Biochemical tests

Shafie Abdulkadir Hassan

 IMViC Test
• IMViC is a series of tests that are useful in
the identification of enteric bacteria
• Tests include:
1. I = Indole test
2. M = Methyl red test (MR)
3. Vi = Voges-Proskauer test (VP)
4. C = Citrate test
 Indole Medium
• Tryptone broth, contains extra tryptophan
• Trp. (tryptophan) can be utilized as a sole carbon and
energy source by some bacteria that produce an enzyme
tryptophanase
(tryptophanase)
• Tryptophan Indole + pyruvic acid + NH3
(C-source) (N-source)

• We test the presence of the bi-product Indole with a

chemical called Korvac’s reagent
• Korvac’s reagent reacts with Indole and turns solution
red
• The alcoholic layer concentrate the red color as a ring at
the top.
 Indole Procedure
• Procedure:
1. Inoculate tryptone broth with
young bacterial culture.

2. Incubate at 37 degrees for 48

hours
 Indole Results
• Results:

• Red ring at the top : + ve ;E.coli

• Coloreless ring at the top: -ve; e.g.
Staphylococcus aureuos,Klebsiella
•  
 Tryptone Broth after addition of Kovacs
(+) Indole test on left --- (-) Indole test on right
 Methyl red

• Strains of E. coli are mixed acid fermenters;

they degrade carbohydrates into acidic end
products such as: lactic acid, acetic acid,
succinic acid, and formic acid
 MR (continued)
– The Methyl-Red tests for acidic products
resulting from the ability of an organism to
produce and maintain stable acid end
products from glucose fermentation.
– These acidic products will drop the pH of the
medium to pH 4.5 or below
– Methyl red is a pH indicator will turn red if
mixed acid fermentation has occurred and
remain yellow in pH over 6.2.
 The principle:
•
Glucose Fermentation (acidic end products) MR+

•
• Hydrogenlyase

• Glucose 2pyrovate (Lactic acid, CO2+H2 (MR+)

• formic acid,
• acetic acid)
Materials for methyl red test:

• Media(MR-VP media ): Peptone ∕

Glucose ∕ K2HPO4 broth

• Reagents: MR reagent: methyl

red solution
The Procedure:

• Incubate 5 ml of the broth with young

bacterial culture for 24- 48 hrs at
37°C.
• Add methyl red reagent into the
broth .
The results:
• Red color solution : + ve ; e.g. E.coli ∕
faint yellow= -ve; Klebsiella
The Voges-Proskauer test= V
test:
The principle:

• This test is used to detect microorganism

that ferment glucose via the butanediol
pathway produce acetoin as an
intermediate which can be further reduced
to 2,3-butanediol.
 The principle:

• Glucose 2pyrovate AcetoIn 2,3 butanediol

•

α -naphthol
• Acetoin + KOH Red color
Material VP test :

Media(MR-VP media ): Peptone ∕ Glucose ∕

K2HPO4 broth
Reagents: VP reagent: α –naphthol+ KOH
which is called (Barritt's reagent).

The procedure:
Incubate 5 ml of the broth with young bacterial
culture for 24- 48 hrs at 37°C.
Add α –naphthol to the broth then add KOH .
The results:

• Red color broth = +ve, e.g. Klebsiella∕

• pale yellow= -ve ,e.g. E.coli
 Citrate Test

Simmon’s Citrate agar is used

to determine an organism’s
ability to use citrate as a sole
carbon source.
 Citrate Test (continued)
• The pH change is induced by CO2, which is
given off as a bi-product of citrate utilization.
When it reacts with Na and H2O in the agar it
raises the pH above 7.6
• The organism must contain the enzyme citrase
to degrade citrate
• EX:
(citrase)
• Citrate CO2 + Na HCO + H2O

(blue color change)

The materials:
Media : Simmon Citrate slant agar.
The Procedure:
• The slant is streaked only with the
required bacteria

• Incubate at 37 degrees for 48 hrs.

 Citrate Results

1. Green color is (-) for Citrase: eg. E.coli

2. Blue color is (+) for Citrase: e.g. Klebsiella
 Urease test
This test is used to detect if the

m.o. possess the enzyme

Urease .
The Principle:
•
•   Urease
Urea+ H2O Ammonia + CO2
• pH 6.8 pH8.1
• Dark Orange Dark pink
(phenol red is a pH indicator)
The materials
• Media: Urea agar base + Urea
solution.
• The indicator: phenol red( pH
indicator ) included within the
media.
The Procedure

• Inculate the urea agar with the

required m.o.

• Incubate at 37°C for 24-48 hrs.

The results:
Pink color = +ve; e.g.Proteus;
orange color = -ve ,e.g. E.coli
 Catalase Test
• This test used to detect the ability
of m.o. ( aerobic bacteria)
produce the enzyme catalase that
degrade H2O2.
 The Principle:
catalase
• H2O2 H2O+ O2 (gas)
• The Procedure:
1)Place a drop of distal water on a clean dry slide
.
2)Transfere a small amount of the bacterial
growth to the slide using a wooden stick.
3)2-3 drops of 3%H2O2 solution is added to the
bacterial growth and mix slightly .
 The results:
• Vigorous bubbling (due to O2 release )= + ve
Staph.

• No bubbling = -ve result (no catalase is

produced) e.g. E.coli