About this test
What is the purpose of the test?
This test determines whether the microbe produces indole from the amino acid tryptophan.
How is the indole production determined?
If indole is produced, it will react with a chemical reagent added after incubation to produce a color change.
What medium is used?
There are two media that are used for this test: Sulfide-Indole-Motility (SIM) medium and Tryptone broth medium. This program uses the Tryptone broth medium. SIM is a nutrient medium which allows the detection of three different traits in bacteria: it contains sulfates to serve as the substrate for detecting sulfide (H2S) production; abundant tryptophan as a substrate for indole production; and its content of 0.5% agar limits bacterial swimming, thereby allowing detection of motility. Tryptone broth serves the same purpose as SIM as far as indole production is concerned, and the means for conducting the test and interpreting results remain the same as SIM.
How is the test performed?
An inoculum from a pure culture is transferred aseptically to a sterile tube of SIM or tryptone broth. An inoculating wire should be used for inoculating SIM and a loop or wire for tryptone broth . SIM should be stabbed all the way to the butt carefully to disturb the medium as little as possible. The inoculated tube is incubated at 35-37 C for 24 hours.
What reagents are added?
Five to ten drops of Kovac’s reagent are added to the tube. The reagent does not mix with water and forms a thin layer above the agar or broth. The reagent reacts with indole to produce a ring that is cherry red in color.
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select the tryptone broth medium.
2. Complete the process of a tube-to-tube aseptic transfer to inoculate the medium.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
4. Press the New Day button to move forward 24 hours.
Addition of Reagents
5. Incubate this test for only 24 hours.
6. Retrieve the incubated culture from the incubator.
7. This is a test that requires addition of reagents to bring about the change in appearance of a positive test. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, select Kovac’s.
8. Remove the cap from the tube.
9. Place the end of the dropper into the tube and add the reagent to the culture.
Determination of Test Results
10. Observe test result. For this test, addition of the appropriate reagent produces a cherry-red ring floating above the culture medium if the test is positive. If negative, the ring that forms is yellow or amber.
11. Record test result.