IMViC Tests (Indole, Methyl red, Voges-Proskauer,

Citrate) + and H2S

These 4 IMViC tests (actually 6 tests if you include motility and H2S) constitute, perhaps, the
most critical tests used for identification of bacteria after the gram stain. The test results
from these 6 tests should carry more weight than almost any other tests, certainly
higher 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), indole
production, and motility. You have already tested for motility via the hanging drop slide and
TTC, and here is an additional way to determine it (although not as good as TTC). The
amino acid tryptophan can be converted by the enzyme tryptophanase into an end product
called indole. This chemical is identified when it reacts with Kovac's reagent. Since the TTC
agar 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 of
hydrogen sulfide. H2S is colorless. Ferric ammonium sulfite is in the medium to reaction with
H2S, producing a black ferrous suflide.

The MRVP tests are run together in the same broth and then split into 2 tubes when ready to
be tested for the end products. The methyl red test determines the use of glucose, with the
subsequent 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 a
neutral product called acetoin (or acetylmethylcarbinol). The VP is really important for
identification 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 other
nutrients in this medium. The basic end products (carbonates, bicarbonates, and ammonium
hydroxide) will cause the brom thymol blue indicator in the medium to turn from forest green
to royal blue.

OBJECTIVES:

Understand the importance of these 4 IMViC tests, plus the addition SIM tests..
Determine the various reactions for these media:.

MATERIALS NEEDED:
1 SIM deep (may use TTC motility agar deep rather than SIM for motility)
1 MRVP broth
1 Simmon citrate agar slants

AFTER INCUBATION: reagents

 Kovac's reagent
 Barritt's reagents A (alpha-naphthol) and B (KOH)
 methyl red reagent
THE INOCULATION PROCEDURES:

Indole test, H2S, and motility

1. 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
because it is easier to interpret..
2. Incubate at 37 degrees C.

Methyl red and Voges-Proskauer tests

1. Inoculate bacterium into the MRVP broth.
2. Incubate at 37 degrees C.

Citrate test
1. Streak up the slant with the inoculum.
2. Incubate at 37 degrees C.

INTERPRETATION: AFTER INCUBATION

SIM: Indole test, H2S, and motility

1. Look for H2S FIRST, a black precipitate within the agar

deep.
2. For SIM: Hold the tube up to the light and look at the stab
line to determine motility. If NONMOTILE, you will see the
intact straight stab line. If MOTILE, the original stab line
will diffuse out into the medium as the bacteria spread
throughout. In fact, you may not even be able to see a
stab line at all: it may be turbid throughout the medium.
FOR THAT REASON, you might want to compare the
inoculated SIM with an uninoculated one.
3. After reading both motility and H2S, add 6-8 drops of
Kovac's reagent to detect the presence of indole produced from tryptophan
breakdown. Within just a few seconds you can see the hot pink color of indole
presence.

Method Red and Voges-Proskauer: AFTER INCUBATION:

Transfer 1 ml of the MRVP broth culture into a clean nonsterile tube: run the MR test
in the tube with broth left in the original tube, and the VP test in the open tube with 1
ml.
 for methyl red: Add 8 drops of methyl red reagent to the tube containing the
original broth. MIX well. This will be an immediate reaction, if positive.
 for Voges-Proskauer: To the tube containing 1 ml of the culture, add 12 drops of
Barritt's A and 4 drops of Barritt's B, mixing gently for 1 minute to aerate the
solution (the reaction requires O2, so no cap on it). Let sit, uncovered and
undisturbed, for 30 minutes.

2
Methyl red
Within just a few seconds after adding methyl red
reagent, you can see the red-pink color of acid
presence from glucose use.

Voges-Proskauer
The reagents MUST be added in the correct order, in the correct amounts, and the
tube must sit undisturbed, and open to the air (no cap) for at least 20 minutes as
the light pink color intensifies at the top of the tube (the
reagents react with acetoin). DO NOT shake the tube
AFTER setting it down for the waiting period.

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

Citrate test
The alkaline by-products of citrate use will cause the pH
indicator to turn royal blue.

BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS

OF ALL OF THESE TESTS BY VISITING OTHER TABLES IN
LAB.

3
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 organism
can use 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 ______.

LAB MANUAL: TABLE OF CONTENTS

Fall 2018 – Jackie Reynolds, Richland College, BIOL 2421