Dr.P.B.PRAVEENKUMAR FIRST YEAR POST GRADUATE DEPARTMENT OF MICROBIOLOGY THANJAVUR MEDICAL COLLEGE THANJAVUR

1. IMViC TEST
Dr. P.B.PRAVEENKUMAR
FIRST YEAR MICROBIOLOGY PG RESIDENT
GOVERNMENT THANJAVUR MEDICAL
COLLEGE

2. INDOLE TEST - PRINCIPLE
• To determine the ability of an organism by
means of trytophanase enzyme to split indole
from the tryptophan molecule.

3. LIST OF INDOLE +VE AND –VE
ORGANISMS
INDOLE POSITIVE
• Escherichia coli
• Proteus vulgaris
• K.oxytoca & Ornitholytica
• Vibrio cholerae
• Edwardsiella
• All Proteus species except Proteus
mirabilis
• Bacillus alvei
• Haemophilus haemoglobinophilus
and Haemophilus influenzae
• Pasteurella multocida and
Pasteurella pneumotropica
INDOLE NEGATIVE
• Klebsiella species except two
• Proteus mirabilis
• Pseudomonas
• Salmonella
• All bacillus species except
Bacillus alvei
• Remaining Haemophilus
species
• Pasturella hemolytica and
Pasteurella ureae

4. MECHANISM OF INDOLE TEST

5. MEDIA USED FOR INDOLE TEST
1. Tryptophan (or) Peptone broth
*Contains basal medium(Peptone water and
Nacl)
*Tryptophan 1%
Commercial products
*BBL: Trypticase Peptone broth (Tryptophanase
broth)
*Difco tryptone broth

6. METHOD OF PREPARATION IN
PEPTONE BROTH
• Fill the test tube with 4 ml of peptone water
• Autoclave it at 121 degree celsius for 15
minutes.
• Light inoculum is added into tube from 18 to
24 hour pure culture / Kligler’s iron agar(KIA)
• Incubate at 35 degrees celsius for 24 to 48
hours.
• Add kovac’s or Ehrlich’s reagent.

7. MEDIA USED FOR INDOLE TEST
(cont)
• 2. Casein medium
*A pancreatic digest of casein solution may
be prepared.
*Protein casein contains 1.2 grams of
tryptophan per 100 grams.
*Dissolve by moderate heating and tube
contains 4 ml
*Autoclave it at 121 degree celsius for 15
minutes

8. INGREDIENTS OF CASEIN
MEDIUM
PANCREATIC DIGEST OF CASEIN 2 GRAMS
SODIUM CHLORIDE 0.5 GRAMS
DISTILLED WATER 100 ml

9. REAGENTS USED FOR INDOLE
TEST
• 1. Ehrlich’s indole test
p-DIMETHYL AMINO
BENZALDEHYDE
2 GRAMS
ABSOLUTE ETHYL ALCOHOL 190 ml
CONCENTRATED HCL 40 ml

10. HOW TO UTILISE EHRLICH’S
INDOLE REAGENT????
• Add 1 ml of ether or xylene to a 24 hour or 48
hour incubated broth tube
• Shake well
• Wait for the ether to rise to the surface of the
medium.
• Add 0.5 ml of Ehrlich’s reagent to the tube and
tilt it.

11. REAGENTS FOR INDOLE TEST
(cont)
• 2. Kovac’s indole test
PURE AMYL / ISOAMYL /
BUTYL ALCOHOL
150 ml
p-Dimethyl amino
benzaldehyde
10 grams
CONCENTRATED HCL 50 ml

12. METHOD OF PREPARATION &
UTILIZATION OF KOVAC’S REAGENT
• Dissolve the aldehyde in the alcohol
• Slowly add the acid in the aldehyde-alcohol
mixture
• Add 5 drops of kovac’s reagent directly into 24
hour or 48 hour incubated tube
• Shake the tube gently.

13. INDOLE TEST HYPOTHESIS
ACCORDING TO GADEBUSH & GABRIEL
• Addition of Isoamyl alcohol makes Kovac’s
reagent more stable.
• With ether reagent in Ehrlich’s method,
dissolving aldehyde in solvent require gentle
heating.
ACCORDING TO EWING AND DAVIS
• Use Kovac’s for Indole and Ehrlich’s for
nonfermenters and anaerobes

14. Why we want to separate 2ml from peptone
broth before we adding reagent after 24 hours??
• Remove 2ml aseptically from 4ml containing
peptone broth to check with reagent.
• Positive means ok.........Finish the test with
interpretation.
• If no change while mixing with reagent
means, then incubate that 2ml broth for
further 24 hours and retested.

15. QUALITY CONTROLS

16. STORAGE OF INDOLE REAGENT
• Store it in refrigerator when not in use
• Each week quality control check should be
done and then discarded.
• Stored in brown bottles with glass toppers.

17. CHEMISTRY OF REAGENT
Due to pyrrole
structure in indole
Quinoidal red
violet compound
Due to tryptophan
splitting of acid
If concentrated Hcl is used no
heating is required

18. INTERPRETATION
INDOLE TEST +VE INDOLE TEST -VE

19. INTERPRETATION IN DETAIL
POSITIVE TEST RED RING AT THE SURFACE OF
THE MEDIUM
NEGATIVE TEST TAKES THE COLOUR OF KOVAC’S
REAGENT
ORANGE COLOUR DUE TO FORMATION OF SKATOLE
WHICH IS A PRECURSOR FOR
INDOLE

20. RAPID INDOLE TESTS
• Indole spot test
• Indole spot test for anaerobic bacteria
• Indole microtechnique
• Reagent impregnated indole test strip

21. INDOLE SPOT TEST
(Method of Vracko and Sherris)
KOVAC’S
REAGENT
DMABA DMACA
3ml OF AMYL ALCOHOL
IN 1ml OF
CONCENTRATED Hcl
1% SOLUTION IN 10%
v/v CONCENTRATED Hcl
1% SOLUTION IN 1% v/v
CONCENTRATED Hcl
PREPARE FRESH EVERY
FOUR MONTHS;
DETECTS 6-12
MICROGRAM OF
INDOLE PER ml
PREPARE FRESH EVERY
TWO MONTHS;
DETECTS 3
MICROGRAMS OF
INDOLE PER ml
MOST STABLE AND
MOST ECONOMICAL
REAGENT
MOST SENSITIVE
AGENT

22. PREPARING INOCULUM AND
PROCEDURE
• Take a 18 to 24 hour pure culture from sheep
blood agar
• Preferably Trypticase soy blood agar(5% SBA)
• Take a whatmann no.1 filter paper and put it in
petri dish
• Moisten the filter paper with 1 to 1.5 ml of
reagent.
• Smear inoculum onto moistened filter paper.

23. INDOLE SPOT TEST
INTERPRETATION
INTERPRETATION
KOVAC’S
REAGENT DMABA DMACA
INDOLE +VE
BROWN
REDDISH-
PURPLE
RED COLOUR
PINK TO RED
COLOUR
BLUE OR GREEN
COLOUR
INDOLE -VE
WHITE TO
YELLOW
COLOUR
WHITE TO
YELLOW
COLOUR
WHITE TO
YELLOW
COLOUR

24. INDOLE SPOT TEST FOR
ANAEROBIC BACTERIA
• Same procedure as previously mentioned
• Exception is that 1% DMACA in 10%
Concentrated Hcl.
• Positive: Immediate formation of blue colour
around growth.
• Negative: No colour change or pinkish colour.

25. INDOLE MICRO TECHNIQUE
(Method of Arnold and Weaver)
BROTH 1 BROTH 2
Tryptone 1% Tryptophane 0.03%
Beef extract 0.3% Peptone 0.1%
Dipotasium phosphate
0.5%

26. INDOLE MICRO TECHNIQUE (cont)
• Preheated broth tubes are used.
• Adding heavy inoculum from tryptone agar.
• Using kovac’s reagent
• Waterbath at 37 degree celsius for 6 minutes to
2 hours.
• Same interpretation as previously mentioned
slide............

27. REAGENT IMPREGNATED
INDOLE TEST STRIP
• Results within 4 hours incubation
• Many false negatives especially when the
inoculum is taken from TSI broth.
• Occasional false positives

28. Why glucose containing broth not
suitable for indole test?????
• Because glucose inhibits indole production.
• Formation of indole will occur with organisms
which is fermenting carbohydrates but not
utilising carbohydrates.

29. Which organism will break down indole
rapidly as soon as it is formed?????
CLOSTRIDIUM
SPECIES

30. Then what’s the optimal pH for indole
production????
SLIGHT ALKALINE
(7.4 TO 7.8)

31. Indole producing organisms interferes with
antimicrobial activity than non-indole
producing organisms???
YES
(ACCORDING TO
MARTIN AND
KLEIN)

32. METHYL RED TEST - PRINCIPLE
• To test the ability of an organism to produce
and maintain stable acid end products from
glucose fermentation and to overcome the
buffering capacity of the system.

33. LIST OF METHYL RED +VE AND
–VE ORGANISMS
METHYL RED +VE
• Escherichia coli
• Yersinia species
• Listeria monocytogenes
METHYL RED -VE
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella
Other gram negative
Non enteric bacilli

34. MECHANISM OF METHYL RED
TEST
• Based on the use of pH indicator, methyl red to
determine the hydrogen ion concentration (pH)
present when an organism ferments glucose.
HYDROGEN ION CONCENTRATION IS
DEPENDS ON GAS RATIO
1:1  MR +VE
2:1MR -VE

35. Why we have to incubate the broth
atleast for 48 hours????
• If we test it before 48 hours by adding methyl
red reagent, false positives will occur......
Since we have to check the capability of bacteria
to retain it’s acid producing capability even in
the presence of phosphate buffer
Just wait for 48 hours and then check with
reagent

36. CHARACTERISTICS OF MR +VE
AND MR –VE ORGANISMS
METHYL RED +VE
• FERMENTED PRODUCTS
PRODUCES ACIDIC
METABOLITES
AFTER
TWO TO FIVE DAYS
MORE ACIDS
LOW TERMINAL pH
MAINTAINING ACIDITY IN
PHOSPHATE BUFFER
METHYL RED -VE
• FERMENTED PRODUCTS
DECARBOXYLATION
NEUTRAL ACETYL METHYL
CARBINOL (ACETOIN)
HIGH TERMINAL pH
NEUTRAL pH

37. METHYL RED +VE 1:1 GAS RATIO
2 Glucose + H2O
2 Lactic acid + Acetic acid + Ethanol + 2Co2 +
2 H2

38. METHYL RED –VE 2:1 GAS RATIO
Glucose + ½ H2O
2,3 butanediol + H2O + 2CO2
Acetoin 2,3 butanediol

39. CLARK AND LUBS MEDIUM
(MR/VP BROTH)
POLYPEPTONE OR
BUFFERED PEPTONE
7.0 GRAMS
DEXTROSE (GLUCOSE) 5.0 GRAMS
DIPOTTASIUM PHOSPHATE BUFFER 5.0 GRAMS
DISTILLED WATER 1000 ml

40. INOCULUM AND INCUBATION
• Light inoculum from 18 to 24 hours pure
culture of kligler’s iron agar in 5 ml broth (MR
and VP)
• Incubated at 35 degree celsius for 48 hours (or)
Incubated at 30 degree celsius for 3 to 5 days

41. REAGENT PREPARATION AND
USAGE
• Dissolve 0.1 gram of methyl red in 500 ml of
95% ethyl alcohol.
• Add 200 ml of distilled water to the alcohol
indicator mixture
• Add 5 drops of methyl red indicator into
aliquot.

42. QUALITY CONTROL FOR
METHYL RED TEST
POSITIVE CONTROL
Escherichia coli (ATCC 25922)
NEGATIVE CONTROL
Klebsiella (ATCC 13883)

43. INTERPRETATION OF MR TEST
COLOUR pH INTERPRETATION
RED LESS THAN OR EQUAL
TO 4.4
METHYL RED
POSITIVE
ORANGE
(Delayed reaction)
5 TO 5.8
FI (FURTHER
INCUBATION) FOR 4
DAYS AND REPEAT
THE TEST
YELLOW AT A pH of 6.0 METHYL RED
NEGATIVE

45. RAPID TEST
(Method of Barry, Bernsohn, Adams and Thrupp)
• 0.5 ml aliquot into small test tubes
• Pick a centre of colony from either
EMB/MAC/BAP
• Incubate it for 18 hours
• After incubation add one drop of MR reagent
in each tube setup
• Interpretation is same as previously mentioned.

46. VOGES PROSKAUER TEST -
PRINCIPLE
• Depends on the production of acetyl methyl
carbinol (Acetoin) from pyruvic acid from
glucose fermentation.
• In the presence of alkali and atmospheric
oxygen, acetoin is oxidised to diacetyl which
reacts with alpha-naphthol to give red colour.

47. LIST OF VP +VE AND VP –VE
ORGANISMS
VOGES PROSKAUER +VE
• Klebsiella pneumoniae
• Enterobacter
• Staphylococcus
• Klebsiella oxytoca
• Hafnia alvei (Enterobacter
alvei at 25 degree celsius)
• Listeria monocytogenes with
coblentz reagent
• Yersinia enterocolitica at 25
degree celsius
• Serratia group
VOGES PROSKAUER -VE
• Escherichia coli
• Micrococcus
• Klebsiella ozaenae
• Klebsiella rhinoscleromatis
• Yersinia enterocolitica at 37
degree
• Listeria monocytogenes
with O’Meara reagent

49. MECHANISM INVOLVED
• Same mechanism as already explained in
methyl red test in exact opposite way (Acetyl
methyl carbinol from BUTYLENE GLYCOL
PATHWAY)

50. MEDIUM AND INOCULUM
• Same MR/VP broth....
• Light inoculum for all except for Coblentz
method where heavy inoculum is used.

51. VP REAGENTS
REAGENTS BARRIT’S VP
REAGENT
COBLENTZ VP
REAGENT
O’MEARA’S
REAGENT
(MODIFIED
SINGLE VP
REAGENT)
REAGENT A
5% ALPHA
NAPHTHOL IN
ABSOLUTE
ETHANOL
5% ALPHA
NAPHTHOL IN
95% ETHANOL
REAGENT B 40% KOH 0.3% CREATINE
IN 40% KOH
REAGENT 0.3% CREATINE
IN 40% KOH

52. METHOD OF UTILISATION
(In MR/VP broth)
REAGENTS
ALIQUOT
DIVIDING
LEVEL
REAGENT
ADDING
PROCESS
SHAKING
THE TUBE
WAITING
TIME
BARRIT’S VP
REAGENT
(0.6 ml and 0.2 ml)
2.5 ml
REAGENT A
f/b
REAGENT B
FOR 30
SECONDS TO
ONE MINUTE
ATLEAST 10
TO 15
MINUTES
COBLENTZ
REAGENT
(0.6 ml and 0.2 ml)
2.0 ml
REAGENT A
f/b
REAGENT B
FOR 30
SECONDS TO
ONE MINUTE
ATLEAST 10
TO 20
MINUTES
O’MEARA’S
REAGENT
1.0 ml Add one ml of
reagent
FOR 30
SECONDS TO
ONE MINUTE
35 degrees/ RT
Final reading
only after 4
hours, If
equivocal
Repeat at 25
degrees
incubation

53. BIOCHEMICAL MECHANISM

54. COMPOUNDS CONTAINING
GUANIDINE NUCLEUS
• Arginine (Here it is present)
• Agmatine
• Creatine
• Dicyanamide
• Guanidine acetic acid

55. ROLE OF REAGENT CONTENTS IN
VP TEST
DIACETYL COMPOUND COLOUR FORMING AGENT
ALPHA NAPHTHOL COLOUR INTENSIFIER
KOH (POTTASIUM HYDROXIDE) OXIDIZING AGENT TO OXIDIZE
ACETOIN TO DIACETYL

56. QUALITY CONTROLS
• Positive control – Escherichia coli
(ATCC 25922)
• Negative control – Enterobacter cloacae
Enterobacter aerogenes

57. HOW LONG WE CAN WAIT FOR
VP TEST INTERPRETATION????
Maximum one hour otherwise after one hour
copper like colour due to the action of KOH
on alpha naphthol
(FALSE POSITIVE)

58. INTERPRETATION
• Pinkish red colour
• Acetoin present
VP
POSITIVE
• Yellow colour
• No acetoin
VP
NEGATIVE

60. ALTERNATE TESTS
• Gas liquid chromatography (Diacetyl
measurement)
• Electron capture gas liquid chromatography
(EC-GLC)
• Gas chromatography chemical ionization mass
spectrometry
• Colorimetric method for measurement of
diacetyl

61. RAPID TESTS
REAGENT IMPREGNATED VP STRIP
TEST
RAPID VP TEST OF BARRY AND
FEENEY
 Results within 4 hours of incubation
 Two step procedure
 False positives with proteus and klebsiella
species
 Growth from MAC, BA, TSI and KIA
 Add it in MR/VP broth
 35 degree celsius waterbath for 4 hours
Add creatine solution 0.3% in 2 drops
Add Barrit’s reagents A and B: 2 to 3 drops
each
Positive – Cherry red colour within 15
minutes

62. Sometimes acetoin positive organisms fails to
give positive VP test.......What will we do????
GENTLE HEATING OF CULTURE
CONTAINING VP REAGENT

63. What will happen if overnight incubation
done more than 3 days (OR) reverse the
pouring of reagents A and B??
WEAKLY POSITIVE OR FALSE
NEGATIVE

64. WHICH ORGANISM GIVES BOTH
MR AND VP POSITIVE???
PROTEUS MIRABILIS
HAFNIAALVEI
(BUT STILL VP DELAYED)

65. Shall we use routine clark and lubs
medium even for bacillus and
staphylococcus???
No......In presence of phosphate and sodium
chloride, it may interfere with acetoin
production.....So use plain glucose peptone
broth without Nacl and phosphate
(ACCORDING TO ABD-ED-MALEK AND GIBSON)

66. Meat infusion broth should not be used
for VP test....Why????
Because of the presence of acetoin, diacetyl
and related substances in muscle extract
FALSE POSITIVE

67. CITRATE TEST - PRINCIPLE
• To determine if an organism is capable of
utilising citrate as the sole source of carbon for
metabolism with resulting alkalinity.

68. LIST OF CITRATE UTILISED AND
CITRATE UTILISED NOT ORGANISMS
CITRATE UTILISED
• Klebsiella species
• Salmonella species except
S.typhi
• Citrobacter species
• Enterobacter species
• Serratia liquefaciens
• Aeromonas hydrophilia
• Bordetella species except
B.pertussis
• Proteus/Providencia rettegeri
• Pseudomonas cepacia
CITRATE NOT UTILISED
• Escherichia coli
• Edwardsiella
• Salmonella typhi
• Yersinia
• Actinobacillus equui
• Aeromonas salmonicida
• Moraxella species
(M.urethralis – variable)
• Morganella morganii (Proteus
morganii)
• Pseudomonas maltophilia

69. MECHANISM IN CITRATE TEST
• Citrate metabolism occurs via Tricarboxylic
acid (TCA Cycle)/Krebs cycle/Citric acid
cycle in presence of Coenzyme A.
• But in bacteria NO COENZYME A.
• Instead bacteria can use Citritase/Citrate
oxaloacetate lyase/Citrate demolase.
• Citrate breakdown will happen well in basic
pH comparing with acidic pH.

70. MECHANISM (cont.)
CITRATE
Mn2+ (or) Mg2+
Co2 + Formic acid + Acetate

71. BASED ON pH CITRATE TEST
LOOKS LIKE......
•BASIC pH
MORE ACETIC
ACID &
FORMIC ACID
FORMED
•ACIDIC pH
LESS ACETIC
ACID &
FORMIC ACID
FORMED

72. This is how citrate test works???
Citrate utilised
Citrate not
utilised

73. Is this citrate test using citrate alone as
a sole source???
• NO.............It’s using citrate as a sole source
for carbon.
• It is using ammonium salts also as a sole
source of nitrogen which is giving
alkalinity.........(By breakdown of ammonium
salts to ammonia)

74. MEDIA USED FOR CITRATE TEST
• Simmons citrate medium – pH 6.9
(Modification of Koser’s medium with 1.5%
agar and pH indicator bromothymol blue)
• Christensen’s citrate sulfate medium – pH 6.7

75. SIMMONS CITRATE MEDIUM
Magnesium sulphate 0.2 Grams
Monoammonium phosphate /
Ammonium dihydrogen phosphate
1.0 Grams
Dipottasium phosphate 1.0 Grams
Sodium citrate (2.77 grams of sodium
citrate in 5 ½ molecules of water)
2.0 Grams
Sodium chloride 5.0 grams
Agar 15 to 20 Grams
Bromothymol blue (1.5% alcoholic) 0.08 Grams
Distilled water 1000 ml

76. METHOD OF PREPARATION OF
MEDIA
• Weigh it properly
• Rehydrate it with distilled water
• Heat gently
• Place approximately 4 to 5 ml in each tube
(Long slant, short butt)
• Autoclave it for 10-15 minutes
• Allow medium to solidify in a slanted position
• Light inoculum is used

77. CHRISTENSEN’S CITRATE
SULFIDE MEDIUM
Sodium citrate 3.0 Grams
Glucose 0.2 Grams
Yeast extract 0.5 Grams
Cysteine monohydrochloride 0.1 Grams
Ferric ammonium citrate
(Will be eliminated if H2S didn’t
formed)
0.4 Grams
Monopotassium phosphate 1.0 Grams
Sodium chloride 5.0 Grams
Sodium thiosulfate (Will be eliminated
if H2S didn’t formed)
0.08 Grams
Phenol red 0.012 Grams
Agar 15.0 Grams
Distilled water 1000 ml

78. QUALITY CONTROL CHECKS FOR
CITRATE TEST
• POSITIVE CONTROL – Klebsiella
aerogenes (ATCC 13048)
• NEGATIVE CONTROL – Escherichia coli
(ATCC 25922)

79. INTERPRETATION OF CITRATE
TEST
INTERPRETATION SIMMONS CITRATE
MEDIUM
CHRISTENSEN’S
CITRATE SULFIDE
MEDIUM
CITRATE UTILISED
INTENSE BLUE
COLOUR ON THE
SLANT
PINK RED COLOUR ON
THE SLANT
CITRATE NOT UTILISED NO COLOUR CHANGE
(GREEN COLOUR)
NO COLOUR CHANGE

80. SIMMONS CITRATE TEST -
INTERPRETATION

81. RAPID CITRATE BLOOD TEST
(Method of Cordaro and Sellers)
• To test the ability of an organism to utilise the
citrate present in citrated blood to form a clot.

82. CORDARO AND SELLER’S
CITRATE BLOOD MEDIA
INOCULUM DILUENT TEST MEDIUM
DEHYDRATED BHI BROTH
(12.5 GRAMS/LITER)
FOR ONE BOTTLE OF BHI BROTH
WE SHOULD ADD 15 ml OF
OUTDATED CITRATED BLOOD
BANK BLOOD
CALCIUM CHLORIDE
(CaCl2 100 mg)
MIX ASEPTICALLY AND PIPETTE IT
1 ml AMOUNTS INTO SMALL
STERILE TEST TUBES BASED ON
OUR REQUIREMENT
SODIUM CHLORIDE
(2.5 g)
REMAINING OUTDATED BLOOD WE
CAN KEEP IT TILL 2 MONTHS IF
IT’S REFRIGERATED

83. RAPID CITRATE TEST –
INOCULUM AND AUTOCLAVING
• Growth from an 18 hour pure culture from TSI
media (Heavy inoculum)
• Incubate it at 35 degrees for 1 to 6.5 hours
(Hourly monitoring is must)

84. INTERPRETATION OF RAPID
CITRATE TEST
• Citrate utilised – Presence of firm clot
• Citrate not utilised – No clot formation

85. What will happen if large inoculum is
used in citrate slant test???
(LIGHT YELLOW TO TAN COLOUR ON SLANT)
FALSE POSITIVE

86. Why it is advisable to diluting the inoculum with
normal saline prior to inoculating citrate???
TO PREVENT CARRY OVER OF OTHER
CARBON SOURCES LIKE GLUCOSE,
PEPTONE AND ACETATE
FALSE POSITIVE

87. Sometimes organisms will grow on citrate slant
but not producing colour
change......Intrepretation????
CITRATE IS UTILISED
(POSITIVE CITRATE TEST)
ORGANISM ENTERED LOG PHASE SO
ONLY GROWTH IS VISIBLE IF CARBON
AND NITROGEN IS ASSIMILATED.......

88. Till how long we can wait to interpret
citrate test???
TILL 7 DAYS
(For pH to change)

89. REFERENCES
• Bailey scott diagnostic microbiology
• Macfaddin biochemical reactions
• Mackie and Mcartney diagnostic microbiology

90. THANK YOU