Fish Microbiology and Quality Assurance

1. Indole Test

Indole test is used to determine the ability of microorganisms to degrade the essential aminoacid tryptophan to form indole by the enzyme tryptophanase.
The test organism is grown in broth medium containing tryptophan and the presence of indole is detected by adding Kovac’s reagent which produces cherry red colour in the form of a ring on the top layer of the medium.

Materials required:
Test bacterial cultures (E. coli, Proteus sp, Pseudomonas etc)

• Trytone broth
  
• Kovac’s reagent

Procedure:
The test microorganisms are inoculated in to tryptone broth, incubated at 37 ⁰C for 24 h, and 3-4 drops of Kovac’s reagent (para-dimethyl aminobenzaldehyde prepared in amyl alcohol) and observed for colour development. The positive reaction is characterized by the development of a pink coloured ring that will float on the surface of the broth.

Tryptone broth

Tryptone : 10.0 g
Sodium chloride : 5.0 g
Distilled water : 1000.0 ml
pH : 7.1 ± 0.1

Disolve the ingredients by boiling , distribute to tubes in 10 ml volume and sterilize at 121⁰Cfor 15 minutes

Kovac’s reagent

Para-dimethyl aminobenzaldehyde (DMAB) :5.0 g
Iso-amyl alcohol :75.0 ml
Concentrated HCl acid : 25.0 ml

Dissolve DMAB in iso-amyl alcohol (if it does not readily dissolve, warm it at 50 -60⁰C in water bath) and add slowly concentrated HCl acid by drop by drop. Store the reagent at 4⁰C.