The indole test is a biochemical test demonstrating a bacteria’s ability to break down the amino acid tryptophan to indole. Microbiologists use the test to narrow down on Enterobacteria that can break down the amino acid.
The test is commonly used after gram staining to narrow down on specific gram-negative or gram-positive bacteria.
In clinical settings, the test is used to differentiate Klebsiella pneumoniae (negative) from Klebsiella oxytoca (positive), Citrobacter freundii (negative) from Citrobacter koseri (positive), and Proteus mirabilis (negative) from other Proteus species.
In the human body, Tryptophan is an essential amino acid for average growth in infants and for maintaining the body’s proteins, enzymes, muscles, and neurotransmitters.
Examples of tests that detect Indole production
There are three tests that can detect Indole production in bacteria. These are the Indole tube test, Indole spot test, and Ehrlich test.
Indole tube test
The Indole tube test is the most widely used test for indole. We shall focus on the test for this article. We call it the ‘tube test’ because it uses a centrifuge or test tube to hold the preparation.
Indole spot test
Unlike the tube test, the spot test uses a filter paper on which the Indole produced combines with p-Dimethylaminocinnamaldehyde (DMACA) at an acidic pH to create a blue-green compound.
Indole-positive bacteria, therefore, form a blue-green compound that is easily visible.
When the acid splits the protein through condensation, it forms the blue-green compound
Indole-producing bacteria release the enzyme tryptophanase, which aids in the breakdown of tryptophan into Indole, pyruvic acid, and ammonia.
Some strains of Aeromonas spp, Providencia, and Proteus vulgaris give false positive results.
Microbiologists have reported the test to help detect Indole producing bacteria of some anaerobic species and the family Enterobacteriaceae.
The spot test is, however, less sensitive than the tube test.
Ehrlich test
The Ehrlich test is a test variation that uses p-dimethylamino benzaldehyde to detect the amino acid tryptophan. The test work under the principle of electrophilic substitution whereby the substitution occurs at the benzyl pyrrole ring of tryptophan to yield a blue-violet condensation product under acidic conditions. The addition of NaNO2 enhances the condensation product.
The Erlich test is more sensitive than the other tests in detecting anaerobes and non-fermenters that can break down the amino acid tryptophan.
Indole test principle
The test uses the principle of deamination. In the presence of water, certain bacteria remove the amino group from the tryptophan molecule, and the enzyme tryptophanase catalyzes the process. The bacteria also make use of the Pyridoxal phosphate coenzyme.
The final products of the reaction are energy, ammonium, pyruvic acid, and indole.
When you add Kovak’s reagent to the solution, its active ingredient, para-dimethylaminobenzaldehyde, reacts with Indole to form a pink-red product.
Kovak’s is an essential ingredient that consists of isoamyl alcohol, para-dimethylaminobenzaldehyde (DMAB), and concentrated hydrochloric acid.
The red product is insoluble in water, so it floats to the top of the solution.
In the case that the bacteria do not break down tryptophan, there is no formation of Indole. The solution maintains its yellow color after the addition of the Kovak reagent.
| Ingredient | Quantity |
|---|---|
| Hydrochloric Acid, 37% | 250 ml |
| p-Dimethylaminobenzaldehyde | 50 g |
| Amyl Alcohol | 750 ml |
Test guidelines
Microbiology tests should be made under sterile conditions to prevent contamination.
Culture media provide the ideal conditions for the growth of microorganisms. A perfect culture media for Indole testing should be:
- Tested with known positives and negatives since microbiologists have shown peptones to vary with indole testing.
- It should not contain glucose. Bacteria break down glucose into acid by products that limit Indole production.
- Should not be Mueller Hilton agar since it destroys tryptophan during the acid hydrolysis of casein.
- Should not contain a dye due to possible carryover of the dye during inoculation, which interferes with the interpretation of color after adding the Kovak reagent. Medias with dyes include Macconkey and Eosin Methylene Blue (EMB) agar.
- Should not be used with a nitrate disk.
Before inoculation, select colonies of distinct morphologies at least 5 mm apart. The diffusion of Indole in the media can result in false positives.
Fastidious gram-negative rods like C hominis require a heavy inoculum.
Microbiologists have documented Indole positive colonies causing adjacent indole-negative colonies.
Test requirements: tryptophan broth, Kovak’s reagent, centrifuge tube, incubator, inoculating loop, and a pipette. And, of course, your test colony is prepared on the appropriate culture media.
Indole test procedure
- Put 4 ml of tryptophan broth into a centrifuge tube.
- Innoculate the test bacteria colony into the broth.
- Incubate the preparation for 24 hours at 37 ℃.
- Pipette 5ml of Kovak’s reagent into the preparation
- Hold the tube against a white background and observe for the formation of a ring and color change.
Indole-positive bacteria
Examples of Indole Positive bacteria are Vibrio sp, Enterococcus faecalis, Pasteurella pneumotropica, Pasteurella multocida, Plesiomonas shigelloides, Klebsiella oxytoca, Haemophilus influenzae, Flavobacterium sp, Edwardsiella sp, Bacillus alvei, Aeromonas punctata, and Aeromonas hydrophila.
Proteus species are indole positive except Proteus mirabilis and Proteus penneri.
Most strains of Escherichia coli, P vulgaris, Providencia sp, M morgani, and P rettgeri also break down tryptophan to indole.
Indole-negative bacteria
Examples of Indole Negative bacteria are Yersinia sp, Serratia sp, Salmonella sp, Pseudomonas sp, Pasteurella ureae, Neisseria sp, Pasteurella haemolytica, Lactobacillus spp, Enterobacter sp, Bordetella sp, Alcaligenes sp, Actinobacillus spp, and Aeromonas salmonicida.
Most Bacillus, Haemophillus, and Klebsiella species are indole negative.Proteus mirabilis and Proteus penneri are indole negative.
