| Technique | INDOLE 09/02/01 |
| Principle | Microbes break tryptophan down into
indole, pyruvic acid and ammonia in a single step by the
use of the enzyme tryptophanase. Bacteria deaminate
tryptophan to indole and skatole, the two volatile
substances that give the characteristic foul odour to
feces. Indole is volatile and can be detected with para dimethylamino benzaldehyde or with paper strips soaked with oxalic acid held near the mouth of the tube. Occasionaly all the indole volatilizes and only the paper strip is positive. Some isolates are indole negative at 37 C but are indole positive at 30 C. Some organisms may appear indole nedgative because the indol in subsequently broken down. Indole is produced from tryptophan. |
| Cautions | Since the indole is volatile and
degradable, old cultures cannot be used. In cultures that contain both protein and carbohydrate (e.g. l% tryptone and l% glucose broth) rapid acid production from the carbohydrate inhibits the formation or the activity of enzymes that attack protein. The protein sparing action of carbohydrates indicates this relationship. It is shown by the lack of indole formation from tryptone when fermentable carbohydrate is available. A pink colour in the aqueous phase does not indicate indole. |
| Method | Peptone water or nutrient broth is
inoculated and incubat ed for 48 hours. Then 0.5 ml of
Kovac reagent is added and the broth shaken. In a rapid
method a broth is inoculated heavily with an actively
growing culture from nutrient agar. The broth is
incubated at 37 C for 1 hour and then 4 drops of Kovac's
reagent is added and the mixture is shaken. . Inoculate a
tube of peptone broth which contains tryptophan and
incubate the culture for 48 hr at 37`C. Test for the
presence of indole by adding 4- 5 drops of Kovacs
reagent. The paradimethylaminobenzaldehyde reacts with
the indole to give a red colour. The Enhrlich indole test is a more sensitive modification. The indole is extracted by the addition of l ml of xylene to the 48 hr broth cultures. Shake well and allow the solvent to rise to the surface. Carefully pour 0.5 ml of Ehrlich's reagent (paradimethylaminobenzaldehyde) down the side of the tube. If indole is present a red ring will appear just below the solvent. |
| Results | Indole production is shown by the development of a pink colour in the kovac's reagent. |
| Positive control | Escherichia coli |
| Negative control | Enterbacter cloacae |
| Reagents | |
| Reference |