Bacteria that produce the enzyme tryptophanase can convert the amino acid tryptophan to by-products that include indole. This test is among a suite of tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate) that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.
When indole is combined with Kovac's Reagent (which contains hydrochloric acid and dimethylaminobenzaldehyde in amyl alcohol), the solution turns from yellow to cherry red. Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the top of the broth.
+ = Positive (Cherry red coloration in oil layer on top of broth)
- = Negative (Not red in oil layer on top of broth).
V = Variable
1. Obtain two TRYPTONE broths from the back shelf.
2. Inoculate one broth using aseptic technique. Leave the other broth uninoculated (this will be a control).
3. Incubate at appropriate temperature (whatever temperature your organism grows well at). Incubate for 24 to 48 hours (do not exceed 48 hours for this test).
4. Obtain your broths from the incubator. Add a dropperful of Kovac's Reagent (in small brown dropper bottles on bottom shelf or in door of refrigerator) to each broth.
5. Allow the broths to sit for 15 minutes (shake gently every few minutes).
6. A red coloration in the top oil layer at any time within 15 minutes indicates a positive results. A negative result can only be concluded if there is no red color formation in the top oil layer after 15 minutes.