---
title: Indole production test
description: "## 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…"
url: "https://vumicro.com/docs/indole-production-test/"
updated: "2022-11-01"
category: Reference Library / Biochemical tests
---

# Indole production test

## 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](https://vumicro.com/docs/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](https://vumicro.com/docs/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](https://vumicro.com/docs/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](https://vumicro.com/docs/kovacs-reagent/) 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](https://vumicro.com/docs/tryptone-broth/) medium.  

2. Complete the process of a [tube-to-tube aseptic transfer](https://vumicro.com/docs/perform-aseptic-transfers-tube-to-tube/) 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](https://vumicro.com/docs/use-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](https://vumicro.com/docs/kovacs-reagent/).  

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](https://vumicro.com/docs/record-test-results/).

[Uninoculated Tryptone Broth:](https://vumicro.com/wp-content/uploads/2022/07/ambybnnnnn-2018_07_01-17_21_39-UTC.jpg)

[Positive Indole test:](https://vumicro.com/wp-content/uploads/2022/07/amrybynnnn-2018_07_01-17_21_39-UTC.jpg)

[Negative Indole test](https://vumicro.com/wp-content/uploads/2022/07/amyybynnnn-2018_07_01-17_21_39-UTC.jpg):

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