Enterobacteriaceae are the gram negative bacteria that are common in the intestinal flora of human and other animals. The IMViC tests are commonly used for the identification and differentiation of these bacteria including Klebsiella, Enterobacter, Proteus and E. Coli.
IMViC is an acronym that stands for Indole, Methyl Red, Voges-Proskauer and Citrate utilization.
Aim: To detect the production of indole from the degradation of the amino acid tryptophan.
Basic Principle: Oxidation of tryptophane to indole by the action of the enzyme tryptophanase is the basic principle of this test. The presence of indole is detected by Kovac’s reagent, which produces a cherry red color.
Test: Indole test is performed by inoculating a bacterium into tryptone broth which contains tryptophane. The members of enterobacteriaceae have the enzyme tryptophanase, that oxidize the tryptophane into indole, pyruvic acid and ammonia. In this test media, when Kovac’s reagent (dimethylaminobenzaldehyde) is added, a cherry red color is produced in the top layer of the solution.
Kovac’s Reagent:
Reactions:
Tryptophane → Indole + Pyruvic acid + Ammonia
Indole + Kovac’s reagent → Rosindole (Cherry red color) + H2O
The entire microorganisms do not show this reaction, therefore this test serves as a biochemical marker.
Aim: The test is used to check the ability of microorganisms for utilization of citrate.
Basic Principle: Some enteric organisms are able to use citrate as their sole carbon source for energy in the absence of glucose or lactose. The enzyme (citrase) present in the bacterial cell metabolizes citrate and produces some alkaline products that change the color of bromothymol blue indicator from green to blue. The change in color indicates a positive reaction.
Test: The citrate test is performed by inoculating microorganism into an organic synthetic media, “Simons Citrate broth” where sodium citrate is the only source of Carbon and energy. Bromothymol blue is used as an indicator. When the citric acid is metabolized, carbon dioxide is generated which combines with sodium and water to form sodium carbonate (alkaline), responsible for change in color from green to blue, indicating positive test.
Aim: To detect the production of acids from glucose.
Basic Principle: Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. The bacterium is inoculated into glucose phosphate broth. Due to the presence of bacteria, glucose is converted to acids which lower the pH and change the color of the indicator.
Test: The bacteria are inoculated in glucose phosphate broth and incubated at 37°C for 48 hours. If enterobacteriaceae are present, they produce sufficient acid to overcome the phosphate buffer. When 4–5 drops of Methyl red indicator are added, the color changes. The indicator turns red at acidic pH (<4.4), indicating positive result. (At neutral pH it is orange, at acidic pH it is red.)
Aim: To detect the production of acetyl methyl carbinol (acetoin) from glucose in bacterial broth culture.
Basic Principle: The test depends on the digestion of glucose to acetylmethylcarbinol. If glucose is being broken down, it reacts with alpha-naphthol (VP reagent #1) and potassium hydroxide (VP reagent #2) to form a red color.
Procedure: Bacterium is inoculated into glucose phosphate broth and incubated for at least 48 hours. 0.6 ml of alpha-naphthol is added to the test broth and shaken. 0.2 ml of 40% KOH is added and shaken. The tube is allowed to stand for 15 minutes. Appearance of red color is a positive test.
Reactions:
Glucose → Acetoin
Acetoin → Diacetyl
Diacetyl + Alpha-naphthol → Pink color
| Microbes | Indole Test | Methyl Red Test | Voges-Proskauer Test | Citrate Utilization Test |
|---|---|---|---|---|
| Escherichia coli | + | + | - | - |
| Enterobacter aerogenes | - | - | + | + |
| Klebsiella | + / - | - | + | + |
| Citrobacter freundii | - | + | - | + |
| Citrobacter diversus | + | + | - | + |
| Shigella species | Varies | + | - | - |
Table: Comparative table for test results and microbial strain