The IMViC series is a group of four individual tests that are commonly used to identify bacterial species, especially coliforms. The capital letters
in ‘IMViC’ each stand for one of the four tests: I for Indole test, M for
Methyl Red test, V for Voges-Proskauer test, and C for Citrate test. The Indole test tests for whether or not a strain can convert tryptophan to indole. To perform this test, we use deeps of SIM agar. SIM stands for sulfide indole motility, and in addition to the indole test SIM tubes can also test for motility and for the reduction of sulfur to hydrogen sulfide. To inoculate a SIM tube, sterilize a needle and let it cool. Touch a pure colony of the species of interest on a plate, then stab the needle straight down into the SIM agar, and remove it by pulling it straight up along the stab path. Incubate for 24-48 hours. After incubation, to perform the indole test, use an ampule of
Kovac’s reagent, here labelled “Indole”. Break the glass capsule inside by
squeezing or bending the ampule, and use it to add 7-8 drops to the SIM tube. If a ring of red or pink liquid appears on the surface of the agar, the organism in the tube is indole positive. It was able to convert the tryptophan in the agar to indole, and this indole reacted with Kovac’s
reagent to produce a red color. If there is no color change, the test is negative. Dispose of the ampule and SIM tube appropriately.
The Methyl Red and Voges-Proskauer tests use a single tube of MRVP broth that is split after incubation to perform both tests separately. Sterilize a loop and let it cool. Use it to touch a pure colony of the strain of interest on a plate, and inoculate a fresh MRVP tube. Incubate for 2-5 days, then use a sterile pipette to transfer about one third of the liquid to a sterile, empty test tube. Use the original tube to perform the Methyl Red test, which tests for the strain’s ability to perform mixed acid fermentation. Add 5-6 drops of Methyl Red to the tube. Methyl Red is a pH indicator, and the top of the broth will turn red if mixed acid fermentation has reduced the pH in the tube, a positive result. If the top of the broth turns yellow or slightly orange, the result is negative. Use the second tube with the smaller volume to perform the Voges-Proskauer test, which tests for the 2,3-butanediol fermentation pathway. There are two Voges-Proskauer reagents for this test, and both come in ampules with a glass capsule inside that must be broken before dispensing, as in the indole test. Both the order and the proportions in which these reagents are added are important. First add 15 drops of reagent A, and then add 5 drops of reagent B. Cap the tube and use a vortex to mix the contents. Over the course of 10 to 15 minutes, vortex
several more times and observe the color in the tube. If the liquid turns red, the reagents have reacted with acetoin, which is an intermediate product of 2,3-butanediol fermentation. This is a positive result. If the liquid does not turn red, even after 15 to 20 minutes, the test is negative. Dispose of both tubes and the reagent ampules appropriately after checking the results.
The last test is the citrate test, which tests for whether or not a bacterial strain can use citrate as its sole carbon source. It uses slants of Simmon’s citrate agar. As you can see, the uninoculated agar is green.
To inoculate a Simmon’s citrate tube, sterilize a needle and let it
cool. Again, touch a pure colony of the strain of interest on a plate, then use the needle to streak the surface of the slant. When replacing the cap on the tube, screw it enough so that it does not fall off, but leave it loose. The test will not be accurate if the cap is on tight. Incubate the tube for 18 to 48 hours. If an organism is able to grow well in the tube, the agar will turn blue after incubation due to a rise in pH due to citrate catabolism. Because citrate is the sole carbon source in the medium, blue agar is a positive result for citrate utilization. If the agar remains green, the test is negative. Dispose of the tube appropriately after noting its color.
The IMViC series is a group of four individual tests that are commonly used to identify bacterial species, especially coliforms. The capital letters in ‘IMViC’ each stand for one of the four tests: I for Indole test, M for Methyl Red test, V for Voges-Proskauer test, and C for Citrate test.