How to Identify Enterobacteria Using the Indole Test

The indole test is a very handy laboratory test used in the identification and isolation of enterobacteria. Testing for indole production is important in the identification of enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgretti, M. morganii, and Providencia species break down the amino acid tryptophan with the release of indole.

Principle Behind Indole Test

The test organism is cultured in a medium which contains tryptophan. Indole production is detected by Kovac’s reagent is recommended in preference to Ehrlich’s reagent for the detection of this substance from enterobacteria.

Ways of Performing an Indole Test

• As a single test using tryptone water and Kovac’s reagent.
• As a combined beta-glucuronidase-indole test using a Rosco PGUA/ Indole identification tablet and Kovac’s reagent. This is useful with identifying E. coli.
• As a combined lysine decarbonxylase-indole test using a Rosco LDC/Indole identification tablet. This is useful in helping to identify salmonellae and shigellae.

Detecting Indole Using Tryptone Water

1. Inoculate the test organism in a bijou bottle containing 3ml of sterile tryptone water.
2. Incubate at 35 – 37^oC for up to 48hrs.
3. Test for indole by adding 0.5ml of Kovac’s reagent. Shake gently. Examine for red color in the surface layer within 10 minutes.

Kovac’s indole reagent: This can be economically purchased as a ready-made reagent from Merck/BDH. A bottle of 100ml (53650-2R) costs about £13.0 or 30ml in a dropper bottle (53640-2X) costs £5.46 (1999). It has long shelf-life when stored at 4 – 8^oC.

Results

Red surface layer ……………… Positive indole test

No red surface layer ……………. Negative indole test

Detecting Indole Using Rosco PGUA/Indole Tablet

PGUA/Indole tablets (code 590 – 21) are available from ROSCO Diagnostica (see Appendix 11), priced about £7.50 for 25 tests (1999). The tablets have a long shelf life (3 – 4y).

1. Prepare a dense suspense of the test organism in 0.25ml physiological saline in a small tube.
2. Add a PGUA/Indole tablet and close the tube. Incubate at 35 – 37^oC for 3 – 4 hours (or overnight).
3. First read the beta-glucuronidase (PGUA) reaction:

Results

Yellow colour ……………… Positve PGUA test

Colourless …………………. Negative PGUA test

4. Add 3 drops of Kovac’s reagent (Rosco code 920-31 or other Kovac’s reagent) and shake.
5. Wait 3 minutes before reading the indole reaction. Examine the colour of the surface layer.

Results

Red surface layer ………………. Positive indole test

Yellow surface layer ……………. Negative indole test

Note: About 94% of E. coli strains are PGUA positive and 99% are indole positive.

Detecting Indole Using Rosco LDC/Indole Tablet

LDC/Indole tablets (code 584 – 21) are available from Rosco Diagnostica (see Appendix 11) priced about £7.50 for 25 tests (1999). The tables have long shelf-life (3 – 4y).

1. Prepare a dense suspension of the test organism on 0.25ml physiological saline in a small tube.
2. Add an LDC/Indole tablet. Add 3 drops of paraffin oil and close the tube.
3. Incubate at 35 – 37^oC for 3 – 4 hours (or overnight).
4. First read the lysine decarboxylase (LDC) reaction:

Results

Blue/violet colour ……………… Positive LDC test

Yellow, green or grey colour ….. Negative LDC test

5. Add 3 drops of Kovac’s reagent (Rosco code 920 – 31 or other Kovac’s reagent) and shake.
6. Wait 3 minutes before reading the indole reaction. Examine the colour of the surface layer.

Results

Red surface layer …………….. Positive indole test

Yellow surface layer ………….. Negative indole test