Professional Documents
Culture Documents
R.Kavitha, M.Pharm Lecturer Department of Pharmaceutic SRM College of Pharmacy Kattankulatur
R.Kavitha, M.Pharm Lecturer Department of Pharmaceutic SRM College of Pharmacy Kattankulatur
R.Kavitha, M.Pharm Lecturer Department of Pharmaceutic SRM College of Pharmacy Kattankulatur
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:
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
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%AlphaNaphtholðanol,
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
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
()
()
()
()
(+)
()
(+)
()
()
()
(+)
()
()
(/+)
()
()
()
()
()
()
(+)
()
()
(+)
(+)
(+)
()
(+)
()
(+)
()
(+)
(/+)
(+)
(+)
()
(+)
(/+)
()
(/+)
H2
()
()
()
(+
()
(+
()
(+
(+
()
G:Glucose,L:Lactose,S:Sucrose,M:Manitol,MR:MethylRed,VP:VogesProskauer