The indole test is a biochemical test used in microbiology to determine whether a bacterium can break down the amino acid tryptophan into indole. It’s one of the most common tests for identifying and differentiating bacteria, particularly among the large family of gut-related organisms known as Enterobacteriaceae. The test works by detecting indole, a byproduct that only certain bacteria can produce, using a chemical reagent that turns a visible color when indole is present.
How the Test Works
Some bacteria produce an enzyme called tryptophanase, which splits the amino acid tryptophan into three products: indole, pyruvate, and ammonia. This enzyme requires vitamin B6 (in the form of pyridoxal 5′-phosphate) as a helper molecule to function. Bacteria that carry this enzyme are called “indole-positive,” while those that lack it cannot perform this breakdown and are “indole-negative.”
The test exploits this difference. You grow the bacterium in a medium rich in tryptophan, give it time to break down the amino acid if it can, then add a chemical reagent that reacts specifically with indole. If indole is present, the reagent changes color. If it’s not, the reagent stays unchanged.
Media and Growth Conditions
The bacterium must be grown in a medium that provides plenty of tryptophan, since that’s the raw material the enzyme acts on. The standard choice is tryptone water (also called peptone water), which contains tryptone, an enzymatic digest of the milk protein casein. Tryptone is naturally high in tryptophan, making it an ideal substrate.
One important detail: the growth medium should not contain fermentable sugars like glucose. When bacteria ferment sugars, they produce acids that drop the pH below about 5.1 to 5.3. At that pH, the enzyme stops working properly, which can cause a false-negative result, making an indole-positive organism look negative. Media with adequate buffering capacity help avoid this problem.
Reagents Used
Two main reagents are used for the tube version of the test, and both rely on the same active chemical: p-dimethylaminobenzaldehyde, often abbreviated DMAB. This compound reacts with indole to form a red-colored complex.
Kovac’s reagent is the more common choice for routine aerobic testing. It dissolves 10 grams of DMAB in 150 ml of amyl (or isoamyl) alcohol and 50 ml of concentrated hydrochloric acid. The alcohol layer floats on top of the broth, concentrating any indole at the surface where the color change is easy to spot.
Ehrlich’s reagent uses the same active ingredient but in different proportions: 1 gram of DMAB dissolved in 95 ml of absolute ethyl alcohol and 20 ml of concentrated hydrochloric acid. Ehrlich’s reagent is generally preferred when testing anaerobic bacteria.
For rapid spot testing, a third reagent based on p-dimethylaminocinnamaldehyde (DMACA) is available. Studies comparing spot reagents have found DMACA to be the most sensitive of the options, producing results that are easiest to read.
Reading the Results
With Kovac’s or Ehrlich’s reagent in a tube test, a positive result appears as a cherry-red ring at the top of the broth within seconds of adding the reagent. A negative result leaves the reagent layer pale yellow with no color change.
The spot test works differently. A colony is smeared onto filter paper saturated with DMACA reagent. A positive reaction produces a blue to blue-green color within 10 seconds. A negative result stays colorless or turns a faint light pink. The color difference between the tube test (red) and the spot test (blue-green) reflects the different chemical complexes formed by each reagent.
Which Bacteria Are Indole-Positive?
The most clinically important indole-positive organism is Escherichia coli, the species most commonly tested with this method. Other notable indole producers include Proteus vulgaris, Providencia species, Klebsiella oxytoca, Vibrio species, Pasteurella multocida, and Haemophilus influenzae.
Common indole-negative organisms include Enterobacter species, Klebsiella pneumoniae, Salmonella, Serratia, Pseudomonas, and Proteus mirabilis.
Key Bacterial Pairs the Test Separates
The indole test is especially valuable for distinguishing closely related species that are otherwise hard to tell apart:
- Proteus mirabilis vs. other Proteus species: P. mirabilis is indole-negative, while most other Proteus species are positive.
- Klebsiella pneumoniae vs. Klebsiella oxytoca: K. pneumoniae is negative, K. oxytoca is positive.
- Citrobacter freundii vs. Citrobacter koseri: C. freundii is negative, C. koseri is positive.
These distinctions matter because species within the same genus can differ significantly in antibiotic resistance patterns and clinical behavior. A single, inexpensive biochemical test can point identification in the right direction within seconds.
Common Sources of Error
The most frequent cause of false negatives is using media that contain fermentable carbohydrates. The acid produced during fermentation inhibits tryptophanase activity, preventing indole production even in organisms that normally test positive. Using tryptone water or another sugar-free, tryptophan-rich medium avoids this problem.
Another potential issue is reading the spot test too late. The blue-green color from DMACA should appear within 10 seconds. Delayed color changes can be misleading and should not be interpreted as positive. For tube tests, using colonies grown on blood agar, trypticase soy agar, or MacConkey agar all work, though sensitivity can vary slightly depending on the plate medium and the reagent chosen.