5 Jan 2012

INDOLE TEST


INDOLE TEST
Testing for indole production is important in the identification of enterobacteria. Most strains of E.Coli , P.Vulgaris, and P.Rettgeri, M.Morganii, and Providencia species break down the amino acid tryptophan with the release of indole.

PRINCIPLE:
The test organism is cultured in the medium which contains tryptophan. Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4 (p)-dimethylaminobenzaldehyde. This reacts with the indole to produce a red colored compound.
In the following method the use of the combined motility Indole Urea (MIU) is described. A Kovac’s reagent paper strip is inserted in the neck of the tube and indole production is indicated by a reddening of the strip. Indole is a volatile substance (easily vaporized). The tube must be well stoppered during incubation.
The indole test can also be carried out by culturing the organism in tryptone water or peptone water containing tryptophan, and detecting indole production  by adding Kovac’s or Ehrlich’s reagent  to an18-24 h culture.

REQUIREMENTS:

1. MOTILITY INDOLE UREA MEDIUM (MIU):

MIU semi solid medium is used to differentiate enterobacteria species by their motility, urease and indole reactions.
Formula and Preparation…………… To make 1 litre of MIU base medium
Tryptone………………………………………………………….30g
(or pancreatic digest of casein)
Potassium dihydrogen Phosphate……………………………       1g
Sodium Chloride………………………………………………….5g
Agar……………………………………………………………     4g
Phenol red 2.5g/l (0.25%)……………………………………… 2ml
Distilled water………………………………………………… 1 liter

                                I.            Mix the dry ingredients in the water and heat to 100oC to dissolve the chemicals (place the flask in a container of boiling water).
                              II.            Allow to cool to 50-55oC and then add the phenol red solution. Mix well.
                            III.            Dispense in 95ml amounts in screw-cap bottles. Sterilize by autoclaving (with caps           loosened) at 121oC for 15 minutes. When the medium has cooled, tighten the bottle            caps.


2. KOVAC’S REAGENT STRIPS

METHOD:
a.      Using a sterile straight wire, inoculate 5 ml of sterile MIU medium with a smooth colony            of the test organism.
b.      Place an indole paper strip in the neck of the MIU tube above the medium, and stopper the tube. Incubate at 35-37 oC overnight.
c.       Examine for indole production by looking for a reddening of the lower part of the strip.

RESULTS:

Reddening of strip…………………………Positive test (Indole produced)
No red color……………………………… .Negative test ( No Indole produced)


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