult.
Positive:Klebsiella pneumoniae
Negative:E.coli

Left Positive:Right Negative

OxidationFermentation(OF)test
(Hugh&Leifson)
Principle:
OxidationFermentationtestisusedtodeterminethe
oxidativeorfermentativemetabolismofacarbohydrateorits
nonutilization.
Fermentationisaanaerobicprocessandbacterialfermenters
ofcarbohydratesareusuallyfacultativeanaerobes.Oxidation
isaaerobicprocessandbacterialoxidisers areusuallystrict
aerobes
Procedures:
Themethoddescribed,sometimesreferredtoastheHugh
andLeifson testemploysasemisolidmediumintubes
containingthecarbohydrateundertest(usuallyglucose)anda
pHindicator
Twotubesareinoculatedandoneisimmediatelysealedwith
paraffinoiltoproduceanaerobicconditions

Resultinterpretation:
y Oxidisingorganisms,egPseudomonas
species,produceanacidreactioninthe
opentubeonly
y Fermentingorganisms,eg
Enterobacteriaceae,produceanacid
reactionthroughoutthemediuminboth
tubes
y Organismsthatcannotbreakdownthe
carbohydrateaerobicallyor
anaerobically,eg.,Alcaligenesfaecalis,
produceanalkalinereactionintheopen
tubeandnochangeinthecoveredtube

MotilityTest
y Propertyittestsfor:Thistestisdonetohelpdifferentiate

speciesofbacteriathataremotilefromnonmotile.
y MediaandReagentsUsed:Motilitymediacontains
tryptose,sodiumchloride,agar,andacolorindicator.
y HowtoPerformTest:Stabmotilitymediawithinoculating
needle.
y ReadingResults:Ifbacteriaismotile,therewillbegrowth
goingoutawayfromthestabline,andtestispositive.If
bacteriaisnotmotile,therewillonlybegrowthalongthe
stabline.Acoloredindicatorcanbeusedtomakethe
resultseasiertosee.

Motility

Fromlefttoright:

LactoseFermentation
y Propertyittestsfor:Thistestsforthebacteriasabilitytoferment

lactose.
y MediaandReagentsUsed:Lactosebrothcontainsbeefextract,gelatin
peptone,andlactose.Aphenolredindicatorisaddedtoindicateacid
productionfromfermentation.
y HowtoPerformTest: Inoculatelactosebrothwithinoculatingloop.
y Results
y Apositiveresultisyellowafterindicatorisadded(indicatinglactose
fermentation)
y Anegativeresultwillhavenocolorchangeorwillbereddish.

SucroseFermentation
y Propertyittestsfor:Thistestisdonetohelpdifferentiatespeciesofthe

familyEnterobacteriaceae.Thisteststhebacteriasabilitytoferment
sucroseandproductionofacidendproduct
y MediaandReagentsUsed:Sucrosebrothcontainsbeefextract,gelatin
peptone,andsucrose.Phenolredindicatorisaddedtoindicateanacid
endproduct.
y HowtoPerformTest:Inoculatesucrosebrothwithinoculatingloop.
y Results
y Apositiveresultisyellowafterindicatorisadded(indicating
sucrosefermentation)
y Anegativeresulthasnocolorchangeorisreddish.

GlucoseFermentation&Gas
Production
y Propertyittestsfor:Thistestisdonetohelpdifferentiatespeciesof
thefamilyEnterobacteriaceae.Thistestsforthebacteriasabilityto
fermentglucoseandproducegasand/oranacidendproduct..
y MediaandReagentsUsed:Glucosebrothcontainsbeefextract,
gelatinpeptone,andglucose.Aphenolredindicatorisaddedto
indicateanacidendproduct.ADurhamtubeisaddedtoindicategas
production.
y HowtoPerformTest:Inoculatebrothwithinoculatingloop.
y Results
y Apositiveresultforacidisyellowafterindicatorisadded
(indicatingglucosefermentation)
y ApositiveresultforgasisabubbleintheDurhamtube.
y Acompletelynegativeresulthasnocolorchangeorreddishcolor
andnobubble.

SugarFermentationTests

Tube1:Negativeacid/Negativegas
Tube2A:Mustincubatelonger(ambiguousresult)
Tube2B:Positiveacid/Negativegas
Tube3A:Positiveacid/Positivegas

TripleSugarIronAgar(TSI)test
y Principle:
TSIagarisusedtodeterminewhetheragramnegativerodutilizesglucose
andlactoseorsucrosefermentatively andformshydrogensulphide (H2S).
TSIcontains10partslactose:10partssucrose:1partglucoseandpeptone.
Phenolredandferroussulphate servesasindicatorsofacidificationand
H2Sformation,respectively.TheformationofCO2andH2isindicatedby
thepresenceofbubblesorcracksintheagarorbyseparationoftheagar
fromthesidesorbottomofthetube.TheproductionofH2Srequiresan
acidicenvironmentandisindicatedbyblackeningofthebuttofthe
mediuminthetube.

y Method:
1.
2.
3.

Withastraightinoculatingwire,touchthetopofawellisolatedcolony.
InoculateTSIbyfirststabbingthroughcenterofthemediumtothe
bottomofthetubeandthenstreakingthesurfaceoftheagarslant.
Leavethecaponlooselyandincubatethetubefor1824hoursat35oCin
anincubator.

TripleSugarIronAgar(TSI)test(contd)
y Resultinterpretation:
1.

2.
3.

4.

bcd

Alkalineslant/nochangeinthebutt
(K/NC)=Glucose,lactoseandsucrose
nonutilizer (alkalineslant/alkalinebutt)
[figure:1(d)]
Alkalineslant/acidbutt(K/A)=Glucose
fermentationonly.[figure:1(b)]
Acidslant/acidbutt(A/A),withgas
production=Glucose,sucrose,and/or
lactosefermenter.[figure:1(a)]
Alkalineslant/acidbutt(K/A),H2S
production=Glucosefermentationonly.
[figure:1(c)]
Qualitycontrol:
A/A,withgas:E.coli
K/A,H2S:Salmonellatyphi
K/NC:Pseudomonasaeruginosa

Figure(1):TSIresults

MannitolSaltAgar(MSA)
y Propertyittestsfor:This testsforthebacteriasability
totolerate7%saltconcentrationandfermentmannitol.
Themediaisselectivebecauseitselectsforsalttolerant
bacteria.

y MediaandReagents:MSAmediacontainsnutrient
agar,mannitol,7%sodiumchlorideandphenolred
indicator.

y HowtoPerformTest:InoculateanMSAplateusing
streakplatemethodandincubate2448hours.

MSAResults
y ReadingResults:
y Iftheorganismistoleranttosaltitwillgrow.
y Iftheorganismisnottoleranttosaltitwillnotgrow.
y Ifthesalttolerantorganismcanfermentmannitolthentherewillbe

yellowzonesaroundthecolonies.
y Ifthesalttolerantorganismcannotfermentmannitolthenthemediawill
remainpink.

Growthwithnomannitolfermentation.

Growthwith+mannitolfermentation.

Testforenzymes
y Catalasetest
y Oxidasetest
y Ureasetest
y Coagulasetest
y Nitratereduction

Catalasetest
Principle:
Thistestdemonstratesthepresenceofenzymecatalasein
theorganism.Theenzymecatalasemediatesthe
breakdownofhydrogenperoxide(H2O2)intooxygenand
water.Thepresenceoftheenzymeinabacterialisolateis
evidentwhenasmallinoculumisintroducedinto
hydrogenperoxide(30%forslidetest),andtherapid
effervescenceofO2bubblesoccurs.Thelackofcatalaseis
indicatedbyalackofbubbleproduction.lase
2H2o2

catalase

2H2o+o2(gasbubbles)

Catalasetest.
Catalaseispresentinmostcytochromecontaining

aerobicandfacultativeanaerobicbacteria(except
streptococcus spp).
Hydrogenperoxideformsasoneoftheoxidativeend
productofaerobiccarbohydratemetabolism.Ifthisis
allowedtoacculmulateinthebacterialcellsitbecomes
lethaltothebacteria
CatalasethushelpsinconvertingH2o2 toH2oando2
OptimalpHforcatalaseactionis7.

Catalasetest.
Reagents:
3%hydrogenperoxidestoredindarkbrownbottle
underrefrigeration
18to24hrscultureoftheorganismtobetested
Controlorganismsused:
Positivecontrol Staphylococcusaureus
Negativecontrol Streptococcusspp.

Catalasetest.
Methods:

1.Slidemethod

2.Tubemethod

3.Directplatemethod

Catalasetest.
Precautions
y Avoidcoloniesfrombloodagar
y 18to24hrscolonyonlyshouldbeused
y Reagenttobefairlyfreshasitisveryunstableand
easilybreaksdownonexposuretolight
y Reagenttobestoredinbrownorambercoloredbottle
inrefrigeratorat4c

Catalasetest.
Positive
Micrococcus
Staphylococcus
Bacillus
Listeriamonocytogenes
Enterobacteriacae
Gonococcus&
Meningococcus
Vibriocholerae
Pseudo/Aero/Plesiomonas

Negative
Streptococcus
Clostridium

OxidaseTest
Principle
Oxidasetestisusedtodeterminethepresenceof
bacterialcytochromeoxidaseenzymeusingthe
oxidizationofthesubstratetetramethylp
phenylenediaminedihydrochloridetoindophenola
darkpurplecoloredendproduct.Apositivetest
(presenceofoxidase)indicatedbythedevelopmentofa
darkpurplecolour.Nocolourdevelopmentindicatesa
negativetestandtheabsenceoftheenzyme.

OxidaseTest.

Cytochromesareironcontainghemoprotiensandinaerobicrespirationthey
transferelectrons(H)tooxygentoformwater.
Thereagentactsasanartificialelectronacceptorsubstitutingtheoxygen.Inthe
reducedstagedyeiscolorless,butinthepresenceofenzymecytochrome
oxidasedyeisoxidisedtoindophenolblue

Methods

1.Moistfilterpaper
method

Qualitycontrols
Positivecontrol Pseudomonasspp
Negativecontrol E.coli

2.Directplatemethod

OxidaseTest..
Positive

Negative

Pseudomonasspp.

y Enterobacteriaceae

Aeromonasspp.

y Acenitobacterspp.

Vibriospp.
Alcaligenesspp.
Neisseriaspp.
Haemophilus sps

OxidaseTest..
Precautions
Useglassrodorwoodenstick
Resultshouldbereadwithin10secs
Donotperformthetestfromcoloniesgrowingin
mediumhavingglucoseasitsfermentationwillinhibit
theoxidaseenzymeactivity
Reagentshouldbefreshlyprepared
Tobestoredindarkbottle
DonotusepigmentedcoloniesorcoloniesfromMac
conkey agar

UreaHydrolysis(Ureasetest)
y Propertyittestsfor:Thistestisdonetodetermineabacterias

abilitytohydrolyzeureatomakeammoniausingtheenzyme
urease.
y MediaandReagentsUsed:StuartsUreabroth(pH6.8)
containsayeastextract,monopotassiumphosphate,disodium
phosphate,urea,andphenolredindicator.
y Principle
Todeterminetheabilityoftheorganismtosplitureaforming2
moleculesofammoniabytheactionoftheenzymeUreasewith
resultingalkalinity
y HowtoPerformTest: InoculateUreabrothwithinoculating
loop.

ReadingResults:
y Ureabrothisayelloworangecolor.

Theenzymeureasewillbeusedto
hydrolyzeureatomakeammonia.If
ammoniaismade,thebrothturnsa
brightpinkcolor,andispositive.Iftest
isnegative,brothhasnocolorchange
andnoammoniaismade.
y Figureintherightshowsnegative(left)and

Positive(right)results.

Coagulasetest
y Principle:

ThistestisusedtodifferentiateStaphylococcusaureus (positive)from
coagulasenegativeStaphylococci.S.aureus producestwoformsofcoagulase:
boundandfree.

Boundcoagulaseorclumpingfactor,isboundtothebacterialcellwalland
reactsdirectlywithfibrinogen.Whenabacterialsuspensionismixedwithplasma,
thisenzymecausesalterationinfibrinogenoftheplasmatoprecipitateonthe
staphylococcalcells,causingthecellstoclump.

Freecoagulaseisproducedextracellularly bythebacteriathatcausesthe
formationofaclotwhenS.aureus coloniesareincubatedwithplasma.

y Method:
A.

B.

Slidetest:(forboundcoagulase)
Placeadropofcoagulaseplasmaonaclean,dryglassslide.
Placeadropofdistilledwaterorsalinenexttothedropofplasmaasa
control.
Withalooporwoodenstick,emulsifyaportionoftheisolatedcolonybeing
testedineachdrop.
Mixwellandrocktheslidegentlyfor5to10seconds.
Tubetest:(forfreecoagulase)
Emulsifyseveralcoloniesin0.5mlofrabbitplasma(withEDTA)togivea
milkysuspension.
Incubatetubesat35oCinambientairfor4hrs.Checkforclotformation.
Ifnegativeat4hrs,incubateatroomtemperatureovernightandcheckagain
forclotformation.

CoagulaseResults
y ReadingResults:
A. Slidetest:
Positive:Macroscopicclumpingin10seconds

orlessincoagulatedplasmadropandno
clumpinginsalineorwaterdrop.
Negative:Noclumpingineitherdrop.
Note:Allnegativeslidetestsmustbe
confirmedusingthetubetest.
B. Tubetest:
Positive: Clotofanysize(a)
Negative: Noclot(b)

a
CoagulasePositive:Staphylococcusaureus
Coagulasenegative:Staphylococcusepidermidis

Nitratereductiontest
Principle:
Thistestisusedtodeterminetheabilityoftheorganismto
reducenitratetonitritesorfeenitrogengas.Thereductionof
nitratetonitriteisdetectedbyaddingsulphanilic acidand
alphanaphthylamine.Thesulphanilic acidandnitritereacts
toformadiazonium saltwhichthenreactswithalpha
naphthylamine toproduceared,watersolubleazodye.
Purpose:
y Aidinthespeciesdeferentiation of
i)Haemophilus duceryi()andHaemophilus vaginalis()from
otherHaemophillus spp.
ii)Neisseria mucosa(+)fromotherNeisseria spp
y AidintheidentificationofEnterobacteriaceae(+)

Nitratereductiontest.
Procedure:
Inordertodetermineifabacteriacanreducenitrate,the

testorganismisinoculatedintonitratebroth[an
undefinedmediumthatcontainslargeamountsofnitrate
(KNO3)].Afterincubation,alphanaphthylamine and
sulfanilic acidareadded.Thesetwocompoundsreactwith
nitriteandturnredincolor,indicatingapositivenitrate
reductiontest
Ifthereisnocolorchangeatthisstep,nitriteisabsent.If
thenitrateisunreducedandstillinitsoriginalform,this
wouldbeanegativenitratereductionresult.However,itis
possiblethatthenitratewasreducedtonitritebuthasbeen
furtherreducedtoammoniaornitrogengas(whichevolved
out).Thiswouldberecordedasapositivenitratereduction
result

Nitratereduction.
Todistinguishbetweenthesetworeactions,zincdust

mustbeadded.Zincreducesnitratetonitrite.Ifthetest
organismdidnotreducethenitratetonitrite,thezinc
willchangethenitratetonitrite.Thetubewillturnred
becausealphanaphthylamineandsulfanilicacidare
alreadypresentinthetube
Thusaredcolorafterthezincisaddedindicatesthe
negativenitratereductiontest.

Nitratereductiontest.

Negative
Negative

Nitratenot
reduced

Posiitive

Nitratereducedto
nitrite

Positive
Nitratereducedto
NH3orN2gas,
nitriteabsent

Nitratenot
reduced

Nitratereductiontest.

AdditionofZndust

or

Grampositiveflowchart

present

Gramnegativeflowchart

Keyidentificationcharacteristicsfor
Enterobacteriaceae
GENUS/SPECIES

Escherichiacoli
Shiegella
Shiegellasonnei
Salmonella
KlebsiellaPneumo.
Enterobacter
Serratia
Proteus
morganella
Yersinia

FermentationofGas
G
L
S
M

(+) (+) (+) (+)

(+)
(+) () () (+)
()
(+) (+) () (+)
()
(+) () () (+)
(+)
(+) (+) () (+)
(+)
(+) () (+) (+)
()
(+) (+) () (+)
(+)
(+) () () (+)
(/+)
(+) () () (+)
(+)
(+) () () (+)
()

MR

(+)
(+)
(+)
(+)
()
(+)
(/+)
(+)
(+)
(+)

VP

()
()
()
()
(+)
()
(+)
()
()
()

Indole Citrate Urease

(+)
()
()
(/+)
()
()
()
()
()
()
(+)
()
()
(+)
(+)
(+)
()
(+)
()
(+)
()
(+)
(/+)
(+)
(+)
()
(+)
(/+)
()
(/+)

H2

()
()
()
(+
()
(+
()
(+
(+
()

G:Glucose,L:Lactose,S:Sucrose,M:Manitol,MR:MethylRed,VP:VogesProskauer