The indole test is a common biochemical procedure used in microbiology to identify and differentiate various types of bacteria. It assesses a bacterium’s ability to produce indole, an organic compound, from the amino acid tryptophan. This test helps characterize bacterial species and identify unknown isolates in clinical and environmental settings.
Understanding the Indole Test Principle
The principle of the indole test centers on the activity of an enzyme called tryptophanase. Many bacteria possess this enzyme, which allows them to break down the amino acid tryptophan. Tryptophanase facilitates the hydrolysis and deamination of tryptophan. This enzymatic reaction yields three main products: indole, pyruvic acid, and ammonia.
Indole is the metabolic byproduct detected by the test. The enzyme tryptophanase requires pyridoxal phosphate as a coenzyme to carry out this reaction. The presence of indole in the bacterial culture medium indicates that the tested organism has the tryptophanase enzyme and can metabolize tryptophan.
Conducting the Indole Test
Performing the indole test involves culturing the bacterial sample in a tryptophan-rich medium, such as tryptone broth or peptone water. The bacterial culture is inoculated into the broth and then incubated, usually at 37°C, for 24 to 48 hours. After incubation, a reagent is added to the broth to detect any indole produced.
The most common reagent is Kovac’s reagent, containing para-dimethylaminobenzaldehyde (DMAB). An alternative, Ehrlich’s reagent, uses ethyl alcohol or 1-propyl alcohol instead of isoamyl alcohol. For a rapid spot test, the reagent can be applied directly to a bacterial colony on filter paper.
Deciphering Indole Test Outcomes
The interpretation of the indole test relies on a visible color change after adding the detection reagent. A positive indole test is indicated by a distinct color in the reagent layer. When Kovac’s reagent is used, a positive result appears as a cherry-red or pink-red ring at the top of the broth culture. This color forms because indole reacts with the para-dimethylaminobenzaldehyde in the reagent to produce a red compound.
Conversely, a negative indole test shows no color change, and the reagent layer remains yellow or light brown. This indicates the bacterium does not possess the tryptophanase enzyme and cannot produce indole from tryptophan. For example, Escherichia coli and Proteus vulgaris are indole-positive bacteria, yielding a red color. In contrast, Klebsiella pneumoniae and Proteus mirabilis are indole-negative, showing no color change.
Role of the Indole Test in Microbiology
The indole test is a fundamental tool in microbiology, primarily used for the identification and differentiation of various bacterial species. It is a component of the IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate) tests, which are useful for distinguishing among members of the family Enterobacteriaceae. These tests help microbiologists classify bacteria based on their metabolic capabilities.
The ability to produce indole provides a distinct characteristic that aids in presumptive identification in clinical and environmental laboratories. For instance, it differentiates indole-positive Escherichia coli from indole-negative Enterobacter and Klebsiella species, and Proteus vulgaris (indole positive) from Proteus mirabilis (indole negative). This differentiation is important for accurate diagnosis and effective treatment strategies in healthcare settings.