IMViC Indole Test Method principle, procedure, Observation and interpretation

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Indole test is a  biochemical test performed  to detect the ability of bacteria to convert tryptophan into indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase". It is used as the part of the IMViC tests, a set of four useful reactions that are commonly designed for the differentiation of enterics (members of family Enterobacteriaceae). The IMViC series stand for the  following four tests

• Indole test
• Methyl red test
• Voges-Proskauer test
• Citrate test

Indole test can also be use to  differentiate micro-organism species such as 

Proteus Species:  Proteus mirabilis  (indole negative) differentiate from other  Proteus species (indole positive)

Klebsiella species: same as Klebssiella pneumoniae (indole negative) from Klebsiella oxytoca (indole positive)

Citrobacter species: and Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive)

Preparation of reagents 

1. Tryptone broth 

• Tryptone = 10gm
• Sodium chloride = 5gm

Procedure 

1. Dissolve the ingredients in 1 liter of sterile water. 
2. Dispense 4 ml per tube. 
3. Cap tube and autoclave at 121oC under 15 psi pressure for 15 minutes. 
4. Store the tubes in the refrigerator at 4 to 10°C.

  2. Kovac’s reagent 

• Amyl alcohol : 150ml
• p-dimethylaminobenzaldehyde (DMAB) : 10gm
• conc. HCl : 50ml

Procedure 

(Dissolve DMAB in the alcohol. Slowly add the acid to the aldehyde-alcohol mixture.)

The Principle of Indole Test

The ability of certain bacteria to split amino acid tryptophan into indole and pyruvic acid using the enzyme called tryptophanase makes them Indole Positive Bacteria. Indole can be detected with Kovac’s reagent or Ehrlich’s reagent. Indole reacts with the aldehyde in the reagent to give a red color which concentrates as a ring at the top of the test tube.

Procedure

• Bacterium/Bacteria to be tested is inoculated in typtone broth.
• Then Incubate overnight at  temperature 37°C.
• Add few drops of Kovac’s reagent. Ehrlich’s reagent is more sensitive in detecting indole production in anerobes and non-fermenters.
• Do not shake the tube and observe the result.

Observation  Interpretation

Indole Positive (A) Indole Negative (B)

Interpretation

• The Formation of red or pink colored ring at the top of the tube is an indication of indole Positive

• If there is No color change after addition of reagent,This  is an indole negative as seen above

Here is a List of Indole positive Mircro-organisms

• Most strains of E.coli
• P. vulgaris, 
• M. morganii 
•  Providenica 

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