Biochemical test of bacteria> indole test

1. INDOLE TEST
• Principle
• To differentiate between members of enterobacteriaceae that can catalyse te essential amino acid
tryptophan into Ammonia, Indole and pyrovic acid using the enzyme tryptophanase.
• Differentiation based on detection of indole production
• Indole + Kovac‘s reagent→ red compound
• Kovac’s reagent: -Para-diamethylamino benzyldehyde
- Isoamyl alcohol
- Conc. Hydrochloric acid

2. How to Perform the Test
Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric
acid, dimethylaminobenzaldehyde, and amyl alcohol.
Procedure :
 Inoculate Tryptophan broth with organism by stab method using inoculating loop( 2tubes for positive
and negative test).
 Incubate tubes at 37℃ for 24 to 48 hrs
Tryptophan Indole +pyruvic acid + Ammonia
tryptophanase
 Add few drops of kovacs reagent and wait for few seconds.
Reading Results: Kovac’s reagent reacts with indole and creates a
red color at the top part of the test tube:
 The red ring on top confirms the presence on indole.
 Yellow or no ring formed confirms there is no indole.

3. Examples of -ve & +ve Indole bacteria
Indole positive
 Flavobacterium sp.
 Haemophilus influenzae
 Klebsiella oxytoca
 Proteus sp.
 Pasteurella
pneumotropica
 Enterococcus faecalis
 Vibrio sp.
 Lactobacillus reuteri
 Plesiomonas shigelloides
 Aeromonas hydrophila
 Aeromonas punctata
 Bacillus alvei
Indole negative
 Actinobacillus spp.
 Aeromonas salmonicida
 Alcaligenes sp.
 most Bacillus sp.
 Bordetella sp.
 Enterobacter sp.
 most Haemophilus sp.
 most Klebsiella sp.
 Neisseria sp.
 Mannheimia haemolytica
 Pasteurella ureae
 Proteus mirabilis
 Pseudomonas sp.
 Salmonella sp.
 Serratia sp.
 Yersinia sp.