Download Microbiology lab TAs/BIO 351

Laboratory Exercise 19
IMViC tests
Expectations of this lab:



Know what the IMViC acronym stands for and which bacteria can be
differentiated using these tests.
Understand the basic biochemistry of these tests.
Understand that these tests can be used to differentiate coliforms that may be used
as indicator organisms in monitoring food safety and waste water management
industry.
The acronym IMViC stands for Indole production from tryptophan, Methyl red test,
Voges-Proskauer test, and the Citrate test. This series of tests is used to differentiate
among enteric bacteria (Enterobacteriaceae). These tests are important in detecting
faecal contamination. Escherichia coli, Enterobacter aerogenes, Klebsiella oxytoca, and
Proteus vulgaris are enterics that can be differentiated using these tests.
Introduction: Indole test (I)
Typtic Soy Broth (TSB) contains tryptophan and if a bacterium produces
tryptophanase then it can hydrolyze tryptophan to indole, pyruvic acid, and ammonia.
Indole accumulates in the medium and can be tested with Kovac’s reagent (paraDimethylamino benzaldehyde). Using the SIM medium discussed earlier or the use of
TSB can easily detect bacteria that produce tryptophan.
Bacteria:
Tryptophan
tryptophanase
Test tube:
Indole + Kovac’s reagent
indole + pyruvic acid + ammonia
rosindole dye (cherry red)
Since p-DMBA (Kovac’s reagent) contains iso-amylalcohol, the indole produced here
will be extracted and rise to the top of the tube, giving a cherry red band in a positive
tube.
Materials:
Uninoculated TSB (2 test tubes)
Microincinerator
Inoculating Loop
Sharpie
Unknown Species
1
Kovac's reagent
Potassium hydroxide
Demos: Escherichia coli, Enterobacter aerogenes
Procedure:
1.
2.
3.
4.
Obtain two uninoculated test tubes with TSB. Label with the date and microbe names.
Inoculate each broth aseptically with the unknown species using a loop.
Incubate the tubes for 48 hours at 35±2˚C.
To both tubes (at the next lab session), add 6-8 drops of Kovac's reagent, one at a time.
Mix the tubes well by tapping the bottom of the tubes. A cherry red colored band
formation on top of the broth indicates a positive test result for Indole while a different
color or no color change is a negative result.
5. Record results and compare to the provided demos.
Data/Results:
Maintain detailed notes of your results in your lab notebook. Clearly labelled drawings
denoting the details and colors are recommended.
Figure 19-1 Escherichia coli (L) and Enterobacter aerogenes (R) grown in TSB at 30oC
for 48 hours and Kovac's reagent added. E.coli positive and E. aerogenes negative for
indole production.
Introduction: Methyl red test (MR) and Voges Proskauer (VP) test
When bacteria obtain energy from food, they can use either oxidation or fermentation.
Oxidative bacteria use the electron transport chain, a terminal electron acceptor, and the
cytochrome system. The terminal electron acceptor is oxygen in aerobic respiration and
2
inorganic compounds such as NO3- and SO4 2- in anaerobic respiration. By using organic
compounds as electron donors and oxygen as the terminal electron acceptor oxidative
bacteria produce CO2 and H2O as the end products.
Bacteria that ferment don’t have the cytochrome system. They use organic compounds as
both electron acceptors and donors. MR/VP tests can be utilized to differentiate
facultative anaerobic enteric bacteria. After all the available oxygen is utilized these
bacteria will choose one of the two pathways mentioned below to ferment glucose.
Mixed acid fermenters (mixed acid fermentation pathway) will produce large amounts
of lactic acid, formic acid, propionic acid, succinic acid, and ethanol. Butanediol
fermenters (butanediol/butylene glycol fermentation pathway), also described as
butylene glycol fermenters, will produce a little quantity of the above mentioned acids
and a large amount of butanediol and ethanol. These are neutral products and will not
alter the pH of the medium. Acetoin is an intermediate product in the butylene glycol
(butanediol) pathway and the VP test is designed to detect acetoin. Various gases such as
CO2 and H2 are also produced here.
Methyl Red test (MR) will detect acidic end products and Voges Proskauer (VP) test
will detect neutral end products from glucose fermentation. Production of large
quantities of succinic, lactic, acetic, propionic, and formic acids will lower the pH to 4 in
MR test. Large quantities of 2, 3, butanediol, ethanol, and small quantities of lactic and
formic acids will leave the pH around 6 in the VP test. Methy red pH indicator is used to
detect mixed acid production in the MR test. Methyl red pH indicator is red at pH 4.2 and
yellow at pH 6.3.
Insert mpf 1708, mpf 1706
3
Materials:
Uninoculated MRVP broth
2 clean test tubes
Microincinerator
Inoculating Loop
Sharpie
Unknown Species
Methyl Red
ɑ-Naphthol (TOXIC)*
Potassium hydroxide
Demos: Escherichia coli, Enterobacter aerogenes (UTAs)
Procedure:
1. Obtain one uninoculated MRVP broth tube. Label with the necessary information.
2. Inoculate the broth aseptically with the unknown species using the techniques outlined in
Exercise 3.
3. Incubate for 48 hours at 35±2˚C.
4. At the next lab session divide the broth in half into a clean test tube. Label one "MR" and
the other "VP".
5. To the tube marked MR, add 10 drops of methyl red. An immediate red color change is a
positive result for the MR test. No color change is a negative result.
6. To the other tube (VP), add 5 drops of KOH and 15 drops of ɑ-naphthol. These reagents
must be added under the hood. Let it sit after vigorous mixing. Results may take 15 -20
minutes to occur. A red color change is a positive result for the VP test.
7. Record results and compare to the provided demos.
Data/Results:
Maintain detailed notes of your results in your lab notebook. Clearly labelled drawings
denoting the details and colors are recommended.
MR test tube - add methyl red pH indicator
The positive result is a red color seen immediately throughout the tube.
4
Figure 19-2 MR test. MRVP broth inoculated at 30oC for 48 hours, divided in half, and a
few drops of methyl red reagent added to each half. Escherichia coli (L) positive and
Enterobacter aerogenes (R) negative.
VP test tube – add α-naphthol and KOH/creatine
Acetoin spontaneously oxidizes to diacetyl in the presence of oxygen and KOH.
Addition of alpha-naphthol increases the sensitivity of this reaction, and diacetyl in the
presence of creatine forms the red dye.
Vigorous mixing is extremely important here. The tube should be mixed for about 30s
and left about 15-20 minutes for the color to develop.
acetoin (acetylmethylcarbinol) + alpha-naphthol*
5
diacetyl
red dye
Most tests will give immediate color changes (e.g., Nitrate test), and VP is one test where
the color change happens slowly with the formation of the red dye from diacetyl.
Figure 19-3 VP test. MRVP broth inoculated at 30oC for 48 hours, divided in half, and a
few drops of alpha-naphthol and KOH/creatine added to each half. Top: Enterobacter
aerogenes: positive. The color will develop slowly within 20 minutes with vigorous
mixing. Bottom: E.coli negative (L) and E. aerogenes positive (R).
6
MR/VP test summary:
1) These two tests are based on the different end products resulting from glucose
fermentation. Divide the MR/VP broth from the previous week and conduct the two
tests separately.
2) MR test: Mixed acid fermentation of glucose-end products are large amounts of
mixed acids such as formic, lactic, acetic, and succinic, which lead to a drop in
pH, and ethanol.
3) VP test: Butanediol fermentation of glucose-end products are a small amount of the
above acids (not significant) and significant amounts of ethanol and butanediol
which are neutral products that would not affect the pH.
4) Escherichia, Enterobacter, Serratia, Klebsiella, Salmonella, Shigella, & Erwinia, can
be identified by the MR/VP test results.
5) Not useful for the identification of the genus Citrobacter.
6) VP test: Mixing is very important to the development of color. The intermediate
acetoin is what we detect by adding alpha naphthol, oxygen (mixing), and
KOH/creatine. Acetoin spontaneously oxidizes to diacetyl when you add
KOH/creatine and mix (O2). Alpha-naphthol makes this reaction more sensitive.
7) Mix the VP tube really well for about a minute, and then let the VP tube sit for
another 15-20 minutes. You can mix the tube a few seconds every five minutes
during the 15-20 minutes if you want the color development to go faster. You can
read results in about 15-20 minutes.
*Αlpha naphthol is toxic! It is a possible carcinogen. Leave the tubes in hood after
the students read results.
Introduction: Citrate utilization (C)
Bacteria can be differentiated based on their metabolism. Citrate is a carbon compound
that can be used in testing a bacterium's ability to utilize citrate as the only available
carbon source in the medium. Other carbon sources also may be tested in a similar
manner. Simmons'citrate medium is a defined medium containing sodium citrate as
the sole carbon source and NH4H2PO4 (ammonium dihydrogen phosphate) as the only
nitrogen source. A pH indicator bromothymol blue is added to the medium to visualize
the biochemistry that occurs with the utilization of citrate. Minimizing carrying over
carbon and nitrogen from the original medium that the bacteria are initially growing in
is important to accurately identify the use of citrate as the sole carbon source. Using a
needle instead of a loop helps achieve this purpose. Cells can also be rinsed in saline
before streaking a citrate slant when using a loop. The stab and streak method using a
needle and a slant works like a rinsing step in removing any excess C and N carried over
7
with the inoculum from the previous medium. Citrate fermentation test can be utilized to
differentiate members of the family Enterobacteriaceae. Klebsiella and Enterobacter
are two genera that can grow utilizing citrate as the only carbon source.
Large molecules such as starch need to be hydrolyzed into smaller components to be able
to get transported into a cell. This is discussed in the next exercise. These bacteria
produce extracellular enzymes to breakdown the larger molecules. However, even small
molecules such as citrate may not be able to get into a cell if the required specific binding
proteins and translocating proteins cannot be made by the bacterium. For a bacterium to
utilize any compound, it has to have the ability to transport it to the cell, make necessary
enzymes to break down the transported molecule, and utilize the products as building
blocks and energy for its own use. This is only possible if the bacterium possesses the
genes to make all these necessary proteins. The bacteria that are capable of utilizing
citrate are able to produce citrate permease and citrate lyase, both essential enzymes in
utilizing citrate as the sole carbon source. Cirate permease is responsible for
translocating citrate into the cell. In the cell citrate is converted to pyruvate through a
series of conversions starting with citrate lyase converting citrate to oxaloacetate and
acetate.
Insert mpf 1529
8
Figure 19-4 Citrate test. Citrate slants inoculated with Escherichia coli and Enterobacter
aerogenes at 30oC for 4 days. E. aerogenes (L) positive and E. coli (R) negative.
9
Materials:
Uninoculated Simmons' citrate slants (2 test tubes)
Microincinerator
Inoculating needle
Sharpie
Unknown Species
Separate rack
Demos: Escherichia coli, Enterobacter aerogenes (UTAs)
Procedure:
1. Obtain two uninoculated test tubes with Simmons' citrate agar medium. Label with the
necessary information.
2. Inoculate each tube aseptically with the unknown species using the needle. Pick up a little
inoculum using a sterile needle and aseptically make a stab in the agar deep bottom
(about 5 mm) and streak in a zig-zag motion on the slant surface using the needle.
3. Incubate for a minimum of 48 hours at 35±2˚C. Some bacteria may need a longer
incubation period. If there is partial color change, incubate the tube a day or two longer
in the incubator.
4. Record results and compare to the provided demos.
Data/Results:
Maintain detailed notes of your results in your lab notebook. Clearly labelled drawings
denoting the details and colors are recommended.
10
Common problems and tips:
Indole test
 TSB or a medium containing tryptophan has to be inoculated with the bacterium
to see if the bacterium produces tryptophanase.
 Fresh Kovac's reagent should be added and thoroughly mixed to see the cherry
red ring layer formation. Kovac's reagent needs to stay in the refrigerator.
 Any other colors (e.g., green) should be considered negative for indole.
MR/VP tests
 Divide the culture after the MR/VP broth has been incubated 24-48 hours.
 No color development. Mix thoroughly after adding the VP reagents because
oxygen is needed for the color change. A red color is positive and this color
darkens with time. Read results after about 15-20 minutes.
 The positive result cannot be seen. MR tubes should be read immediately after
adding methyl red reagent.
Citrate test
 Growth should be associated with the color change (a heavy inoculum may look
like growth with no color change).
 Any C and N carried over from the original medium may give false positive
results. Therefore, rinsing the cells in a saline tube may wash the cells to give
more accurate results, especially if you are using a loop. A needle is
recommended for this procedure.
 If there is partial color change the tube needs to be incubated longer.
 A positive test indicates that the microbe can utilize citrate as the sole C source.
 It also indicates that the bacterium was capable of producing the enzymes citrate
permease and citrate lyase.
 To see the positive color change (blue) the pH indicator bromothymol blue has to
be present in the medium.
References:
Claus GW. 1989. P263-268,277-284,291-296. In Understanding microbes: A
laboratory textbook for microbiology, W. H. Freeman and Company, New York.
Leboffe MJ, Pierce BE. 2015. P311-316,339-341. In Microbiology laboratory theory
and application, 4th ed, Morton publishing Company, Englewood, Colorado.
11