INDOLE TEST

INDOLE TEST

AIM

       To detect the ability of the microorganisms to degrade the amino acid Tryptophan and produce Indole.

PRINCIPLE   

       As far back as 1889, the Indole test was used to distinguish Escherichia coli (Indole positive) from Enterobacter aerogenes and Klebsiella sp. (Indole negative). The numerous variations of the Indole test alone and in combination with other Biochemical tests attest to the central role this test has played in the characterization of Gram negative coliforms. Tryptophan is an Essential amino acid that can undergo Deamination and Hydrolysis by bacteria that express Tryptophanase enzyme. The amino acid Tryptophan present in the Culture media is acted upon by the enzyme Tryptophanase produced by bacteria and converted into Indole, Pyruvic acid and Ammonia. Indole reacts with the Indicator Kovac’s reagent or Ehrlich’s reagent (which contains 4 (p)-dimethylamino benzaldehyde) and produce Pink to Cherry Red colour ring at the top. Ehrlich’s method may also be used to differentiate organisms under Anaerobic conditions.

Figure – 1: Conversion of Tryptophan into Indole, Pyruvate and Ammonium by Tryptophanase

Indole Positive Bacteria – Escherichia coli, Proteus vulgaris, Enterococcus faecalis, Klebsiella oxytoca, Flavobacterium sp., Haemophillus influenzae, Pasteurella multocida, Pasteurella pneumotropica, Edwardsiella sp., Bacillus alveli, Aeromonas hydrophila, Aeromonas punctata, Pleisomonas shigelloides and Vibrio cholerae.

Indole Negative Bacteria – Bacillus sp., Lactobacillus sp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella sp., Shigella sonnei, Pseudomonas sp., Serratia sp., Yersinia sp., Pasteurella haemolytica, Pasteurella ureae, Haemophillus sp., Enterobacter sp., Aeromonas  salmonicidia and Alcaligenes sp.

MATERIALS REQUIRED

1. Sterile Test tubes
2. Tryptone or Tryptophan broth
3. Inoculation loop
4. Bunsen burner
5. Incubator
6. Kovac’s or Ehrlich’s reagent.

PROCEDURE

a)     Take sterilized test tubes containing 5 ml of Tryptone or Tryptophan broth.

b)     Inoculate the tube with Tryptophan broth aseptically by taking bacterial culture.

c)     Incubate at 37 °C for overnight or 24 hours.

d)     Add 0.5 ml of Kovac’s reagent or Ehrlich’s reagent to the Bacteria incubated Tryptophan broth.

e)     Observe the presence or absence of Red colour ring.

OBSERVATION AND RESULTS

·       Positive Indole Test – Formation of Pink to Cherry red ring.

·       Negative Indole Test – No Cherry red ring.

(Left – Red ring positive; Right – No red ring negative)

Figure – 2: Indole test