BIOCHEMICAL

TESTING
FOR MICROORGANISM
IDENTIFICATION

Learning Outcome
At the end of this lecture student
should be able to:
1-know the principle of each
biochemical test used.
2-elaborate the function of
biochemical based on the reaction
occur during bacterial identification.
3- select the suitable biochemical test
toward the target organism.

WHAT IS
BIOCHEMICAL
TEST?

BIOCHEM TEST
Is a measuring the amount or activity
of a particular enzyme or protein in a
sample of blood or urine or other
tissue from the body.

LIST OF BIOCHEMICAL TEST:

Indole
Methyl-red (MR)
Voges-Proskauer (VP)
Citrate Utilization
Triple Sugar Iron (TSI)
Urease
Phenylalanine Deaminase (PD)
Motality

INDOLE TEST :

PURPOSE OF INDOLE TEST:

To determine the ability of
microorganisms to break
down the amino acid
tryptophan with the
release of indole.

PRINCIPLE OF INDOLE TEST:

Organism is cultured in a medium which
contains tryptophan. Indole production is
detected by Kovacs or Ehrlichs reagent
which contains 4-(p)-dimethylaminobenzaldehyde.
4-(p)-dimethylamino-benzaldehyde reacts
with the indole to produce a red coloured
compound.

PROCEDURE OF INDOLE
TEST:
The test is performed by
inoculating a colony of the
test organism in a medium
which contains
tryptophan.

Broth then
observed
if there is a
change in color

Incubate
over
night at
35-37 C

After overnight
incubation at 35-37
C, a drop of
Ehrlichs reagent or
Kovacs solution is
added to the
cultured organism.

RESULT OF INDOLE TEST:

Positive result (+)

red ring compound will

appear

Negative result (-)

remain with same

color

EXAMPLE:
POSITIVE
INDOLE:

NEGATIVE
INDOLE:

Shigella flexneri

Salmonella
paratyphi

Escherichia coli
Shigella sonnei
Proteus vulgaris
Vibrio
parahemolyticus

Klebsiella
pneumonia
Proteus mirabilis

METHYL-RED (MR) TEST:

PURPOSE OF METHYL-RED
(MR) TEST:
To determine the ability of
microorganisms to ferment
glucose by mixed acid
pathway

PRINCIPLE OF METHYL-RED
(MR) TEST:
Glucose is the major substrate
oxidized by all enteric organisms for
energy production
If glucose is fermented, it will
produced acidic as end product & it
can be detected by the methyl red
pH indicator.

PROCEDURE OF METHYLRED (MR) TEST:

The test is
performed by
inoculating a colony
of the test organism
in 0.5 ml of Tryptic
Soy Broth (TSB).

Broth then
observed
if there is a
change in color

Incubate
over
night at
35-37 C

After overnight incubation

at 35-37 C, a drop of
methyl red solution is
added to the cultured
organism.

RESULT OF MR TEST:
If the methyl red turns red, it is indicative of a positive reaction
If the indicator remain yellow, it is a negative reaction

EXAMPLE:
POSITIVE MR:

NEGATIVE MR:

Shigella flexneri

Vibrio cholerae

Escherichia coli

Klebsiella
pneumonia

Proteus vulgaris
Vibrio
parahemolyticus

Pseudomonas
aeruginosa
Enterobacter
aerogenes

voges-proskauer (vp) tEST:

PURPOSE OF vogesproskauer (vp) TEST:

To determine the ability of
microorganisms to ferment
glucose by the detection
of acetoin.

PRINCIPLE OF vogesproskauer (vp) TEST:

To detect the presence of acetylmethyl carbinol.
By adding alpha naphtol & 40%
NaOH, acetyl-methyl carbinol is
converted to diacetyl, which is red
complex

PROCEDURE OF vogesproskauer (vp) TEST:

The test is
performed by
inoculating a colony
of the test organism
in 0.5 ml of Tryptic
Soy Broth (TSB).

Broth then
observed
if there is a
change in color

Incubate
over
night at
35-37 C

After overnight incubation

at 35-37 C, a few drop of
creatine and 3 drops of
potassium hydroxide
solution is added to the
cultured organism.

RESULT OF voges-proskauer (vp)

TEST:
If the VP turns pink-red, it is indicative of a positive reaction
If the indicator remain yellow, it is a negative reaction

EXAMPLE:
POSITIVE VP:

NEGATIVE VP:

Vibrio cholerae

Shigella flexneri

Klebsiella
pneumonia

Escherichia coli
Proteus vulgaris

Pseudomonas
aeruginosa
Enterobacter
aerogenes

Vibrio
parahemolyticus

Citrate utilization tEST:

PURPOSE OF Citrate
utilization TEST:
To determined the ability of
organism to use citrate as
it only source of carbon for
their energy.

PRINCIPLE OF Citrate
utilization TEST:
If sodium citrate is utilized, alkaline
products (NaOH) are produced.
This is indicated by bromothymol blue,
which is blue colour at alkaline pH.
Bromothymol blue use to detect alkaline
Ferment citrate = alkaline product
alkaline product+ bromothymol
blue=blue color

PROCEDURE OF Citrate
utilization TEST:
Using a straight
wire, the top of
the well isolated
colony was
touch to transfer
the organism to
citrate tube agar
Citrate agar
then observed if
there is a
changing in
color on the
next day

Citrate agar was

inoculated by first
stabbing through the
center of the medium to
the bottom of the tube
and then was streaked
at the surface of the
agar slant

Incubate
over
night at
35-37 C

RESULT OF Citrate utilization TEST:

If the Citrate agar turns blue, it is indicative of a positive
reaction
If the citrate agar remain light green, it is a negative reaction

EXAMPLE:
POSITIVE CITRATE:

NEGATIVE CITRATE:

Klebsiella
pneumonia

Salmonella
paratyphi

Proteus mirabilis

Shigella flexneri

Pseudomonas
aeruginosa

Vibrio
parahemolyticus

Enterobacter
aerogenes

Escherichia coli

TRIPLE SUGAR IRON

(tsi) tEST:

PURPOSE OF TRIPLE SUGAR IRON

(tsi) TEST:

To determined the
fementation of glucose,
lactose or sucrose and also
H S production.
2

PRINCIPLE OF
TRIPLE SUGAR IRON (tsi) TEST:
A/A (yellow slunt/yellow butt)

Ferment all sugar, because this organism

produce enough acid to change color to
yellow at both slant and butt

K/A (red slant/yellow butt)

Ferment glucose only

K/K (red slant/red butt)

Non glucose fermentation, no reaction

H S production
2

Black pigmentation

Gas production

Agar crack, present of gas and bubble

PROCEDURE OF TRIPLE
SUGAR IRON (tsi) TEST:
By using a
straight wire,
the top of the
well isolated
colony was
touch to transfer
the organism to
TSI tube agar.
TSI agar then
observed if
there is a
changing in
color on the
next day

TSI agar was inoculated

by first stabbing through
the center of the
medium to the bottom
of the tube and then
was streaked at the
surface of the agar slant

Incubate
over
night at
35-37 C

RESULT OF TRIPLE SUGAR IRON

(tsi) TEST:

RESULT OF TRIPLE SUGAR IRON

(tsi) TEST:
A/A (yellow slunt/yellow butt)

Ferment all sugar, because this organism

produce enough acid to change color to
yellow at both slant and butt (ie: Klebsiella
pneumonia)

K/A (red slant/yellow butt)

Ferment glucose only (ie: Shigella flexneri)

H S production
2

Black pigmentation

Gas production

agar crack, present of gas and bubble (ie:

Proteus mirabilis)

UREASE tEST:

PURPOSE OF UREASE tEST :

To determined the present
of enzyme urease in
microorganism

PRINCIPLE OF
UREASE tEST :
Urea broth medium which contains
phenol red indicator, that will turns
pink at alkaline pH.
When urea is hydrolysed, it will
release ammonia.
This is an alkaline reaction, which
causes pink color to develop.

PROCEDURE OF UREASE
tEST :
The organism is
inoculated into
urea broth
medium

Urease result
then observed if
there is a
changing in
color on the
next day

Incubate
over
night at
35-37 C

RESULT OF UREASE tEST :

Positive (+) Change color

to pink
Negative (-) Remain at
same color

EXAMPLE:
POSITIVE UREASE:

NEGATIVE UREASE:

Klebsiella
pneumonia

Salmonella
paratyphi

Proteus mirabilis

Shigella flexneri

Proteus vulgaris

Vibrio
parahemolyticus
Escherichia coli

PHENYLALANINE
DEAMINASE (pd) Test:

PURPOSE OF PHENYLALANINE
DEAMINASE (pd) Test :

To determine the ability of

an microorganism to
produce phenylpyruvic
acid by oxidative
deamination.

PRINCIPLE OF
PHENYLALANINE DEAMINASE (pd) Test :

The test determines whether the

organisms possesss the enzyme that
deaminates phenylalanine to
phenylpyruvic acid.
Throught the addition of ferric
chloride reagents, will show a green
color as a positive result if
phenylpyruvic acid is present.

PROCEDURE OF PHENYLALANINE
DEAMINASE (pd) Test :
By using a
straight wire,
the top of the
well isolated
colony was
touch to transfer
the organism to
phenylalanine
tube agar.
3 drops of ferric
chloride reagent
is add into the
culture allowing
the reagent to
run down the
slope

Phenylalanine agar was

inoculated by first
stabbing through the
center of the medium to
the bottom of the tube
and then was streaked
at the surface of the
agar slant

Incubate
over
night
(24
hour) at

RESULT OF PHENYLALANINE
DEAMINASE (pd) Test :

Positive (+) Dark to deep

green
color on agar slope
Negative (-) Remain at
same color

EXAMPLE:
POSITIVE PD:

NEGATIVE PD:

Proteus mirabilis

Salmonella
paratyphi

Proteus vulgaris

Shigella flexneri
Vibrio
parahemolyticus
Escherichia coli

Motility Test:

PURPOSE OF motility Test :

To determine the motility of
an organism

PRINCIPLE OF
motility Test :
By inoculating organism in the
motility medium that contains a low
concentration of agar, motility can be
observed as diffuse growth spreading
out from the line of inoculation.

PROCEDURE OF motility Test

:
By using a
straight wire,
the top of the
well isolated
colony was
touch to transfer
the organism
tube agar.
Motility
activity in
the tube
agar is
observed on
the next day

The wire loop was stab

in straight position in
the tube agar

Incubate
over
night
(24
hour) at

RESULT OF motility :

Positive (+) Turbidity can

be seen
Negative (-) growth along
the line
of inoculation

EXAMPLE:
motile:

Non motile:

Escherichia coli

Klebsiella
pneumonia

Proteus mirabilis
Shigella flexneri
Proteus vulgaris
Shigella sonnei
Salmonella
paratyphi

conclusion
Biochemical test are often
required to identify
pathogen.

references