Identification of Gram Negative Bacilli—IMViC Introduction IMViC is a mnemonic to remember the four biochemical tests being used: Indole, Methyl red, Voges-Proskauer, and Citrate. These four tests help divide the Enterobacteriaceae into two major groups—the E. coli group and the Enterobacter-Klebsiella group. Principle Indole Organisms that posses the enzyme tryptophanase can break down the amino acid tryptophan to indole. When indole reacts with para-dimethyl-aminobenzaldehye (Kovac’s reagent) a pink-colored complex is produced. Tryptophan is plentiful in most media, but growth on blood agar or chocolate agar produces the best effects. Methyl Red Some organisms produce acid from the metabolism of glucose in a sufficient quantity to produce a pH of 4.4 in the media. These acids are not further metabolized and are said to be stable acids. At a pH of 4.4 or less the pH indicator methyl red is a bright cherry red. Voges-Proskauer Some organisms initially produce acid from glucose metabolism but further metabolize the acid produced to neutral end products, such as acetoin, and 2,3-butanediol. Initially the pH may drop to 4.4 but the neutral end products raise the pH so the methyl red test will be negative. Acetoin and 2,3 -butanediol under alkaline conditions will react with alpha-naphthol (1-naphthol) to produce a mahogany red color. Citrate Citrate contains carbon. If an organism can use citrate as its only source of carbon the citrate in the media will be metabolized. Bromthymol blue is incorporated into the media as an indicator. Under alkaline conditions this indicator turns from green to blue. The utilization of citrate in the media releases alkaline bicarbonate ions that cause the media pH to increase above 7.4.

Procedure Organisms used E. coli Klebsiella pneumoniae Enterobacter cloacae 1. Students work in groups to complete this exercise. 2. Obtain a DrySlide indole test card. Using a sterile loop transfer cells from

an agar plate or slant to the test area on the card. Observe for the development of a pink color within 30 seconds. Record your results in the table provided (Page 60) and discard the test card in the biohazard container.

3. Obtain an MRVP broth and using aseptic technique inoculate the tube. It

is important to inoculate this test heavily. Incubate for at least 24 hours at 35o C.

4. Obtain a citrate slant. Aseptically inoculate the slant and incubate for at

least 24 hours at 35o C. 5. After incubation of the MRVP broth obtain a spot plate and a sterile

dropper. Observe the MRVP for turbidity. If turbidity is not noted the test results are not reliable. The tube may be re-incubated until growth is evident. Place 3 drops of turbid broth into two of the wells on the spot plate. Methyl Red Test—To one well add 1-2 drops of methyl red reagent. Observe for an immediate cherry red color that indicates a positive test. Orange or yellow is considered negative. Record your results in the table provided (Page 60). Voges-Proskauer Test—To the remaining well add 2 drops of alpha-naphthol and 1 drop of potassium hydroxide (KOH). Observe for the development of a mahogany red color. The color development takes 20 minutes or longer. Be extremely careful with the KOH. It is caustic and may cause burns if it gets on your skin. The mahogany red color is considered positive. Record your results in the table provided (Page 60). Remove all marks from the MRVP tube and discard in the designated area. Clean the spot plate by covering the surface with disinfectant. Allow the disinfectant to sit for a few minutes, then rinse with water, wash and dry. Return the spot plates to their original location.

6. Observe the citrate for a change from green to blue. Blue is considered

positive for this test. Record your results in the table provided (Page 60). Remove all marks from the tube and discard in the designated area.

Organism Indole Methyl Red Voges- Proskauer

Citrate

E. coli

Enterobacter cloacae

Klebsiella pneumoniae

Review Questions What is the IMViC pattern for the E. coli group? What is the IMViC pattern for the Enterobacter-Klebsiella group? What is the difference between a reagent and an indicator? Complete the following table.

Test Substrate Reagent Indicator Positive Result

Indole Methyl Red

Voges- Proskauer

Citrate Is it possible for an organism to be Methyl red and Voges-Proskauer positive? Explain your answer. What might happen if the MRVP tests are read too soon?