Biochemical tests for identification of bacteria

Identification of bacteria by biochemical testing

1. Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Determiningthenutritionalandmetaboliccapabilitiesofa
bacterialisolateisthemostcommonapproachusedfor
determiningthegenusandspeciesofanorganism.
Themethodsavailableuseacombinationofteststoestablish
theenzymaticcapabilitiesofagivenbacterialisolateaswellas
theisolatesabilitytogroworsurvivethepresenceofcertain
inhibitors(e.g.salts,surfactants,toxinsandantibiotics)

3. A.EstablishingEnzymaticCapabilities
Enzymebasedtestsaredesignedtomeasurethepresenceofasingleenzymeaswellasa
completemetabolicpathway.
SINGLEENZYMETESTS:
Catalasetest
Coagulasetest
Pyrasetest
Hippuratehydrolysistest
Oxidasetest
Indoletest
Dnasetest
ONPG(B-galactosidase)test
Ureasetest
AssaysforMetabolicPathways:Carbohydrateoxidationandfermentation
Oxidationfermentationtests
CarbohydratefermentationinTSIA
Methylredtest
VogesProskauertest
Aminoaciddegradation
Decarboxylase-dihydrolasereactions
deaminationreactions
decarboxylationanddeaminationreactionsinLIA
Singlesubstrateutilization
Citrateutilizationtest
Acetateutilizationtest
Acetamideutilizationtest
A.Establishing Enzymatic Capabilities

4. B.EstablishingInhibitorProfiles
Bacitracinsusceptibilitytest
Bacitracinandsulfamethoxazole-trimethoprimsusceptibilitytest
Novobiocinsusceptibilitytest
Vancomycinsusceptibilitytest
Antibioticdisksforpresumptiveidentificationofanaerobes
C.Othermorespecifictests
GrowthinvariousNaClconcentrations-EnterococciandVibriospecies
Susceptibilitytooptochinandsolubilityinbile–Streptococcuspneumoniae
Abilitytohydrolyzeesculininthepresenceofbile–Enterococcusspp.and
GroupDstreptococcus
CAMP–Streptococcusagalactiae

5. PURPOSE
TodifferentiatemembersofthefamilyMicrocococcaceae(including
Staphylococcus)whicharecatalasepositivefromStreptococcusspecies
whicharecatalasenegative.
TodifferentiateListeriamonocytogenesandcorynebacteria(catalase
positive)fromotherGrampositive,non-sporeformingbacilli.
Principle:
The enzyme catalasecatalyzes the release of water and oxygen from
hydrogen peroxide.
catalase
2 H202 --------------2 H20 + O2
bubbles or effervescence
A.Establishing Enzymatic Capabilities

6. Interpretation
Positive–rapid and sustained appearance of bubbles or effervescence
Negative–lack of bubble formation 30 seconds later
Catalase test
A.Positive–Staphylococcus aureus; Negative–Streptococcus pyogenes

7. Coagulase Test
PURPOSE
Todeterminetheabilityoftheorganismtoproducecoagulasewhich
clotsplasma.
Todistinguishthepathogeniccoagulasepositivestaphylococcus
fromthenonpathogeniccoagulasenegativestaphylococcus.
Principle:
Coagulaseisanenzymethatconvertssolublefibrinogeninto
insolublefibrin.
Twoformsofcoagulase:
1.Boundcoagulase(clumpingfactor)–
Detectedinthecoagulaseslidetest
Candirectlyconvertfibrinogentoinsolublefibrinandcausesthe
staphylococcitoclumptogether
2.Freecoagulase
Detectedinthecoagulasetubetest
Reactswithaglobulinplasmafactor(coagulasereactingfactor-CRF)
toformathrombinlikefactor,staphylothrombin---catalyzesthe
conversionoffibrinogentoinsolublefibrin

8. Interpretation
Slide Coagulase test
Positive–white fibrin clots in plasma; Negative–smooth suspension
Tube Coagulase test
Positive–formation of fibrin clot; Negative–no clot is formed
Slide coagulase test
A.Negative–Staphylococcus epidermidis
B.Positive–Staphylococcus aureus
Tube coagulase test
A.Positive–Staphylococcus aureus
B.Negative–Staphylococcus epidermidis

9. Purpose:
TodeterminetheabilityoftheorganismtohydrolyzethesubstrateL-
pyrrolidonyl-beta-napthylamide.
TodifferentiateEnterococcusspeciesfromnon-enterococcusspecies.
UsefulforpresumptiveidentificationofGroupAbetahemolytic
streptococcus(Streptococcuspyogenes)
Interpretation:
Positive –pink to cherry red color (after the addition of color developer)
Negative –no color change in inoculated portion of the disk
L-pyrrolidonyl-beta-napthylamide ------------Beta napthylamide +
p-dimethylaminocinnamaldehyde
hydrolysis
Pyrrolidonylarylamidase
Pink to cherry red color
(color developer)

10. Interpretation
Positive:Pinktocherryredcolor(aftertheadditionofcolor
developer)
Negative:Nocolorchangeininoculatedportionofthedisk
PYRase (PYR) test
A.Positive –Enterococcus
B.Negative–Non-Enterococcus

11. Purpose
Todeterminetheabilityoftheorganismtoproducehippuricase
whichhydrolyzesthesubstratehippurate.
UsefulintheidentificationofStreptococcusagalactiae,
CampylobacterjejuniandListeriamonocytogenes.
Principle:
Theendproductsofhydrolysisofthesubstratehippuratebya
constitutiveenzymehippuricaseincludeglycineandbenzoicacid.
Glycineisdeaminatedbytheoxidizingagent,ninhydrin,which
isreducedduringtheprocess.
Theendproductsofninhydrinoxidationreacttoformapurple
coloredproduct.

12. Hippurate hydrolysis test
A B
A.Positive–Streptococcus
agalactiae
B. Negative-Enterococcus
Interpretation
Positive–deep purple
color
Negative–slightly yellow
pink or colorless

13. Purpose:
Toscreencoloniessuspectedofbeingoneof
theEnterobacteriaceae(allnegative).
Toidentifycoloniessuspectedofbelongingto
othergenerasuchasAeromonas,Pseudomonas,
Neisseria,CampylobacterandPasteurella.

14. Principle
Thecytochromeoxidasetestusescertainreagent
dyes,suchasp-phenylenediaminedihydrochloride
thatsubstituteforoxygenasartificialelectron
acceptors.Itiscolorlessinthereducedstate.
Inthepresenceofcytochromeoxidaseand
atmosphericoxygen,p-phenylenediamineisoxidized
formingindophenolblue.
Tetramethyl-p-phenylene diamine hydrochloride -----------purple color
Dimethyl compound (1%)-----------black color
P-phenylenediamine dihydrochloride -----------------Indophenol blue
cytochrome oxidase + atmospheric air

15. A B
Oxidase test
A.Positive–Pseudomonas aeruginosa
B.Negative–Escherichia coli
Interpretation
Positive–blue/ dark purple/black color
Negative –no color development

16. Purpose:
TodistinguishEnterobacteriaceaebasedonthe
abilitytoproduceindolefromtryptophan.
Toidentifylactosefermentingmembersof
Enterobacteriaceae,Escherichiacoli(indolepositive)
fromKlebsiellapneumoniae(indolenegative).
TospeciateProteus:
Proteusmirabilis:Indolenegative
Proteusvulgaris:Indolepositive

17. Principle
Bacteriathatpossesstheenzymetryptophanaseare
capableofhydrolyzinganddeaminatingtryptophan
withtheproductionofindole,pyruvicacidand
ammonia.
Aredcomplexisformedwhenindolereactswiththe
aldehydegroupofp-dimethylaminobenzaldehyde,the
activechemicalinKovac’sandEhrlich’sreagent.
Tryptophan --------------indole + pyruvic acid + NH3
tryptophanase
Indole + p-dimethylaminobenzaldehyde red complex

18. Reagents used to detect indole
Ehrlich’s –to detect indole in anaerobic and
non-fermentative bacteria
Kovac’s –to identify members of Enterobacteriaceae
Media used with tryptophan
Sulfide indole motility (SIM)
Motility indole ornithine(MIO)
Indole nitrate
Rapid spot tests –filter paper strips impregnated
with p-didimethylaminocinnamaldehyde reagent –
useful in screening bacteria that are prompt indole
producers

19. INTERPRETATION
Positive:Redringattheinterfaceofreagent
andbroth(orreagentandxyleneorchloroform)
Negative: No colordevelopment
Variableresults:Orangecolor,indicates
productsofskatole,amethylatedintermediate
thatmaybeaprecursortoindoleproduction
Rapid spot test
Paradimethylaminocinnamaldehyde: blue green
Paradimethylaminobenzaldehyde: bright pink
color

20. A B
Indole test
A.Positive–Escherichia coli
B.Negative–Klebsiella pneumoniae

21. Indole spot test
A B
A.Negative-Klebsiella pneumoniae
B.Positive-Escherichia coli

22. Purpose:
TodetectDNaseactivityinspeciesofaerobic
bacteria.
Todifferentiatenon-fermentingGram-negative
bacteriaaswellasStaphylococcusaureusand
Serratiamarcescens.

23. Metachromaticdyes
ToluidineblueiscomplexedwithDNA.
HydrolysisofDNAbytheinoculatedmicroorganism
causeschangesofstructureofthedyetoyieldapink
color.
MethylgreenisalsocomplexedwithDNA.Ifthe
organismgrowingonthemediumhydrolyzesDNA,the
greencolorfadesandthecolonyissurroundedbya
colorlesszone.
Principle
INTERPRETATION
Positive rose pink clear zone
Negative no change no clearing
Toluidine blue Methyl green

24. Positive –S. aureus
Serratia marcescens
Negative –S. epidermidis
Enterobacter cloacae
Deoxyribonuclase test
A.Positive –S. aureus
B.Positive–S. marcescens
C.Negative–S. epidermidis

25. Purpose:
Todeterminethepresenceoflateorslow
fermentingstrains.
Todetectthelatelactosefermentingstrainsof
Escherichiacoli
TodistinguishsomeCitrobacterspeciesand
arizonaesubspecies(ONPGpositive)from
similarSalmonellasubspecies(ONPGnegative)
TospeciateShigella,sinceShigellasonneiis
theonlyONPG-positiveShigellaspecies.

26. Principle
Twoenzymesrequiredforlactosefermentation
Lactosepermease:activelytransferslactose
intothebacterialcell
Betagalactosidase:degradeslactoseinto
glucoseandgalactose
Lactosefermenters:possessbothenzymes
Sloworlatelactosefermenters:nopermease;
onlybetagalactosidase
Nonlactosefermenters:lackbothenzymes

27. ONPG(o-nitrophenyl-beta-D-galactopyranoside)is
usefulindetectinglatelactosefermenters,since
ONPGmoleculeisstructurallysimilartolactose.
Itcanenterthebacterialcellwithoutapermease.
Inthepresenceofgalactosidase,ONPG(colorless)
isconvertedintogalactoseando-nitrophenyl,which
isayellowchromogenandthealkalineend
product.
Interpretation
Positive: yellow colorwithin 20 minutes to 24 hours
Negative:no color change / colorlessafter24 hours

28. ONPG (O-nitrophenyl-beta-D-galactopyranoside) test
A B
A.Negative–Salmonella Typhimurium
B.Positive –Escherichia coli(EHEC)

29. Purpose
Todeterminetheabilityofanorganismtoproduce
theenzyme,urease,whichhydrolyzesurea.
Toidentifytherapidureaseproducers(Proteus
andMorganella)andweakureaseproducers
(KlebsiellapneumoniaeandspeciesofEnterobacter)
Principle
Ureasesplitstheureamoleculeintoammonia(NH3),
CO2andwater(H20).
Ammoniareactsinsolutiontoformanalkaline
compound,ammoniumcarbonate,whichresultsinan
increasedpHofthemediumandacolorchangeinthe
indicatortopinkred.

30. Urea + 2H2O ---------------CO2 + H2O +2NH3
urease
(NH4)2CO3Interpretation
Christensens Urea agar
Positive:Rapid urease activity; redthroughoutthe medium
Positive:Slow urease activity: redin slantinitially gradually
converting the entire tube
Negative: No urease activity; medium remains yellow
Stuart (urea) broth
Positive:-Redcolor inthe medium
Negative: No colorchange (buffto pale yellow)

31. A B C
A.Positive: Proteus spp.
B.Positive: Klebsiella spp.
C.Negative:Escherichia coli
Urease test
(Christensens Urea agar)

32. Urease test
Stuart Urea broth
A BC
A.Uninoculated
B.Strong positive reaction-
Proteus spp.
C. Negative: Escherichia coli

33. Purpose:
Todeterminewhetherasubstrateutilizationisan
oxidativeorfermentativeprocessforthe
identificationofseveraldifferentbacteria.
Toseparateorganismsintotwomajorgroups:
Enterobacteriaceae: fermentative
Pseudomonas: oxidative

34. Composition:
High concentration of carbohydrates (1%)
Small concentration of peptone (2%)
Indicators
Bromcresol purple: purple to yellow
Andrade’s acid fuchsin: pale yellow to pink
Phenol red: red to yellow
Bromthymol blue: green to yellow

35. Principle of glucose oxidative fermentation test

36. INTERPRETATION
Glucosefermenter:Whenacidproductionis
detectedonbothtubessincefermentationcanoccur
withorwithoutoxygen
Glucoseoxidizer:Acidisdetectedbytheopen
aerobictube
Non-utilizer:Somebacteriadonotuseglucoseasa
substrate

37. Open tube Closed tube Metabolism
Acid (yellow)alkaline (green)oxidative
Acid (yellow)acid (yellow) fermentation
Alkaline (green)alkaline (green)non-saccharolytic
(non-utilizer)
Oxidative-Fermentation Medium of Hugh and Leifson

38. Oxidative Fermentative medium (CDC method)
A.Fermenter–
Escherichia coli
B. Oxidizer
Pseud. aeruginosa
C. Nonutilizer-
Alcaligenes faecalis

39. Purpose:Asaninitialstepintheidentificationof
Enterobacteriaceae.
Principle:
1.Theactionofmanyspeciesofmicroorganismsona
carbohydratesubstrateresultsintheacidificationof
themediumwithorwithoutgasformation.
2.Ironsalts(ferroussulfateandferricammonium
citrate)reactswithH2Stoproduceaninsolubleblack
precipitate(ferroussulfide).

40. TSIA –two reaction chamber
Aerobic slant portion
Anaerobic deep portion
Proteinsources–beefextract,peptone,yeast
extract,proteosepeptone
Sugars(lactose,sucrose,glucose)
Indicators
a.phenolred–carbohydratefermentation
b.ferroussulfate–hydrogensulfideproduction
Sodiumthiosulfate–sourceofsulfuratoms
Sodiumchloride–osmoticstabilizer
COMPOSITION

41. BIOCHEMICAL REACTIONS
Carbohydrate fermentation
acid production
Yellow deep –glucose fermented
Yellow slant –lactose and/ or sucrose fermented
Gas formation
Bubble formation
Cracking or splitting of the agar
Upward displacement of the agar
Pulling away of the medium from the walls of test tube
H2S production
Blackening of the butt (FeS–black precipitate)

42. A/@H2S(-)
Acid slant; acid butt; gas
formation; no H2S
all sugars fermented; with
gas formation;
no blackening of the butt
Escherichia
Klebsiella
Enterobacter

43. K/@H2S+
alkaline slant; acid butt; with gas
formation with H2S
glucose fermented; lactose and
or/sucrose not fermented; with gas
formation and black precipitate
Salmonella
Proteus
Citrobacter

44. K/A H2S( –)
alkaline slant; acid butt; no gas; no H2S
glucose is fermented; lactose
and/or sucrose not fermented;
no gas formation; no black
precipitate
Shigella
Providencia
Serratia
anaerogenic Escherichia coli

45. K/KH2S(-)
alkaline slant; alkaline butt; no gas;
no H2S
no sugars fermented; no gas;
no black precipitate in the butt
Pseudomonas
Alcaligenes

46. A/@H2S+
acid slant; acid butt; with gas; with
H2S
all sugars fermented; with gas
formation;
with black precipitate in the butt
Citrobacter freundii

47. Purpose:
Toidentifythelactosefermenting
EnterobacteriaceaesuchasEscherichiacoli(MR
positiveandVPnegative)whereasmostmembersof
theKlebsiella-Enterobacter-Serratia-Hafniagroupare
VPpositive.

48. Metabolism of glucose using MR and VP pathways
Glucose
Acetoin Pyruvic acid Mixed acid fermentation
KOH + air pH less than 4.4 (red)
Diacetyl
Napthol + creatine
pink red complex
Positive VP

49. Inthefirstpathway,
mixedacidproducts
(lactic,acetic,formic
andsuccinic)result,
leadingtoadecreasein
thepHofthemedium
andapositiveMRtest.
ThepHmustdropto
4.4orlessfortheMR
indicatortotakeonits
acidicredcolor.
Principle –Methyl Red Test
A B
Methyl Red test
A.Positive –Escherichia coli
B.Negative –Klebsiella pneumoniae

50. Inthesecondpathway,
acetylmethylcarbinol/acetoin
isanintermediateproductto
butyleneglycol.
Itistheneutralproduct
detectedintheVPreaction.
Inthepresenceofoxygen
and 40%potassium
hydroxide,acetoinis
convertedtothediacetyl
form,whichresultsinared
colorinthepresenceof
alpha-napthol.
Principle –Voges Proskauer Test
A B
Voges Proskauer test
A.Positive–Klebsiella pneumoniae
B.Negative–Escherichia coli

51. Interpretation
Methyl red test
Positive–distinct red colorat surface of the medium
Negative–yellow colorat the surface of the medium
Voges Proskauer test
Positive–pink red colorat surface of the medium
Negative–yellow colorat surface of the medium

52. Purpose:Todeterminetheproductionofdecarboxylaseby
bacteria(Enterobacteriaceae).
Principle
Decarboxylaseenzyme:removescarboxylgroupsfromthe
aminoacidslysineandornithine.
Dihydrolaseenzyme:removesacarboxylgroupfrom
arginine.
Glucosebasewithouttheaminoacidandtubes
containingglucose&aminoacidsubstratesareinoculated.
Decarboxylationanddihydrolationareanaerobic
reactionssoinoculatedtubesareoverlayedwithmineraloil
toexcludeair.

53. Composition–Moellerdecarboxylasemedium
1.Glucose
2.Aminoacidsubstrate(1%lysine,1%arginine
1%ornithine)
3.pHindicator
a.bromcresolpurple
AlkalinepH-Purple
AcidpH-Yellow
b.phenolred
AlkalinepH-Red
AcidpH-Yellow

54. Lysine -----------------cadaverine
Ornithine--------------putrescine
Arginine---------------citrulline-----------ornithine
dihydrolase
reaction decarboxylation
putrescine
Specific amine products

55. Earlyincubation:bothtubesyellowduetoacidificationof
theindicator(bromocresolpurple)bytheacidendproductsof
glucosefermentation.
Ifaminoacidisdecarboxylated,alkalineaminesareformed
andcausetheindicatortoreverttoanalkalinepH.
INTERPRETATION
Controltube–yellow-glucosefermentation;viable
organisms;pHofthemediumhasbeenloweredsufficientto
activatethedecarboxylase
enzyme
Positivetest–purple–decarboxylation;formationofthe
alkalineaminesfromthedecarboxylation

56. A B
A.Positive–purple; decarboxylation
B.Negative–yellow;nodecarboxylation;onlyglucose
fermentation
Moeller decarboxylase medium

57. A B C D
Decarboxylase-dihydrolase reactions–Enterobacter cloacae)
A.Control –without amino acid C. lysine-negative
B.Arginine –positive D. ornithine-positive

58. Enterobacter cloacaeKlebsiella pneumoniae
Arginine +(purple)alkaline -(yellow) acid
Lysine -(yellow)acid +(purple)alkaline
Ornithine +(purple)alkaline -(yellow)acid

59. Purpose:Todeterminethedeaminaseactivityusingtheamino
acidsphenylalanineortryptophan.
OnlyProteus,ProvidenciaandMorganellaspecies
possessthedeaminaseenzyme.
Principle:
Deaminationoftheaminoacidresultsinacolored
compoundwiththeadditionof10%ferricchloride
Phenylalanine----------------PPA+10%FeCl3
Phenylalaninedeaminase greencolour
Tryptophan-------------Indole-pyruvicacid+10%FeCl3
Tryptophandeaminase browncolour

60. INTERPRETATION
Positivedeaminationfor
phenylalanine–intense
greencolor
Positivedeaminationfor
tryptophan–browncolor
Negative–slantretains
itsoriginalcolorafterthe
additionofferricchloride
A. Negative–Escherichia coli
B. Positive–Proteus vulgaris
Phenylalanine deamination test

61. Purpose:Todeterminetheabilityoftheorganismto
deaminatelysine,decarboxylatelysineandproduceH2S.
ToidentifySalmonella,Proteus,Providenciaand
Morganella.

62. COMPOSITION
1.Proteins
2.Sugar-Glucose
3.Amino acid -Lysine
4.Sulfur
5.indicators
a. ferric ammonium citrate –
H2S production
b. bromcresol purple –
carbohydrate
fermentation

63. PRINCIPLE:
Asglucosefermentationoccurs,deepofthetubeturns
yellow.
Lysinedecarboxylationproducesalkalinecadaverineand
leadstoreversionofthedeepfromyellowtopurple.
Lysinedeaminationoccursinthepresenceofoxygen(on
theslant)andresultsintheproductionofaredcolor.
H2Sproductionisnotedbyablackprecipitateinthedeep
asH2Sreactswithferricammoniumcitrate.

64. Interpretation
Lysine decarboxylation -butt
Positive–purple
Negative–yellow
Lysine deamination -slant
Positive–red
Negative–purple

65. K/K alkaline slant/
alkaline butt
H2S(-) purple/ purple
Negative deamination
Positive decarboxylation
No blackening of the
butt
Escherichia coli
K/K alkaline slant/alkaline
butt H2S + purple/purple
Negative deamination
Positive decarboxylation
With black precipitate in
the butt
Salmonella typhimurium
K/A alkaline slant/acid
butt H2S(-)
(purple/yellow)
Negative deamination
Negative
decarboxylation
No black precipitate
in the butt
Shigella flexneri
R/A red slant/acid butt
H2S(-) red/yellow
Positive deamination
Negative
decarboxylation
No black precipitate in
the butt
Proteus vulgaris

66. Purpose:
TodetermineifamemberoftheEnterobacteriaceae
iscapableofutilizingcitrateasthesolesource
ofcarbon.
Usefulintheidentificationofthelactosefermenting
Enterobacteriaceae:Escherichiacoliiscitrate
negative;EnterobacterandKlebsiellaarepositive

67. Principle
SodiumcitrateistheonlycarbonsourceinSimmons
citrateagar.
Iftheorganismcanutilizecitrate,thesodiumcitrateis
convertedtoammonia,whichisthenconvertedto
ammoniumhydroxide.
ThealkalinityofthecompoundformedraisesthepHofthe
medium,andthebromthymolblueindicatortakesonits
alkalinecolorwhichisblue
Interpretation
Positive:Growthwithanintensebluecolorontheslantor
solelythepresenceofgrowth
Negative: Absence of growth and no color change in the
medium (remains green)

68. Citrate Utilization test
A.Positive -Klebsiella pneumoniae
B.Negative -Escherichia coli
A B

69. Purpose:Todeterminetheabilityofanorganismto
useacetateasthesolesourceofcarbon.
Principle:
BreakdownofthesodiumacetatecausesthepH
ofthemediumtoshifttowardthealkalinerange,
turningtheindicatorfromgreentoblue.

70. Interpretation
Positive –Medium
becomes alkalinized
(blue) because of the
growth of the organism
Negative–no growth
or growth with no
indicator change to blue
A B
Acetate utilization test
A.Positive-Klebsiella pneumoniae
B.Negative –Escherichia coli

71. Purpose
Todeterminetheabilityofanorganismtouseacetamideas
thesolesourceofcarbon.
Principle
Bacteriathatcangrowonthismediumdeaminate
acetamidetoreleaseammonia.
TheproductionofammoniaresultsinapH-drivencolor
changeofthemediumfromgreentoroyalblue.
Interpretation:
Positive: Deamination of the acetamide resulting in a blue
color
Negative: No color change

72. Acetamide utilization test
A.Positive–Klebsiella pneumoniae
B.Negative–Escherichia coli

73. Purpose:
TodifferentiateMicrococccusandStomatococcus
fromStaphylococcuswhencombinedwithother
proceduressuchasthemodifiedoxidasetest.
ForpresumptiveidentificationofGroupA
streptococcus
Principle:
Bacitracin(0.04units)inhibitsthegrowthof
MicrococcusandStomatococcusandGroupA
streptococcuswhilehavingnoeffecton
Staphylococcuswhichisresistant.

74. Interpretation
susceptible–zonesof
inhibitiongreaterthanor
equalto10mm
resistant–zonesof
inhibitionlessthanor
equalto9mm.
A. Susceptible: Micrococcus and
Stomatococcus
B. Resistant: Staphylococcus
epidermidis
Bacitracin susceptibility test

75. :
Purpose:ToidentifythedifferentspeciesofStreptococcus
especiallyGroupAandGroupBbetahemolyticstreptococci.
Principle:
GroupAbetahemolyticstreptococci(Streptococcus
pyogenes)aresusceptibleto0.04unitsbacitracinbut
resistantto1.25ugsulfamethoxazole-trimethoprim(SXT)
GroupBbetahemolyticstreptococci–resistanttoboth
bacitracinandSXT

76. Interpretation
Susceptible:anyzoneofinhibitionaroundeither
disk
Resistant:growthuptothedisk(nozoneof
inhibition
Organism BacitracinSXT
Group A susceptible resistant
Group B resistantresistant
Group C,F,G resistant susceptible

77. Purpose
Todifferentiatethedifferentspeciesofcoagulasenegative
staphylococci.
Principle
Afterincubationwith5ugofnovobiocin,Staphylococcus
saprophyticusisnotinhibitedbytheantibioticwhereas
Staphylococcusepidermidisaresusceptibletonovobiocin.

78. Interpretation:
Susceptible –zone
greater than 16 mm
Resistant –zone
diameter less than or equal
to 16 mm
A B
Novobiocin susceptibility test
A.Susceptible -Staphylococcus epidermidis
B.Resistant -Staphylococcus saprophyticus

79. Purpose
TodifferentiatePediococcusfromotheralphahemolytic
streptococcus.
Principle
Afterincubationwith5ugofvancomycin,Pediococcusis
notinhibitedbytheantibioticwhereasViridansstreptococcus
issusceptibletovancomycin.

80. Interpretation:
Susceptible–zone
of inhibition
Resistant–no
zone of inhibition
Vancomycin susceptibility test
A. Susceptible -Viridans streptococcus
B. Resistant -Pediococcus
A B

81. Purpose
Todetermineananaerobe’sinhibitionthatcanbeusedfor
presumptiveidentificationbasedonitscharacteristic
susceptibilitypatterntocolistin(10ug),vancomycin(5ug)
andkanamycin(1mg).

82. Principle
Mostanaerobeshavea
characteristicsusceptibility
patterntocolistin(10ug),
vancomycin(5ug),and
kanamycin(1mg)disks.
Kanamycin-inhibitsfacultative
Gram-negativebacilli
Vancomycin-inhibitsfacultative
andobligateGram-positive
bacteria
Colistin-inhibitsfacultative
Gram-negativebacilli

83. K
Va
Co
Interpretation
Susceptible –zone greater than 10 mm
Resistant –zone of 10 mm or less
Antibiotic Disks for the Presumptive Identification of Anaerobes

84. Purpose
Toclassifybacteriabasedontheirabilitytogrowinthe
presenceof6.5%NaCl,acharacteristicofcertainspecies
ofGrampositiveandGramnegativebacilli.
TodifferentiatetheGroupD(salttolerant)fromthenon-
enterococci(intolerant).

85. PrincipleNutrient broth or 6.5%NaCl
Trypticase broth-salt
free medium
Positive equal equal
Negative good very weak
Interpretation:
Positive : If growth is equivalent to both media -tolerant of salt
Negative-growth on the salt containing medium is
very weak or absent
growth in the salt free medium is good
-intolerant of salt
Indicator: bromocresol purple
Positive: medium turns yellow from purple or the appearance of
growth

86. SALT TOLERANCE TEST
A.Positive -Enterococcus faecalis ( salt tolerant)
B.Negative -Streptococcus bovis (salt intolerant)

87. Purpose:
TodistinguishGroupDstreptococciandEnterococcus
speciesfromotherLancefieldgroupofstreptococci
Principle:
Basedontheorganismsabilitytogrowin40%bileandto
hydrolyzeesculintoproduceescultin
Escultinreactswithferriccitratetoformabrownblack
precipitate.

88. Interpretation
Positive:
growth indicates
tolerance to 40% bile
(40% oxygall)
blackening indicates
hydrolysis of esculin
Negative:
lack of growth indicates
inability to grow in 40%
bile
lack of color change
indicates inability to
hydrolyze esculin
A.Positive -Enterococcus faecalis
B.Negative -Streptococcus viridans
Bile esculin agar
A B

89. Purpose:
TodifferentiateStreptococcuspneumoniaefromother
alphahemolyticstreptococci
Principle:
Inthepresenceofoptochin,coloniesofStreptococcus
pneumoniaeareselectivelylysedindicatedbyazoneof
inhibitionafterincubationunderincreasedCO2.
Otheralphahemolyticstreptococciareresistantto
optochin.

90. Interpretation
Positive –zone of
inhibition at least 14 mm in
diameter using a 10 ugP
disk and at least 10 mm
using a 6 ugP disk
Negative –growth up to
the disk or a zone of
inhibition
less than 14 mm with a 10
ugP disk or less than 10
mm with a 6 ugP disk
Optochin susceptibility
test
A.Positive –Streptococcus
pneumoniae
B.Negative –Viridans streptococci
A B

91. Purpose
TodifferentiateStreptococcuspneumoniae(positive)from
otheralphahemolyticstreptococci.
Principle
Pneumococcalcoloniesarerapidlylysedbybileora
solutionofabilesaltsuchassodiumdeoxycholate.
Lysisdependsonthepresenceofanintracellular
autolyticenzyme.
Bilesaltslowerthesurfacetensionbetweenthe
bacterialcellmembraneandthemediumthusaccelerating
theorganism’snaturalautolyticprocess.

92. Interpretation
Positive –colony
disintegrates; an
imprint of the lysed
colony may remain
within the zone
Negative –intact
colonies
B
A
Bile solubility test
A.Positive –Streptococcus pneumoniae
B.Negative –Viridans Streptococci

93. Purpose
todemonstratethephenomenaofsynergistichemolysis
betweengroupBstreptococcusandbetahemolytic
Staphylococcusaureus.
Principle
Acharacteristic“arrowhead”hemolyticpatternresults
whentheorganismisstreakedperpendiculartobeta
hemolyticStaphylococcusaureus

94. Interpretation
Positive–azoneofenhanced
hemolysisgivenbyan
arrowheadappearanceatthe
junctionofthe
Staphylococcus and
Streptococcus–indicativeof
GroupBstreptococcus
Negative–nozoneof
enhancedhemolysis-
notindicativeofGroupB
streptococcus
CAMP REACTION
A.Positive -Streptococcus agalactiae
B.Negative -Streptococcus bovis

95. Principles:
Many bacteria produce enzymes called hydrolases.
Hydrolases catalyzethe splitting of organic molecules
into smaller molecules in the presence of water.
The starch molecule consists of two constituents:
Amylose, an unbranchedglucose polymer (200 to
300 units)
Amylopectin, a large branched polymer.
Both amylopectin and amylose are rapidly
hydrolyzedby certain bacteria,
Using their α-amylases, to yield dextrins, maltose, and
glucose.

96. Interpretation:
Gram’siodinecanbeusedtoindicatethepresenceof
starch.
Whenitcontactsstarch,itformsabluetobrown
complex.
Hydrolyzedstarchdoesnotproduceacolourchange.
IfaclearareaappearsafteraddingGram’siodinetoa
mediumcontainingstarchandbacterialgrowth:
Amylasehasbeenproducedbythebacteria.
Ifthereisnoclearing,starchhasnotbeenhydrolyzed.

97. 1.Determinetheabilityofbacteriatohydrolyzelipidsby
producingspecificlipases.
Principle:
Lipids are high molecular weight compounds possessing large
amounts of stored energy.
The two common lipids catabolized by bacteria are the
triglycerides (triacylglycerols) and phospholipids.
Triglycerides are hydrolyzedby the enzymes called lipases into
glycerol and free fatty acid molecules.
Glycerol and free fatty acid molecules can then be taken up by
the bacterial cell and further metabolized through reactions of:
Glycolysis, β-oxidation pathway, and the citric acid cycle.
These lipids can also enter other metabolic pathways where
they are used for the synthesis of cell membrane phospholipids.
Since phospholipids are functional components of all cells, the
ability of bacteria to hydrolyzehost-cell phospholipids is an
important factor in the spread of pathogenic bacteria.

98. In addition, when lipase-producing bacteria contaminate food
products. the lipolytic bacteria hydrolyze the lipids, causing
spoilage termed rancidity.

99. The culture medium contains tributyrin as a reactant;
degradation of this compound gives rise to clear zones
surrounding the lipolytic colonies in the otherwise turbid
culture medium.

100. Nitrate reduction Test
It is used to
determine if an
organism is capable
of reducing nitrate
(NO
3
-
) to nitrite (NO
2)
or other nitrogenous
compounds via the
action of the enzyme
nitratase (also called
nitrate reductase).
This test is important
in the identification
of both Gram-
positive and Gram-
negative species.

101. Nitrate reduction Test
Afterincubation,these
tubes are first
inspectedforthe
presenceofgasinthe
Durhamtube.
Incaseofnon
fermenters,thisis
indicativeofreduction
ofnitratetonitrogen
gas.However,inmany
casesgasisproduced
byfermentationand
furthertestingis
necessarytodetermine
ifreductionofnitrate
hasoccurred.

102. Nitrate reduction Test
•Thereductionofnitrateto
nitritewasdetectedwith
dimethyl-a-naphthylamine
(Wallace&Neave,1927)
andsulphanilicacid.
•Thereactionwasrapid
withallthespeciestested;
at30min.theresultswere
consistentwiththeusual
culturalmethod

103. GELATIN LIQUEFACTION TEST
Purpose:Thistestistodeterminean
organism'sabilitytoproduceproteolytic-
likeenzymesandliquefygelatin.
Principle:Gelatinistolargetoentera
bacterialcellwallandthusextracellular
enzymesmustcatabolizethemintosmaller
componentsbeforetheycanbeutilized.
Possessionoftheseextracellular
gelatinasescanaidinthedifferentiationof
bacteria
Thistestisusedtodifferentiatebetween
speciesinthegeneraStaphylococcusand
Clostridiumaswellasaidinthe
identificationofotherspeciesandgenra.
•POSITIVE TEST =
medium liquefied.
NEGATIVE TEST =
medium remains
solid.

104. Thanks
Acknowledgement:Allthematerial/presentationsavailableonlineonthe
subjectaredulyacknowledged.
Disclaimer:Theauthorbearnoresponsibilitywithregardtothesource
andauthenticityofthecontent.