IMViC: Indole, Methyl red, Voges-Proskauer, Citrate

+ and H2S

These 4 IMViC tests (actually 6 tests if you include motility and H2S) constitute, perhaps, themost critical tests used for identification of bacteria after the gram stain. The test resultsfrom these 6 tests should carry more weight than almost any other tests, certainlyhigher priority than sugar results since they are more stable reactions.

The SIM agar deep is a multi-test medium comprising 3 tests: sulfide (H2S gas), indoleproduction, and motility. You have already tested for motility via the hanging drop slide andTTC, and here is an additional way to determine it (although not as good as TTC). Theamino acid tryptophan can be converted by the enzyme tryptophanase into an end productcalled indole. This chemical is identified when it reacts with Kovac's reagent. Since the TTCagar deep is a better way to run motility, we use SIM for indole and sulfide production.Sodium thiosulfate is in this medium and can be utilized by bacteria, with the production ofhydrogen sulfide. H2S is colorless. Ferric ammonium sulfite is in the medium to reaction withH2S, producing a black ferrous suflide.

The MRVP tests are run together in the same broth and then split into 2 tubes when ready tobe tested for the end products. The methyl red test determines the use of glucose, with thesubsequent production of acid, tested for by the pH indicator methyl red. The Voges-Proskauer test also determines glucose use, but for a different end product---not acid but aneutral product called acetoin (or acetylmethylcarbinol). The VP is really important foridentification of many bacteria, but it is a picky test, and MUST be done exactly right.

The citrate test identifies the use of citrate as a sole carbon source, since there are no othernutrients in this medium. The basic end products (carbonates, bicarbonates, and ammoniumhydroxide) will cause the brom thymol blue indicator in the medium to turn from forest greento royal blue.

OBJECTIVES:

Understand the importance of these 4 IMViC tests, plus the addition SIM tests..Determine the various reactions for these media: MRVP broth, SIM, Simmon citrate, andSIM.

MATERIALS NEEDED:1 SIM deep per unknown1 MRVP broth per unknown1 Simmon citrate agar slants per unknownTTC motility agar deep

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AFTER INCUBATION: reagents

Kovac's reagent Barritt's reagents A (alpha-naphthol) and B (KOH) methyl red reagent

THE PROCEDURES:

Indole test, H2S, and motility1. Inoculate bacterium into a tube of SIM media with a NEEDLE, all the way to the

bottom. YOU MAY PREFER TO USE THE TTC MOTILITY AGAR FOR MOTILITY.2. Incubate at 30 or 37 degrees C.3. AFTER INCUBATION: After reading both motility and H2S, add 6-8 drops of Kovac's

reagent.4. Record the results of your bacterial unknown in your journal.

Methyl red and Voges-Proskauer tests1. Inoculate bacterium into the MRVP broth.2. Incubate at 30 or 37 degrees C.3. AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: run

the MR test in the tube with 2/3, and the VP test in the open tube with 1/3. for methyl red: Add 6-8 drops of methyl red reagent. for Voges-Proskauer: Add 12 drops of Barritt's A, 4 drops of Barritt's B, mixgently for 1 minute to aerate the solution (the reaction requires O2). Let sit, undisturbed, for at least 20 minutes, maybe more.

4. Record the results of your bacterial unknown in your journal.

Citrate test1. Streak up the slant with the inoculum.2. Incubate at 30 or 37 degrees C.3. Record the results of your bacterial unknown in your journal.

INTERPRETATION:

SIM: Indole test, H2S, and motility

1. Look for H2S FIRST, a black precipitate within theagar deep.

2. Hold the tube up to the light and look at the stab lineto determine motility. If NONMOTILE, you will see theintact straight stab line. If MOTILE, the original stabline will diffuse out into the medium as the bacteriaspread throughout. In fact, you may not even be ableto see a stab line at all: it may be turbid throughoutthe medium. FOR THAT REASON, you might want tocompare the inoculated SIM with an uninoculated one.YOU MAY PREFER TO USE THE TTC MOTILITYAGAR FOR MOTILITY (see motility exercise for interpretation).

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3. Lastly, you will add the Kovac's reagent to detect the presence of indole produced.Within just a few seconds you can see the hot pink color of indole presence.

Methyl redWithin just a few seconds after adding methyl red reagent,you can see the red-pink color of acid presence fromglucose use.

Voges-Proskauer testsThe reagents MUST be added in the correct order, in the correct amounts, and thetube must sit undisturbed, and open to the air (no cap) for at least 30 minutes (45minutes is even better) as the light pink color intensifies atthe top of the tube (the reagents react with acetoin). DONOT shake the tube AFTER setting it down for the waitingperiod.

NOTE: Some bacteria will have different VP reactions at 30Cvs. 37C (noted in the identification books). You might want to runthis test at different temperatures.

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Citrate testThe alkaline by-products of citrate use will cause the pHindicator to turn royal blue.

BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS OF ALL OF THESE TESTSBY VISITING OTHER TABLES IN LAB.

QUESTIONS:

1. What are the reagents used in the indole test, methyl red test, and Voges-Proskauer test?

2. The purpose of the citrate in the Simmon citrate medium is to determine if the organismcan

used citrate as the sole ___________ source.

3. Why determine motility and H2S before adding Kovac's reagent?

4. What is so picky about the Voges-Proskaeur test?

5. The end product identified in the VP test is a neutral compound called ______.

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2421