ImVIC Test Sugar Fermentation Litmus milk Reactions Nitrate reaction Test Potassium Cyanide Test Cataise production Urease Test Oxidase Test

1. Biochemical test for
Identification of bacteria
Jagruti Marathe

2. 1. ImVIC Test
2. Sugar Fermentation
3. Litmus milk Reactions
4. Nitrate reaction Test
5. Potassium Cyanide Test
6. Cataise production
7. Urease Test
8. Oxidase Test

3. What is Biochemical Test
Presence or absence of certain enzymes, intermediatory
metabolites or end products often give valuable information in
identifying and classifying or differentiating microorganisms.
Some of the most important biochemical reactions are
summarized as:

4. IMVIC TEST
• Theory: The test is used to differentiate the gram
negative enteric bacteria of the family
Enterobacteriaceae on the basis of their biochemical
properties and enzymatic reactions in presence of a
specific substrate. The test involves four different types
of tests as (i) Indole production; (ii) Methyl- red (iii)
Voges-Praskauer and (iv) Citrate utilization. The name
IMVIC stands for the first letter of each test respectively.

5. IMVIC TEST
• 1. Indole Production Test
Principle: Some enteric bacteria (e.g. E.coli) produce indole (a
nitrogen containing compound) during the hydrolysis of
tryptophan. Tryptophan is an essential amino acid which is
oxidized by some bacteria with the help of an enzyme
tryptophanase with the formation of indole, pyruvic acid and
ammonia.
Tryptophan  indole + pyruvic acid + NH3
Tryptophanase

6. IMVIC TEST
• The indole production can be demonstrated by inoculating a
bacterium into tryptophan broth, the indole produced can be
detected by adding Kovac's reagent
(dimethylaminobenzaldehyde) which produces a cherry-red
colour band at the junction of medium and the reagent.
Indole +Kovac’s reagent ---> Rosindole(cherry red
compound)+H2O
HCL
(Butanol)

7. IMVIC TEST
• Medium used: Tryptone broth (1%) or Indole Tryptophan Broth Medium.
• Reagent used: Kovac’s reagent.
• Procedure :
• 1. Prepare 1% tryptone broth and dispense in 5 ml amounts in a sterile
test tubes.
• 2.Inoculate the broth tubes with the test organism.
• 3. Incubate the tubes at 37 °C for 24-48 hours.
• 4. After incubation, add 0.5 to 1 ml of Kovac’s reagent to each tube.
• 5. Shake the tubes gently for 10-15 minutes and allow to stand so that the
reagent comes at the top.
• 6. Examine the colour in the reagent layer.

8. IMVIC TEST
• Results: After 2 minutes, a cherry red or dark red colour band
appears at the junction of medium and the reagent which is
an indicative of a positive test for indole production.

9. IMVIC TEST
2. MR & VP Test:-
A) Methyl Red(MR) Test:- The test is used to detect the
production of acid during the fermentation of glucose.
By production of acid, pH of the medium falls and it is
maintained below 4.5. A red colour signifies positive
while yellow signifies a negative test.

10. IMVIC Test
B) Voges – Proskauer (VP) test: Many bacteria ferment
carbohydrates with the production of acetyl methyl
carbiol (acetoin). In the presence of alkali and
atmospheric oxygen, the small amount of acetyl methyl
carbiol presence in the medium is oxidised to diacetyl
which reacts with the peptone of the broth to give a
red colour.

11. IMVIC Test
Medium used: MR-VP Broth.
Reagents used: Barritt’s Reagent
VP reagent 1 (5% alpha-naphthol dissolved in absolute alcohol)
VP reagent II (40% potassium hydroxide solution).
Procedure:-
1. Prepare MR-VP broth and dispense in 5 ml amounts in a sterile
test tubes making two sets of tubes for two tests (total of 6
tubes, 2 each for the two tests with 2 control tubes).
2. Inoculate the tubes with the test organisms.

12. IMVIC Test
3. Incubate the tubes at 35°C for 48 hours.
4. Add 5 drops of methyl red indicator to the one set of tubes and
observe the change in colour for MR test.
5. Add 10 drops of VP reagent I and 2-3 drops of VP reagent II to the
other set of two tubes.
6. Gently shake the tubes for 30 seconds, remove the plugs/caps to
expose the media to
external oxygen.
7. Allow the tubes to stand for 15-30 minutes to complete the
reaction. Now observe the change in colour for VP test.

13. IMVIC Test
• Observation:-
1.In MR test, the methyl red indicator in the pH range of 4
will remain red (throughout the tube) which is an indicative
of a positive test, while turning of methyl red to yellow is a
negative test.
2.In VP test, the development of a pink-red colour is an
indicative of a positive VP test while as no change in colour
or faint brown colour (copper color) is an indicative of a
negative test.

14. IMVIC Test
+ve MR

15. Citrate Utilization Test
• Principle:- The test is mainly used to differentiate among
enteric bacteria on the basis of their ability to utilize citrate
as the sole source of carbon which depends upon the
presence of an enzyme citrase that breaks down the citrate to
oxaloacetic acid (OAA) and acetic acid(AA). These products are
further enzymatically converted into pyruvic acid and carbon
dioxide.

16. Contd…
The indicator used in the above procedure is bromothymol blue. It is
green when acidic (pH 6.8 or below) and blue when alkaline(pH 7.6 or
higher).
Medium used: Simmon’s Citrate Agar Medium.
Procedure:
1. Prepare Simmon’s citrate agar slants aseptically.
2. Inoculate the slants by stabbing to the base of slant. Thereafter
streak the surface (stab and-streak inoculation).
3. Incubate the slants at 37 °C for 48 hours.

17. Contd
Results: The growth of the organism at stabbed region and
change in colour of the medium from green to blue indicates
citrate utilization by a given organism and hence a positive test.
While as no growth and no colour change of the medium is
assigned as negative test

18. Contd
Before test After test

19. Sugar Fermentation Test
Sugar Fermentation: The ability of microorganisms to
ferment various sugar is tested by inoculation of the
test microorganisms in different sugar media containing
indicators. Acid production is shown by change in the
colour of the medium to pink or red and the gas. If
produced, gets collected in Durham’s tube.

20. Litmus Milk Test
Litmus milk reactions: The major substrates capable of
transformation are milk sugar lactose and the milk
proteins casein, lacto- albumin and lactoglobulin.
Litmus milk forms an excellent differential medium in
which microorganisms can metabolise milk substrates
depending on their enzymatic complement such as
lactose fermentation, gas production, litmus reduction,
curd formation, proteolysis and alkaline reaction.

21. Nitrate Reaction Test
Nitrate reduction test: This test detects the production of enzyme nitrate
reduction which reduces nitrate to nitrite. Inoculate test microorganisms in
5ml medium containing potassium nitrate, peptone and distilled water.
Incubate it at 37 for 96 hours. Then add 0.1ml test reagent which consists of
equal volumes of sulphanilic acid and a naphthylamine in 5N acetic mixed just
before use. A red colour development within a few minutes indicates the
presence of nitrite.

22. Potassium Cyanide Test
Potassium cyanide test: This test is used to detect the ability of
microorganisms to grow in the presence of potassium cyanide.
Inoculate buffered people peptone water medium containing 1
in 13,000 concentration of potassium cyanide with test
microorganisms. Incubate at 37 for 24-48 hours and detect the
development of turbidity in the medium.

23. Cataise Production
• Catalase production: Add a nutrient agar. Alternatively, pick
up a few colonies of the test microorganisms with platinum
loop from nutrient agar plate and dip it in a drop of 10%
hydrogen on a clean glass slide. The production of gas
bubbles from the culture indicates a positive reaction.

24. Urease Test
Urease test: This test detect the ability of a microorganisms to
produce urease enzyme. The test microorganisms is inoculated
on the entire slope of Christensen’s medium which contains urea
and a phenol red indicator. It is incubated at 37 and examined
after 4 hours and after overnight incubation. Development of
purple-pink colour indicates production of urease. The latter in
the presence of water convert urea into ammonia and
carbondioxide. Ammonia makes the medium alkaline and phenol
red indicator changes to purple-red in colour.

25. Oxidase Test
• Oxidase test: This depends on the presence of oxidases (in bactria) that
catayse the oxidation of reduce tetramethyl-p-phenylenediamine
dihydrochloride (oxidase reagent) by molecular oxygen. Add a drp of
freshly prepared 1% solution of oxidase reagent on a piece of filter paper.
Then rub a few colonies of test microorganism on it. If it is oxidase
positive, it will produce a deep purple colour within 10 seconds.
Alternatively, pour oxidase reagent over the colonies of the test
microorganisms on the culture plate. The colonies of oxidase positive
microorganisms repidly develop a deep purple colour.

26. THANK YOU