Table of Contents iii

Table of Contents

Table of Contents………………………………………………………………………………………………………………………iii Introduction .......................................................................................................................... …..v

What you need ........................................................................................................................ v

Terminology ........................................................................................................................... vi

Considerations……………………………………………………………………………………………………………………….vi Chapter One …………………………………………………………………………………………………………….....……….1 Performing the Tests……………………………………………………………………………………………………………..3 Preparation……………………………………………………………………………………………………………………………3 Indole Test Preparation…………………………………………………………………………………………………………3 MRVP Test Preparation…………………………………………………………………………………………………..…….3 Citrate Test……………………………………………………………………………………………………………………………3 Incubation…………………………………………………………………………………………………..………………………..4 Indole Test…………………………………………………………………………………………………………………………….4 MRVP Test…………………………………………………………………………………………………………………………….4 Chapter Two …………………………………………………………………………………………………………………………5 Interpreting the Results…………………………………………………………………………………………………………7 Indole Test…………………………………………………………………………………………………………………………….7 MR Test…………………………………………………………………………………………………………………………………7 VP Test………………………………………………………………………………………………………………………………….7 Determine the Species………………………………………………………………………………………………………….7 Chapter Three – Troubleshooting………………………………………………………………………………………….9 “I Can’t Identify My Species”……………………………………………………………………………………………….11 “The Species Matches Wither Everything Except the VP Test”…………………………………………….11 “The Bacteria Did Not Transfer From the Sample to the Tests”……………………………………........11 Index…………………………………………………………………………………………………………………………..........13

Introduction v

Introduction The purpose of this manual is to explain how to perform an IMViC test. The IMViC test is a very

important tool for environmental research, allowing you to distinguish between coliform bacteria

(such as E. coli), from and other members of Enterobacteriaceae. The results of the test will help

you determine whether the sample has fecal contamination. The IMViC test can also be useful in

identifying the species of bacteria causing an intestinal infection in a patient.

What you need To perform the IMViC you will need to have the following:

Skills A basic understanding of microbiology

A solid grasp on laboratory techniques, such inoculation and aseptic skills

A sample only containing the isolated bacteria species

Test Materials Peptone water broth tube

MRVP broth

An empty sterile test tube

Simmon’s Citrate agar test slant tube

Kovak’s reagent

KOH

Naphthol alpha

Methyl Red

Rack for storing of tubes

Inoculation Materials Bunsen burner (or a different small flame producer)

Gas source for burner (propane or natural gas)

Striker

Gloves

Inoculation loop

Incubator

vi How to Perform an IMViC Test

Terminology In this manual, I will use microbiology-related terms that may be difficult to understand. Here

are some terms and their definitions.

Media – A surface containing a nutrient rich environment for growing micro-organisms.

Inoculate – To purposefully infect a media with the sample bacteria.

Incubate – To place bacteria samples in their optimum growing conditions, often done

with an Incubator.

Sterilize – Kill all micro-organisms.

Considerations Before you start your tests, you should make sure that your sample is completely pure and

uncontaminated. A single bacterial contamination could provide false results after incubation. To

check for a pure culture, perform a four quadrant streak and check for the visual consistency of

the colonies, or prepare a wet slide and check for more than one species of bacteria using a

microscope.

Good aseptic practices will help you prevent contamination. When performing these tests, you

should:

Work near the flame to prevent areal contamination.

Place the inoculation loop and tubes upright in the rack so that the tops to not touch the

table surface.

Keep the lids of the test tubes secured on as much as possible.

Wear gloves.

Wipe down the area with Lysol or another kind of sterilizer.

When performing the inoculation part of this test, you should always work near the flame to

prevent aerial contamination. Working with an open flame brings a host of many dangers to you

and others, as well as the building. Always use caution with an open flame and have emergency

solutions (like a fire extinguisher) prepared

In this manual I will use this caution symbol to show steps where you should proceed with

caution.

Chapter One 3

Performing the Tests

Chapter One 3

Performing the Tests Preparation

1. Clear a large clean working space for the tests.

2. Ignite the Bunsen burner.

3. Gather all the materials at the work area.

4. Put on gloves.

Indole Test Preparation 1. Sterilize the inoculation loop using the flame.

2. Grab the peptone water broth tube.

3. Swirl the loop in the sample to gather specimen.

4. Dip the loop into the peptone water broth tube to

inoculate the media. (See Figure 1.)

5. Sterilize the inoculation loop using the flame.

6. Place peptone water broth tube back in the rack.

MRVP Test Preparation 1. Sterilize the inoculation loop using the flame.

2. Grab the peptone MRVP broth.

3. Swirl the loop in the sample to gather specimen.

4. Dip the loop into the MRVP broth tube to inoculate the media.

(Refer to Figure 1)

5. Sterilize the inoculation loop using the flame.

6. Place MRVP broth tube back in the rack.

Citrate Test 1. Sterilize the inoculation loop using the flame.

2. Grab the citrate agar tube.

3. Swirl the loop in the sample to gather

specimen.

4. Inoculate the media by brushing the loop

against the media in a zigzagging motion. (See

figure 2.)

5. Sterilize the inoculation loop using the flame.

Figure 2

Figure 1

4 How To Perform an IMViC Test

6. Place peptone water broth tube back in the rack.

Incubation 1. Collect the rack containing peptone water broth, MRVP broth, and the citrate agar tube.

2. Using the incubator, incubate the tubes in the rack at 37 degrees Celsius for a day.

Indole Test 1. Grab the peptone water broth tube.

2. Add 3-4 drops of Kovak’s reagent to the top of the solution.

3. Replace tube in rack.

MRVP Test 1. Grab the MRVP broth.

2. Carefully divide the broth between MRVP broth tube and the empty test tube.

3. Label one tube MR

4. Label the other tube VP

5. Add 3-4 drops of Methyl Red into the MR tube.

6. Replace tube in rack

7. Add about 6 drops of Alpha Naphthol to VP tube.

8. Add about 6 drops of KOH to the VP tube.

9. Shake the tube vigorously (make sure to replace the cap first).

10. Note the time (you need to wait at least 15 minutes before reading the results).

11. Replace tube in rack

Chapter Two 5

Interpreting the Results

Chapter Two 7

Interpreting the Results As you are inspecting the tubes, write down the results so that you can compare them with the

established results of possible bacteria later.

Indole Test Inspect the peptone broth tube. A positive result is indicated by a pink/red layer forming on top

of the liquid, and no change suggests a negative result.

MR Test Inspect the tube labeled MR. If the solution has turned red, the test is positive. If the solution

remained yellow, the test is negative.

Citrate Test Inspect the Citrate agar tube. If the color of the media has changed from green to blue, the test is

positive. If the color has remained green, the test is negative.

VP Test Verify that you have waited 15 minutes since you have added the reagents to the VP test. If so,

inspect the tube. A pinkish-red color indicates a positive test result. No change indicates a

negative result.

8 How to Perform an IMViC Test

Determine the Species Compare your results with established results of IMViC tests on specific bacteria species. Use

your results to match up with bacterial tests from a credible chart and identify your species.

Figure 3 shows test results from a few common bacteria.

Figure 3

Chapter Three 9

Troubleshooting

Chapter Three 11

Troubleshooting “I can’t identify my species” One of the best indications that you have made a mistake in this experiment is if you cannot find

a species that fits all of your tests results. If this has occurred, then you might have either

performed a test wrong or your sample has been contaminated with more than one type of

bacteria. In this event, you should throw away your sample and collect a new one. Then make

sure that a single bacteria species is isolated, and begin the testing over again. Another issue may

be that your bacteria may not be recognized from the source you are using. If this is the case,

then you should check other sources such as www.pubmed.com for different bacteria IMViC

results.

“The species matches with everything except the VP test” If you suspect that you have identified your sample, but the VP test does not match up, then you

may have performed the VP test wrong. You must be sure that you have waited at least 15

minutes for the Alpha Naphthol and KOH to take full effect before you record the results.

“The bacteria did not transfer from the sample to the tests” If you suspect that there has not been any bacteria transferred from your sample to the tests, then

you may have not let your inoculation loop cool long enough before dipping it into the broth. An

indication of this problem is no change in any of the tests after inoculation. You should repeat

the experiment and allow about 10 seconds after you sterilize the inoculation loop to cool down

before dipping it into the sample.

Index 13

Index

Citrate, iii, 5, 3, 7

Incubate, 6

Indole, iii, 3, 4, 7

Inoculate, 6, 3

Media, 6

MRVP, iii, 5, 3, 4

Sterilize, 6, 3