31.10: Indole Broth Test
- Page ID
- 165420
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The indole test is used to determine whether a bacterial organism can break down the amino acid tryptophan to produce indole. This biochemical reaction helps identify certain Gram-negative bacteria, especially within the Enterobacteriaceae family. It is commonly used in clinical and environmental microbiology to help differentiate between closely related species, including those responsible for typhoid fever (Salmonella) and bacterial dysentery (Shigella).
The test medium, tryptone broth, contains tryptophan as a substrate. Bacteria that produce the enzyme tryptophanase can hydrolyze tryptophan into three products: indole, pyruvate, and ammonia. Indole, if present, can be detected by adding Kovac’s reagent, which contains para-dimethylaminobenzaldehyde, amyl alcohol, and hydrochloric acid.
When indole is present, it reacts with the aldehyde component of the reagent in the acidic and alcohol-containing environment to form a rosindole dye. This compound is visible as a bright pink or fuchsia color in the top layer of the tube. Because indole is soluble in amyl alcohol and tryptophan is not, the color forms only in the upper alcohol layer. Any pink color observed in the aqueous lower layer is not considered a positive result and may be due to unreacted tryptophan.
Procedure
- Inoculate the tryptone broth with a loopful of your culture
- Incubate at 30°C for 48 hours
- After incubation, carefully add 10 drops of Kovac's reagent down the side of the tube
- Do not shake or swirl the tube to mix the contents
- Allow the tube to stand undisturbed for a few moments
- Observe the upper layer for a color change.
Interpretation of Results
A bright pink or fuchsia color in the top (amyl alcohol) layer indicates a positive result, confirming indole production
No color change, or a yellow layer at the top, indicates a negative result (no indole produced)
Color in the bottom aqueous layer is not used for interpretation.
