BIOCHEMICAL TESTS
These are tests done to differentiate or identify bacteria and most of them are based on the
biochemical reactions and enzymatic activity of the bacteria. There are several variations to some
of the tests but this varies from one laboratory to the other. It is necessary to prepare controls to
confirm that the test media and reagent have been prepared correctly.
Identification tests
1. Catalase test: this test helps to differentiate Staphylococci from Streptococci. It is also
used in differentiating Mycobacterium species.
PRINCIPLE
The enzyme catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen
and water.
METHOD
1. Emulsify few colonies of the organisms in distilled water on a clean slide
2. Add 1-2 drops of H2O2 and observe
3. Gas bubbles indicate a positive test that means catalase positive.
2. Citrate utilization: used in the identification of enterobacteria.
Principle
This is based on the ability of an organism to utilize citrate as its carbon source.
METHOD
1. Make a light suspension of the organism in saline.
2. Inoculate the Koser’s citrate medium with a straight wire or stab inoculate with
Simmon’s citrate agar.
3. A positive result is seen if the Koser’s medium becomes turbid. If Simmon’s citrate
agar turns blue, indicates positive. This means that citrate has been utilized.
3. Hydrogen sulphide (H2S)
This test is used in the identification of enterobacteria.
PRINCIPLE
, Hydrogen sulphide is produced when a sulphur containing amino acid is decomposed by the
enzyme action of the bacterium.
TEST
1. Inoculate test organism in peptone water
2. Insert a lead acetate paper strip in the neck of the tube
3. Stopper the tube well so that the strip is firmly wedged between the tube and the stopper.
4. Incubate at 370C for 24 hours.
5. Examine for blackening of the lower part of the strip of paper.
4. INDOLE TEST: this test is used as an aid in differentiation of Gram negative bacilli.
PRINCIPLE
Some bacteria are able to breakdown the amino acid tryptophan present in peptone water to
release indole.
Test
1. Grow the organism overnight in peptone water
2. Add a few drops of Kovac’s reagent
3. A red ring above the peptone water indicates positive indole production.
5. Methyl red (MR) test:it detects the production of acid during the fermentation of glucose.
PRINCIPLE
In the fermentation of glucose, different organisms produce end products at different pH some
bacteria with extended incubation time, are able to maintain the pH level of 4.5. This is
demonstrated by a change in the colour of Methyl red indicator which is added at the end of the
incubation period.
Test
1. Inoculate the medium from a young culture
These are tests done to differentiate or identify bacteria and most of them are based on the
biochemical reactions and enzymatic activity of the bacteria. There are several variations to some
of the tests but this varies from one laboratory to the other. It is necessary to prepare controls to
confirm that the test media and reagent have been prepared correctly.
Identification tests
1. Catalase test: this test helps to differentiate Staphylococci from Streptococci. It is also
used in differentiating Mycobacterium species.
PRINCIPLE
The enzyme catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen
and water.
METHOD
1. Emulsify few colonies of the organisms in distilled water on a clean slide
2. Add 1-2 drops of H2O2 and observe
3. Gas bubbles indicate a positive test that means catalase positive.
2. Citrate utilization: used in the identification of enterobacteria.
Principle
This is based on the ability of an organism to utilize citrate as its carbon source.
METHOD
1. Make a light suspension of the organism in saline.
2. Inoculate the Koser’s citrate medium with a straight wire or stab inoculate with
Simmon’s citrate agar.
3. A positive result is seen if the Koser’s medium becomes turbid. If Simmon’s citrate
agar turns blue, indicates positive. This means that citrate has been utilized.
3. Hydrogen sulphide (H2S)
This test is used in the identification of enterobacteria.
PRINCIPLE
, Hydrogen sulphide is produced when a sulphur containing amino acid is decomposed by the
enzyme action of the bacterium.
TEST
1. Inoculate test organism in peptone water
2. Insert a lead acetate paper strip in the neck of the tube
3. Stopper the tube well so that the strip is firmly wedged between the tube and the stopper.
4. Incubate at 370C for 24 hours.
5. Examine for blackening of the lower part of the strip of paper.
4. INDOLE TEST: this test is used as an aid in differentiation of Gram negative bacilli.
PRINCIPLE
Some bacteria are able to breakdown the amino acid tryptophan present in peptone water to
release indole.
Test
1. Grow the organism overnight in peptone water
2. Add a few drops of Kovac’s reagent
3. A red ring above the peptone water indicates positive indole production.
5. Methyl red (MR) test:it detects the production of acid during the fermentation of glucose.
PRINCIPLE
In the fermentation of glucose, different organisms produce end products at different pH some
bacteria with extended incubation time, are able to maintain the pH level of 4.5. This is
demonstrated by a change in the colour of Methyl red indicator which is added at the end of the
incubation period.
Test
1. Inoculate the medium from a young culture
