The indole test evaluates an organism’s capacity to break down the amino acid tryptophan and generate indole. It is a component of the IMViC protocols, a set of tests used to differentiate between members of the Enterobacteriaceae family.
What is indole test?
The indole test is a microbiological procedure used to assess bacteria’s capacity to synthesize indole from tryptophan. It is frequently used to distinguish between various bacterial species, notably those belonging to the Enterobacteriaceae family. A bacterial culture is subjected to the test by adding Kovac’s reagent; a good outcome is indicated by the appearance of a pink or red color. Based on the bacteria’s capacity for metabolism, this test aids in their identification and classification.
Principle of indole test?
The tryptophanase enzyme, which can break down the amino acid tryptophan into a number of compounds including indole, pyruvic acid, and ammonia, is produced by certain bacteria, and this enzyme serves as the basis for the indole test. Using a reagent similar to Kovac’s reagent, the chemical indole can be found.
A bacterial culture is cultivated in a medium containing tryptophan during the test. Tryptophan can be broken down into indole, pyruvic acid, and ammonia by the bacterium if it has the enzyme tryptophanase. The resultant indole will then interact with Kovac’s reagent to form a pink or red hue.
Purpose of indole test
The purpose of indole test are discussed below:
- Bacterial Identification: Identification and differentiation of bacteria belonging to the Enterobacteriaceae family, including Escherichia coli, Proteus species, and Klebsiella species, is one of the main goals of the indole test. The ability to generate indole varies between species, which helps with classification.
- Diagnostic Instrument: The test aids in the diagnosis of bacterial infections in clinical laboratories. Medical experts can limit down the possible bacterial origins of an infection by determining if indole synthesis is present or absent.
- Taxonomic Classification: The test helps to categorize bacteria according to their taxonomic group. Larger taxonomic groupings may group together bacteria that produce indole or have comparable metabolic characteristics.
- Quality Assurance: The indole test can be used as a quality control tool in a variety of industries, including assessing the quality of food and water. It aids in the detection of specific bacteria that could point to contamination or deterioration.
- Research and teaching In order to teach students about bacterial identification and metabolic pathways, the indole test is frequently used in educational settings. It also contributes to the study of the variety and traits of various bacterial species. Overall, the indole test is a crucial tool for medical practitioners and microbiologists to learn more about the metabolic capabilities of bacteria, assisting in the identification and diagnostic procedures.
Positive indole test
The appearance of a red or reddish-violet hue in the broth’s surface alcohol layer indicates a successful reaction. A unfavorable outcome is colored yellow. An orange color may appear as a result of a varied result. This is owing to the presence of skatole, another potential byproduct of tryptophan decomposition, also known as methyl indole or methylated indole.
If the result is positive (pink or red color development), the bacterium can make indole, which can be useful for classifying and distinguishing various bacterial species. The test aids in categorizing bacteria according to their metabolic properties since not all bacteria can make indole.
The indole test is a microbiological procedure used to assess bacteria’s capacity to synthesize indole from tryptophan. It is frequently used to distinguish between various bacterial species, notably those belonging to the Enterobacteriaceae family. A bacterial culture is subjected to the test by adding Kovac’s reagent; a good outcome is indicated by the appearance of a pink or red color. Based on the bacteria’s capacity for metabolism, this test aids in their identification and classification.
Negative indole test
When a bacterium is examined for the ability to synthesize indole from the amino acid tryptophan, the test results are negative. In other words, the tryptophanase enzyme, which is required to catalyze the breakdown of tryptophan into indole, pyruvic acid, and ammonia, is absent from the bacterium.
This finding is helpful for identifying bacteria because it helps exclude some species that are known to be indole-negative. For instance, certain Escherichia coli strains are indole-positive while others are indole-negative. The ability to generate indole is not shared by all bacteria belonging to the Enterobacteriaceae family.
Indole test reagents
Use of particular reagents, most notably Kovac’s reagent, is necessary for the indole test in order to identify the presence of indole generated by bacteria. Kovac’s reagent, which comprises compounds that react with indole to generate a color shift, is the main reagent used in the indole test. The reagent commonly consists of hydrochloric acid and p-dimethylaminobenzaldehyde dissolved in amyl alcohol or isoamyl alcohol.
Protocol of indole test
Materials Needed:
1. Bacterial culture to be tested
2. Tryptophan-containing medium (such as Tryptone broth)
3. Kovac’s reagent
4. Inoculating loop or needle
5. Test tubes
6. Incubator (if needed for bacterial growth)
Procedure:
- The organism first grows in a tryptophan-rich media (such as Tryptone broth) until it reaches the required growth phase to create a bacterial culture. Depending on the needs of the particular organism’s growth, incubate as necessary.
- The test tubes is mark off separately for each bacterial culture you’re analyzing.
- Aseptically a tiny amount of bacterial culture is transfer into the test tube with the corresponding label using a sterile inoculating loop or needle. To allow for the addition of reagents, make sure the tube is only half filled.
- Some Kovac’s reagent is drops directly over the liquid surface in the test tube. Avoid combining the reagent with the bacterial culture.
- The test tube is gently shake to combine the bacterial culture with Kovac’s reagent.
- The reagent layer at the test tube’s top takes on color is observed. If indole production is successful, it will be pink or red; if it is unsuccessful, it will be no color change or yellow.
- Keep a record of your findings.
References
https://universe84a.com/collection/imvic-test/
