Shaylyn Robison
Lab Report #4
Micro Lab BIOL 2065
W 10 am-11:50 am
Report #4: IMViC Test
Introduction
The IMViC Test is a series of four experiments that help differentiate between the
groups of Enterobacteriacaea according to their biochemical properties and reactions. The
IMViC test stands for Indole, Methyl-red, Voges-Proskauer, Citrate Utilization tests. The
four series of tests include the Indole Production Test, Methyl Red test, Voges-Proskauer
Test, and the Citrate Utilization test.
Part 1: Indole Production Test
This test will “determine the ability of microorganisms to degrade the amino acid
tryptophan.” (Rudolph 121)
Materials and Methods
 24 to 48-hour Trypticase soy broth cultures of Escherichia coli, Proteus vulgaris
and Enterobacter aerogenes
 4 SIM agar deep tubes
 Kovac’s reagent
 Bunsen burner
 Inoculating needle
 Test tube rack
 Marker
1.) Label 3 of the 4 tubes with one organism name on each tube.
2.) Use sterile technique to inoculate each organism into its corresponding labeled
deep tube. Use a stabbing inoculating technique. The last tube is used as the
control and will not contain any organism.
3.) Incubate tubes for 24 to 48 hours at 37 degrees Celsius.
4.) After incubation, add 10 drops of Kovac’s reagent to all tubes and agitate gently.
5.) Examine the tubes and record results.
Results
Organisms
E. coli
E. aerogenes
P. vulgaris
Color of Reagent layer
Red
Yellow
Red
Trytophan Hydrolysis (+) or (-)
+
+
Discussion
The organism E. coli and P. vulgaris yielded positive results for the Indole test,
meaning they degrade tryptophan using tryptophanase to produce indole. The contents of
the tube containing E. aerogenes turned yellow which means that E. aerogenes does not
degrade tryptophan.
Extension
The Indole production test would be useful in determining and differentiating
between multiple microorganisms in a patient’s sample. It would differentiate between
pathogens and normal intestinal flora, such organisms as indole producing E. coli (
normal intestinal flora) and E. aerogenes.
Part 2: Methyl Red Test
The Methyl Red test will determine microorganisms’ ability to ferment glucose
and “differentiate between all glucose-fermenting enteric organisms.” (Rudolph 123)
Materials and Methods
 24 to 48-hour Trypticase soy broth cultures of Escherichia coli, Proteus vulgaris
and Klebsiella pneumoniae.
 MR-VP broth
 Methyl red indicatior
 Bunsen burner
 Inoculating loop
 4 test tubes
 Marker
1.) Label tubes with organisms’ names, one organism per tube. The fourth tube will
serve as a control.
2.) Use sterile technique to inoculate each organism into the corresponding tube. Do
not inoculate the last tube.
3.) Incubate cultures for 24 to 48 hours at 37 degrees Celsius.
4.) After incubation, transfer one-third of each culture into an empty test tube and set
aside for the Voges-Proskauer test.
5.) Add five drops of methyl red indicator to the remaining cultures.
6.) Examine cultures and record results.
Results
Methyl Red Test
Organisms
Color of Medium
(+) or (-)
E. coli
Pink/rose
+
E. aerogenes
yellow
*Note: The results of the third tube containing K. pneumoniae was not recorded.
Discussion
The Methyl red test differentiated E. coli from E. aerogenes. The results showed
that E. coli does have the ability to oxidize glucose into acid end-products. The color red
was result of a pH < 4.4. The color of E. aerogenes showed that its pH was at or around
6.2. This means that E. aerogenes does not produce acid.
Extension
The Methyl red test can be used to differentiate between acid-producing
microorganisms and non-acid-producers. This differentiation would be useful when
narrowing down a list of potential pathogens.
Part 3: Voges-Proskauer Test
This test will help differentiate between enteric organisms such as E. coli and E.
aerogenes.
Materials and Methods
 24 to 48-hour Trypticase soy broth cultures of E. coli and E. aerogenes.
 Barritt’s reagents A and B
 Bunsen burner
 Inoculating loop
 Marker
1.) Obtain aliquots from the Methyl Red test.
2.) Add 10 drops of Barritt’s reagent A to the aliquots of each broth culture separated
during the methyl red test. Shake cultures. Immediately add 10 drops of Barritt’s
reagent B and shake. Re-shake cultures every 3 to 4 minutes.
3.) After 15 minutes of the addition of Barritt’s reagent, examine and record the color
of the cultures.
Results
Voges-Proskauer Test
Organisms
Color of Medium
(+) or (-)
E. coli
Yellow
E. aerogenes
Pink
+
Discussion
E. aerogenes turned pink after the addition of the Barritt’s reagent which means
the organism produces non-acidic end products. The detection of the non-acidic product,
such as acetylmethylcarbinol will occur due to the presence of a-naphthol catalyst and
guanidine group in the MR-VP medium. A yellow color means that the test proved the
organism does not produce non-acidic end products and therefore was a negative result.
Extension
The Voges-Proskauer Test could be compared next to the Methyl red test to
determine non-acid and acid producers and differentiate between the two. One test could
serve as a control or evidence that will support the other test’s results.
Part 4: Citrate Utilization Test
The Citrate Utilization test will help further differentiate between enteric
microorganisms based of the ability to ferment citrate.
Materials and Methods
 24 to 48-hour Trypticase soy broth cultures of E. coli and E. aerogenes.
 4 Simmons citrate agar slants
 Bunsen burner
 Inoculating needle
 Test tube rack
 Marker
1.) Label all tubes with one organism each. The fourth tube will be the control.
2.) Use sterile technique to inoculate each organism into its corresponding tube. Do
not inoculate the fourth tube.
3.) Incubate all cultures for 24 to 48 hours at 37 degrees Celsius.
4.) After incubation, examine the agar slant cultures for growth and coloration of the
medium. Record the results.
Results
Organism
E. coli
E. aerogenes
Presence or Absence of Growth
(+) or (-)
+
+
Color of Medium
Green
Blue
Citrate Utilization
(+) or (-)
+
Discussion
The blue color of growth of E. aerogenes showed a positive result for citrate
utilization as a carbon source. The green color of growth of E. coli showed a negative
result for citrate utilization. The results show that E. aerogenes is capable of using citrate
as a carbon source for energy. They also mean that E. coli may ferment glucose or
lactose; further tests could prove this theory.
Extension
The Citrate Utilization test would be helpful in figuring out if organisms are able
to ferment citrate. This would help differentiate between organisms who require different
nutrients but are similar in morphology, or those who are alike in biochemistry in other
terms.
References
Rudolph, Dr. Jane. Symbiosis. Boston: Pearson, 2010. 121-130. Print.