Indole Test – Principle, Reagents, Procedure, Result Interpretation And Limitations

Life is everywhere, and even in the most brutal environments, you can be sure to find a host of living creatures that have adapted to survive.

Indole Test - Principle, Reagents, Procedure, Result Interpretation And Limitations

It is a well-known, yet commonly overlooked fact that there are whole worlds of living creatures that we can’t even see. 

Although we can’t see them without the use of a microscope, scientists have slowly been able to learn more and more about bacteria over the years.

One of the ways that they study these microscopic creatures is through chemical tests that allow us to differentiate between various species. 

One such test is the indole production test. This experiment determines the ability of a microorganism to break down the amino acid tryptophan into a compound called indole.

Scientists use this procedure to identify enterobacteriaceae such as E.coli, P.Vulgaris and many, many other species. 

Over the following section, we will break down exactly what the indole test is, the materials it requires, as well as how to perform it and interpret the final result

What Is The Indole Test? 

The indole test is a procedure used in the identification and classification of certain species of enterobacteriaceae.

It works by monitoring the ability of a bacterial culture to break down the amino acid tryptophan.

Many species of enterobacteriaceae such as E.coli and Providencia will be able to make this amino acid undergo the process of deamination.

This is accomplished with the help of an enzyme called tryptophanase, which is capable of removing the amine group from tryptophan. 

The end result of this reaction is pyruvate, ammonium and a compound called indole.

As such, scientists can access a bacterial cultures’ ability to break down tryptophan by testing for the presence of indole in a medium.

To do this, a special solution, called Kovacs reagent, made from hydrochloric acid and  p-Dimethylaminobenzaldehyde dissolved in amyl alcohol is added to the medium. 

In the presence of indole, Kovacs reagent will cause the medium to change color from yellow to bright red.

Since the amyl alcohol used in this reagent is not soluble in water, the red part of the mixture will separate from the rest to form a hydrophobic ring that sits on top of the medium. 

Another way to test for the presence of indole is to do a spot test.

Here, the indole combines with p-Dimethylaminocinnamaldehyde (DMACA) at a low pH to cause a color change from blue to blue/green. 

Reagents Required For The Indole Production Test

There are two different procedures that you can use to test if bacteria can produce indole, including a spot test or using Kovac’s reagent.

An important distinction between these two methods is that the test using Kovac’s reagent requires a tryptophan broth and is testing for the indole itself. 

Meanwhile, the spot test is testing for the presence of tryptophanase, the enzyme responsible for allowing this reaction to occur.

While the tests are similar in nature, they both require different reagents in order to work. 

Below you will find a list of the chemicals required to make both Kovac’s reagent and an indole spot test reagent. 

Composition Of Kovac’s Reagent

  • 50.0gm of p-Dimethylaminobenzaldehyde
  • 250.0ml of Hydrochloric acid (37%). 
  • 750.0ml Amyl Alcohol

Composition Of Indole Spot Reagent 

  • 10.0gm of p-Dimethylaminocinnamaldehyde (DMACA)
  • 100.0ml of Hydrochloric acid (37%) 
  • 900.0ml of deionized water. 

Indole Test Procedure 

For this test, you will need a tryptophan broth along with a bacterial culture that is 18 to 24 hours old.

Once you have both of these components, you can follow the steps below to carry out the test. 

  1. Fill a sterilized test tube with 4ml of your tryptophan broth. 
  2. Using sterilized equipment, inoculate the tube with a sample to take from your culture. 
  3. Place the test tube in an incubator for 24 to 28 hrs. Set the temperature to 37 °C 
  4. Add 0.5 ml of Kovacs reagent to your test tube. 
  5. Observe the solution to see if a ring develops. 

Interpreting Your Results 

The amyl alcohol contained in Kovac’s reagent is hydrophobic, meaning it won’t dissolve in water.

As such, it will cause the solution to rise and form a ring that rests on top of the solution.

For a negative result, the ring will be the same color as the solution below it, which should be pale yellow. 

A successful result, however, will produce a ring with a bright, cherry red coloration, indicating the presence of indole. 

Escherichia coli is the bacteria used as a control for a positive result, with Proteus mirabilis being used as the negative control.

Haemophilus influenzae, Pasteurella multocida, and Enterococcus faecalis are all other species of bacteria that will produce a positive result when subjected to this test

Meanwhile, Pasteurella haemolytica, Aeromonas salmonicida and Salmonella sp. are all known to produce a negative result. 

Procedure For The Indole Spot Test

Procedure For The Indole Spot Test

Unlike the last test we looked at, you won’t need to subject your bacterial culture to any tryptophan to carry out this experiment.

This because the reagent used for this version of the test, is designed to react with the enzyme tryptophanase rather than indole.

For this procedure, you will still need to have a bacterial culture that is between 18 and 24 hours old. 

  1. Place multiple drops of the indole spot reagent we described above onto a piece of filter paper. Make sure to place enough drops that the paper becomes saturated. 
  2. With a plastic inoculating loop or similar implement, smear a sample from your bacterial culture onto the area of the paper that has been saturated with the reagent. 
  3. Wait up to 3 minutes for the results. 

Interpreting Your Results

If tryptophanase is present, then the area where you smeared the bacteria will turn blue/purple within three minutes.

If no color change occurs, then this enzyme is not present and the test is negative. 

You may see the area where you placed your smear turn slightly pink.

This is another sign of a negative test result, and should be treated the same as observing no color change at all. 

However, this is not the case if you notice the smear turning bright red or violet in color.

This is a positive test result that indicates the likely presence of Providencia alcalifaciens. Further tests may be needed to confirm this result. 

What Is The Indole Production Test Used For? 

There are several different situations where the indole production test can be used to differentiate between various species of bacteria.

Sometimes you can use this test to discover the specific species you are working with, although you may need to carry out other tests to get confirmation. 

For instance, Proteus mirabilis is indole negative, which sets it apart from all other Proteus species.

Another good example of how the indole test can be used is telling the difference between Klebsiella pneumoniae, which is indole negative, and Klebsiella oxytoca, which is positive.

What Are The Limitations Of These Tests?  

All scientific experiments have their limitations, and it is important to know what they are before carrying out any kind of test.

The indole tests can be useful for differentiating Gram negative and Gram positive species of bacteria.

However, further biochemical tests will be required to get a positive identification of exactly which species you are working with. 

You can use Kovacs reagent for a spot test instead of the usual spot test reagent, should you need to.

However, be warned that Kovacs reagent is less sensitive when used in this way and may take longer to produce a clear result. 

Overall, the tube test is more sensitive and therefore more accurate when it comes to testing for the presence of indole.

However, Kovac’s reagent will not work well with anaerobic bacteria, which means you will need to use the spot test when identifying anaerobes. 

You should never attempt these tests on mediums containing glucose, as this will lead to acid by-products that will skew your results.

Mueller Hinton’s agar and any medium containing dyes should also be avoided for similar reasons. 

Finally, colonies of indole positive bacteria may contaminate indole negative colonies through the diffusion of indole through the medium.

To avoid false positives caused by this phenomenon, take samples from different morphologies and ensure all colonies are separated by at least 5 mm. 


Indole testing is one of the many ways that scientists can tell the difference between various species of bacteria.

This allows them to better understand the world of microorganisms and identify new species when they appear.

The more we understand about bacteria, the more we can use them to develop new medicines, as well as other useful technology. 

Jennifer Dawkins