Indole Test- Principle, Media, Procedure, Types, Results, Uses

Indole test is a biochemical test that determines whether a bacterial species can produce indole from the amino acid tryptophan. Indole is a nitrogen-containing compound that is formed by the action of an enzyme called tryptophanase, which breaks down tryptophan into indole, pyruvic acid, ammonia, and energy. Indole can be detected by adding a chemical reagent that reacts with it to produce a color change. The indole test is one of the four tests in the IMViC series, which is used to differentiate members of the Enterobacteriaceae family. The indole test is also useful for identifying other bacteria that can produce indole, such as Proteus, Morganella, and some anaerobes. The indole test can be performed in different ways, such as the spot test, the tube test, or the Ehrlich`s reagent test, depending on the type of medium and reagent used. The indole test is a simple and rapid method to characterize bacteria based on their metabolic capabilities.

The indole test has two main objectives:

• To detect the formation of indole from tryptophan by the enzymatic action of tryptophanase. This indicates the presence or absence of the enzyme in the bacterial species.
• To differentiate members of the family Enterobacteriaceae, especially E. coli from Enterobacter and Klebsiella. This helps in the identification and characterization of these bacteria based on their indole production.

The indole test is a simple and rapid test that can be performed as a part of the IMViC test or as a standalone test. It can also be used to distinguish other bacteria that produce indole from those that do not, such as Proteus, Morganella, Citrobacter, etc. The test can be performed on both aerobic and anaerobic bacteria using different reagents and media. The test is useful for clinical and environmental microbiology as it helps in the detection and diagnosis of various infections caused by indole-positive bacteria.

The indole test can be performed on various types of microorganisms, depending on the medium and the reagent used. The test is mainly used for Gram-negative rods, especially the members of the Enterobacteriaceae family. However, some Gram-positive and anaerobic bacteria can also be tested by this method. The following are some examples of microorganisms that can be tested by the indole test:

• Escherichia coli: This is the most common indole-positive organism that produces a strong positive reaction with both Kovac`s and Ehrlich`s reagents. E. coli is a facultative anaerobe that can grow on both broth and agar media containing tryptophan. E. coli is differentiated from other indole-negative coliforms such as Enterobacter and Klebsiella by this test.
• Proteus spp.: These are Gram-negative rods that are motile and produce hydrogen sulfide. Proteus mirabilis is indole-negative, whereas Proteus vulgaris and Proteus penneri are indole-positive. The indole test helps to distinguish these species from each other and from other Gram-negative rods.
• Morganella morganii: This is a Gram-negative rod that is motile and produces hydrogen sulfide. It is indole-positive and can be differentiated from Proteus mirabilis by this test. It can grow on both broth and agar media containing tryptophan.
• Klebsiella spp.: These are Gram-negative rods that are non-motile and produce a mucoid capsule. Klebsiella pneumoniae is indole-negative, whereas Klebsiella oxytoca is indole-positive. The indole test helps to differentiate these species from each other and from other coliforms such as E. coli and Enterobacter.
• Citrobacter spp.: These are Gram-negative rods that are motile and produce citrate as their sole carbon source. Citrobacter freundii is indole-negative, whereas Citrobacter koseri is indole-positive. The indole test helps to differentiate these species from each other and from other Enterobacteriaceae such as Salmonella and Shigella.
• Clostridium spp.: These are anaerobic Gram-positive rods that form endospores and produce toxins. Some species of Clostridium, such as Clostridium perfringens and Clostridium tetani, are indole-positive, whereas others, such as Clostridium difficile and Clostridium botulinum, are indole-negative. The indole test can be performed on anaerobic media with tryptophan using Ehrlich`s reagent.
• Bacteroides spp.: These are anaerobic Gram-negative rods that are part of the normal flora of the human colon. Some species of Bacteroides, such as Bacteroides fragilis and Bacteroides vulgatus, are indole-positive, whereas others, such as Bacteroides thetaiotaomicron and Bacteroides uniformis, are indole-negative. The indole test can be performed on anaerobic media with tryptophan using Ehrlich`s reagent.

The principle of the indole test is based on the ability of some bacteria to produce indole from the amino acid tryptophan by the action of an enzyme called tryptophanase. Tryptophanase catalyzes the cleavage of tryptophan into indole, pyruvic acid, ammonia, and energy. The indole produced can be detected by adding a reagent that reacts with it to form a colored compound.

There are different reagents that can be used to detect indole, such as Kovac`s reagent, Ehrlich`s reagent, or p-dimethylaminocinnamaldehyde (DMACA). These reagents contain an aldehyde group that reacts with indole to form a quinoidal compound. The color of the quinoidal compound depends on the type of reagent and the pH of the medium. For example, Kovac`s reagent produces a pink-red color, Ehrlich`s reagent produces a blue-green color, and DMACA produces a blue color.

The indole test can be performed in different ways, such as the spot test or the tube test. The spot test is a rapid method that involves rubbing a colony from a tryptophan-rich medium onto a filter paper moistened with the reagent. The tube test is a more sensitive method that involves inoculating a broth or an agar medium with tryptophan and adding the reagent after incubation. The presence or absence of indole is indicated by the color change at the interface between the medium and the reagent.

The indole test is useful for differentiating bacteria that can or cannot produce indole from tryptophan. It is especially important for identifying members of the family Enterobacteriaceae, such as Escherichia coli, Proteus, Morganella, etc. It is also used to distinguish some anaerobic bacteria and non-fermenters that have tryptophanase activity. The indole test is often combined with other biochemical tests to provide more information about the metabolic characteristics of bacteria.

Media Used

The media used for the indole test should contain tryptophan as a substrate for the enzyme tryptophanase. The media can be either liquid or solid, depending on the method of testing. Some of the commonly used media are:

• Tryptone broth: This is a simple liquid medium that contains tryptone as a source of tryptophan. It is used for the tube test with Kovac`s reagent or Ehrlich`s reagent.
• Motility-indole-ornithine (MIO) agar: This is a semi-solid medium that contains tryptone, glucose, ornithine, and bromocresol purple as a pH indicator. It is used for the tube test with Kovac`s reagent. It also tests for motility and ornithine decarboxylase activity of the bacteria.
• Sulfide-indole-motility (SIM) agar: This is a semi-solid medium that contains casein and animal tissue as sources of tryptophan, ferrous ammonium sulfate as a source of iron, and sodium thiosulfate as a source of sulfur. It is used for the tube test with Kovac`s reagent. It also tests for hydrogen sulfide production and motility of the bacteria.
• Heart infusion broth or anaerobic medium with tryptophan: These are liquid media that contain heart infusion or other nutrients as sources of tryptophan. They are used for the tube test with Ehrlich`s reagent for anaerobic bacteria or weak indole producers.

Reagents Used

The reagents used for the indole test are either aldehydes or ketones that react with indole to form colored compounds. The reagents can be either added directly to the culture medium or to a filter paper moistened with the culture extract. Some of the commonly used reagents are:

• Kovac`s reagent: This is a solution of p-dimethylaminobenzaldehyde (DMAB) in isoamyl alcohol and hydrochloric acid. It is used for the tube test with aerobic bacteria. It forms a pink-red coloration with indole at the interface of the reagent and the medium.
• Ehrlich`s reagent: This is a solution of p-dimethylaminobenzaldehyde (DMAB) in ethyl alcohol and hydrochloric acid. It is used for the tube test with anaerobic bacteria or weak indole producers. It forms a red coloration with indole below the xylene layer.
• p-Dimethylaminocinnamaldehyde (DMACA): This is a solution of DMACA in hydrochloric acid. It is used for the spot test with aerobic or anaerobic bacteria. It forms a blue to blue-green coloration with indole on the filter paper.

Supplies Used

The supplies used for the indole test are:

• Sterile loop, swab, or stick for inoculation
• Filter paper (optional)

There are two methods to perform the indole test: a rapid spot test and a tube test. The spot test is a simple and quick method that can be done directly on a culture plate, while the tube test is more sensitive and requires incubation and reagent addition.

A. Rapid Spot Test

1. Take a piece of filter paper and moisten it with 5% p-dimethylaminobenzaldehyde or 1% p-dimethylaminocinnamaldehyde in 10% (v/v) concentrated HCl.
2. Pick a well-isolated colony from an 18-24-hour culture with an inoculating loop and rub it onto the moist filter paper.
3. Observe the paper for the appearance of color within 20 seconds.

B. Tube Test

1. Inoculate a broth medium or an agar medium in a tube with tryptophan with the colony taken from an 18-24-hour culture. Stab the agar medium with a needle or loop.
2. For the liquid medium, transfer a small portion of the inoculated broth to a separate tube.
3. Incubate the tubes at 37°C for 24 hours.
4. For Kovac’s reagent, add three drops of Kovac’s reagent down the side of the tube and observe the color change at the meniscus.
5. For Ehrlich’s reagent, add 0.5 ml of xylene to the tube and invert to mix well. Then add six drops of Ehrlich’s indole reagent down the side of the tube and observe the color below the xylene layer.

The indole test results are indicated by a change in color following a reaction with an added reagent. The reagent used depends on the type of bacteria being tested and the method of testing.

Spot Test

The spot test is a rapid method that detects indole directly from a colony growing on a medium rich in tryptophan. The indole combines with the p-dimethylaminocinnamaldehyde (DMACA) present on the filter paper at an acid pH to produce a blue to blue-green compound.

• A positive result is indicated by the development of a blue color within 20 seconds, which means that the bacteria can produce indole from tryptophan.
• A negative result is indicated by no color or the appearance of a slightly pink color on the filter paper, which means that the bacteria cannot produce indole from tryptophan.

Tube Test

The tube test is a more sensitive method that detects indole from a broth culture inoculated with bacteria. The indole combines with Kovac’s reagent or Ehrlich’s reagent, depending on the type of bacteria being tested, to form a colored compound.

• A positive result is indicated by the formation of a pink-red coloration (cherry red ring) in the reagent at the point of contact between the reagent and the medium, which means that the bacteria can produce indole from tryptophan.
• A negative result is indicated by the absence of color or the appearance of a slightly yellow color in the reagent, which means that the bacteria cannot produce indole from tryptophan.

Some bacteria may produce variable results, showing an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.

Some examples of indole-positive and indole-negative bacteria are:

• Indole-positive bacteria: Escherichia coli, Proteus vulgaris, Morganella morganii, Klebsiella oxytoca, Enterococcus faecalis, etc.
• Indole-negative bacteria: Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Serratia spp., etc.

The indole test is one of the four tests of the IMViC series, which tests for evidence of an enteric bacterium.

• The indole test is used to test an organism`s ability to utilize tryptophan and produce indole.
• The test is used to differentiate members of the Enterobacteriaceae family as a part of the IMViC test.
• The test also differentiates Proteus mirabilis from other Proteus species.
• This test differentiates indole-positive E. coli from indole-negative Enterobacter and Klebsiella.
• The test further differentiates K. pneumoniae (indole negative) from K. oxytoca (indole positive) and Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive).
• The test plays a central role in the characterization of coliforms, which are indicated by the various variations of the indole test and in combination with other biochemical tests.
• A variation of the test, called Ehrlich`s reagent (using ethyl alcohol instead of isoamyl alcohol), is used when the test has to be performed on non-fermenters and anaerobes.

• Detectable indole will diffuse to colonies within 5 mm of a 2- to 3-mm colony, giving false-positive results.
• Media that contain dyes (e.g., EMB, MAC) shouldn`t be used.
• The growth medium must contain an adequate amount of tryptophan. Do not use Mueller-Hinton agar for the test, because tryptophan is destroyed during the acid hydrolysis of casein.
• Only the cinnamaldehyde reagent can be used for spot testing of anaerobic microorganisms. It is a more sensitive reagent, but it is less stable.
• Do not use a plate with a nitrate disk to perform the indole test, as nitrate can interfere with the spot indole test by inducing false-negative results.
• If the rapid indole test is negative, the isolate could be positive in the more sensitive tube test. Extraction with xylene is the most sensitive test. Xylene substitutes are less sensitive.
• For fastidious Gram-negative rods, such as C. hominis, a heavy inoculum and extraction are necessary.

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