INDOLE TEST

 

Aim

To test whether the given bacterial culture can produce indole from tryptophan.

 

Principle

The indole test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole. Tryptophanase is an enzyme produced by certain bacteria to degrade the tryptophan in the peptone and other components of media to indole, pyruvate, indole acetic acid and ammonia. Indole combines p-dimethylaminobenzaldehyde in  Kovac’s reagent and the solution turns from yellow to cherry red colour. Kovac’s reagent also contains  amyl alcohol which is  not water soluble, the red coloration will form in an oily layer at the top of the broth.

 


Materials required

·       24-48 hour old bacterial culture

·       Kovac’s reagent

·       Tubes containing 1% Tryptone broth(5 ml in each tube)

·       Dropper

·       Inoculating loop

·       Bunsen burner

Procedure

Ø  The organism was inoculated into broth that contains tryptophan.

 

Ø  The broth was then incubated at 37oC for 24-48 hours.

Ø  0.5 ml(5 drops) of Kovac’s reagent added to 24-48 hour old broth culture.

Ø  The tube was shaken and pink colour was observed in a ring around the interphase between the broth and alcohol reagent which rises to the surface.

Ø  If the test was negative, the remaining broth may reincubated for additional 24 hours.

 

Result

The culture shows indole positive result yields a cherry red ring in the lower portion of the alcohol phase layer above the medium. No cherry red ring formation in negative result.

Examples

Positive: E coli

Negative: Staphylococcus sp, Proteus sp, Peudomonas sp, Bacillus sp

 

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