BIOCHEMICAL REACTIONS

FOR THE IDENTIFICATION

OF BACTERIA
Presented by
Nusrat Aga
MSC Final
year
Department
of
Microbiology

INDOLE TEST
MEDIA AND REAGENTS
MEDIA

REAGENT

KOVACS REAGENT
Tryptophan broth
Peptone
Sodium chloride
Distilled water

P- Dimethyl aminobenzaldehyde
Pure amyl or iso amyl
Alcohol
Conc. Hcl

EHRLICHS REAGENT
P-dimethyl amino benzaldehyde
Ethyl alcohol
Conc. Hcl

PRINCIPLE
tryptophan
pyruvic acid

tryptophanase
indole + ammonia +

indole + p-Dimethyl
amino

benzaldehyde

quinidol + water
red violet
complex

PROCEDURE
Tryptophan broth + test organism .
18-24 hrs
Add 15 drops of indole reagent
inner wall of the tube.

Red violet coloured ring

INTERPRETATION
POSITIVE TEST:
Red violet
colour at the
interface
between the
broth and the
reagent.

 NEGATIVE TEST:
No colour
change. after the
addition of the
reagent.

INDOLE POSITIVE ORGANISMS WITH

EXCEPTIONS

Escherichia coli except Escherichia

vulneris.
Proteus except Proteus mirabilis.
Citrobacter except citrobacter frueundii.
Vibrio cholera except vibrio fluvialis.

INDOLE NEGATIVE ORGANISMS WITH

EXCEPTIONS

Klebsiella except Klebsiella oxytoca.

Edwardsiella except Edwardsiella
tarda.
Salmonella.
Pseudomonas.

CITRATE TEST
MEDIA AND REAGENTS
SIMMONS CITRATE MEDIUM

Ammonium
dihydrogen phosphate

Sodium chloride
Sodium citrate
Magnesium sulphate
Bromothymol blue

KOSERS MEDIUM

Sodium chloride
Magnesium sulphate
Ammonium
dihydrogen
Sodium citrate
Distilled water

CITRATE UTILIZATION TEST

PRINCIPLE :
citrate as sole source of carbon and energy

sodium citrate
Bromothymol blue
blue
(green)
ph 6.9

alkaline (production)
bromothymol
(blue)
ph 7.6

PROCEDURE
Citrate agar tube

Test organism inoculated on slant

surface
18-24 hrs
Blue colour appears on slant

INTERPRETATION
SIMMONS CITRATE
MEDIUM
Positive_____ Blue colour
Negative _____ Green
colour

 KOSERS MEDIUM
POSITIVE
Turbidity
NEGATIVE No
Turbidity

CITRATE POSITIVE ORGANISMS WITH

EXCEPTIONS

Klebsiella except Klebsiella oxytoca,

Klebsiella planticola, Klebsiella
ornithinolytica.
Salmonella except Salmonella typhi.
Proteus.
Pseudomonas.

CITRATE NEGATIVE ORGANISMS

Escherichia coli.
Salmonella typhi.
Edwardsiella.

UREASE TEST
MEDIA AND REAGENTS
STUARTS UREA BROTH CHRISTENSENS UREA
Yeast extract
AGAR
Potassium phosphate Peptone
Di- sodium phosphate Glucose
Hydrogen phosphate Sodium chloride
Urea
Di potassium hydrogen
Phenol red
phosphate
Distilled water
Urea
Phenol red
Distilled water

PRINCIPLE
Urea is a diamide of carbonic acid.
urease

Urea

PHENOL RED
(colourless)

ammonia +CO2

PHENOL RED
(pink red)

17

PROCEDURE
The broth medium is inoculated with a
loop ful of a culture of the test
organism.

The surface of the agar slant is

streaked with the organism.
18-24
hrs
Pink colour appears

INTERPRETATION
CHRISTENSENS AGAR :
Rapid urea splitters pink
throughout the medium.
Slow urea splitters red
colour initially in slant
only and then gradually
in entire
tube.

No urea hydrolysis
Negative yellow or straw
colour.

 STUARTS BROTH :
Positive pink colour
through out the
broth.

UREASE POSITIVE ORGANISMS WITH

EXCEPTIONS

Proteus
Klebsiella except Klebsiella
planticola.
Nocardia.
Cryptococcus.
Helicobacter.

UREASE NEGATIVE ORGANISMS WITH

EXCEPTIONS

Providencia except Providencia

alcalifaciens.
Escherichia coli.
Aeromonas.
Salmonella typhi.

TRIPLE SUGAR IRON (TSI)

MEDIA

Beef extract
Yeast extract
Nutritionally rich
Peptone
Sodium chloride
Glucose
1%
Lactose
10%
Sucrose
10%
Sodium thiosulphate
Phenol red
Indicator

TRIPLE SUGAR IRON AGAR

(TSI)
PRINCIPLE :
Attack carbohydrates incorporated in a basal
growth medium

with or without the production of gas

Phenol red
Alkaline (Yellow)
(6.8)

Acidic (Red)
(8.2)

PROCEDURE
With a sterlised straight inoculation
needle touch the top of a well
isolated colony.
First stabbing through the centre of
the medium to the bottom of the
medium and then streaking on the
surface of the slant.
Incubate the tube for 18-24 hrs.

PRINCIPLE
glucose is
fermented
small amount of
acid is produced.
Peptone is utilized
(aerobically)
forming an alkaline
by- product, so the
top of the slant
where oxygen is
present may turn
deeper red.

 If lactose and sucrose

are fermented large
amount of acid is
produced which turns
phenol red indicator
both butt and slant
will be yellow.
High level of acid
products produced
from abundant sugars
Alkaline neutralises

 H2S is produced
For hygrogen sulfide
production sodium
thiosulfate is the source of
sulfur atoms which is
colourless.
Iron salts (ferrous sulfate
and ferric ammonium
citrate)react with hydrogen
sulfide to produce an
insoluble black precipitate
(ferrous sulfide).

EXAMPLES OF TSI
A/A
Escherichia coli, klebsiella,
staphylococcus aureus.
A/A with H2S

Citrobacter freundii,
Proteus mirabilis,
Proteus vulgaris.

 K/K

Pseudomonas, Acinetobacter.

K/A
morganii.

Citrobacter koseri , Morganella

K/A with Gas

Enterobacter cloacae.

K/A With H2S Salmonella,

Citrobacter freundii,
Edwardsiella tarda