VU Lab M9: Differential Media – IMViC
Virtual Unknown Software:
For the best results on these labs, do the exercises in order and do not skip any exercises. The
exercises build and if you skip any, there is a good chance you will be lost and overwhelmed with
the later activities. Be sure to use the instructor-developed directions, not the directions supplied
by the program company.
Lab M9 Activities:
1. Open the Virtual Unknown software.
2. Review the procedures covered in the previous exercises. As you learn new procedures,
it may help to make index cards for future reference.
3. Select a new unknown (click on the “New” button with the orange biohazard symbol) with the
subgroup Media Tests 1 (make sure to pick #1 and not a different number or these directions
won’t work correctly!!).
* Give the unknown a name you will remember (no spaces).
* Do NOT click auto-inoculation (this will show up on your lab report).
4. Read through the case study and assign the Gram Stain status.
5. In this lab we will continue to explore selective and differential media.
6. Click on the T? button at the top of the lab page and read through the background and
procedures for the Indole Production Test. Note, this test requires the addition of Kovac’s
reagent after incubation.
7. The Indole test is the first test in a series of tests commonly performed on Gram negative
enteric bacteria (bacteria that cause intestinal infections). The series of tests is called the
IMViC and it includes the Indole, Methyl-Red, Voges-Porkauer, and Citrate tests.
Results reference: http://www.uwyo.edu/virtual_edge/lab25/imvic_results.html
8. From the Media dropdown box, select Tryptone Broth.
9. Using the tube to tube transfer procedure, inoculate the media and place it in the 37C incubator.
10. Click “New Day” one time and remove the sample from the incubator.
11. Using the dropper tool, add Kovac’s reagent to the tube and record the results.
Formation of a red ring is a positive result, a yellow ring is a negative result.
12. Click on the T? button at the top of the lab page and read through the background and
procedures for the Methyl Red Test. Note, this test requires the addition of Methyl Red reagent
after incubation.
13. From the Media dropdown box, select MRVP Broth.
14. Using the tube to tube transfer procedure, inoculate the media and place it in the 37C incubator.
15. Click “New Day” one time and remove the sample from the incubator.
16. Using the dropper tool, add Methyl Red reagent to the tube and record the results.
A color change to red indicates a positive result. No color change indicates a negative result.
17. Click on the T? button at the top of the lab page and read through the background and
procedures for the Voges-Proskauer Test. Note, this test requires the addition of two reagents –
Barritt’s A and B - after incubation.
18. From the Media dropdown box, select MRVP Broth (note this same broth is used for both
the Methyl Red (MR) test and the Voges-Proskauer (VP) test, hence the name of the broth).
19. Using the tube to tube transfer procedure, inoculate the media and place it in the 37C incubator.
20. Click “New Day” one time and remove the sample from the incubator.
21. Using the dropper tool, add Barritt’s A reagent then Barritt’s B reagent to the tube and
record the results. A color change to red indicates a positive result. No color change indicates
a negative result.
22. This next test we completed in lab #M8 – please review the procedures and background
from that lab if you do not remember them.
23. From the Media dropdown box, select Simmon’s Citrate Agar Slant.
24. Using the tube to tube transfer procedure, and a wire instead of a loop, inoculate the media
and place it in the 37C incubator.
25. Click “New Day” one time then remove the tube from the incubator and record your results.
26. Identify the organism as Escherichia coli and view your lab report for errors. Save your lab report.
NOTE: If you get an "All microbes eliminated" error, watch this video for corrections:
https://www.youtube.com/watch?v=Pu3h8cZgOhU
27. Repeat steps #8-25 for a new unknown from the subgroup Media Tests 2 (make sure to choose
#2!).
28. After completing the tests, identify your unknown as Enterobacter aerogenes and view your lab
report for errors. Save your lab report.
Questions: Answer the following questions in a Word document and upload the
document to the correct dropbox, along with your .pdf lab reports saved from the VU
program. Both the .pdf reports AND the question answers must both be uploaded for
the lab to be graded. PDF reports must show that they were completed during this term
(noted in the bottom left corner of the report) and by the person submitting the report for
a grade (noted in the top left corner of the report).
1. Case Study Number (click View --> Case Study to see the number):
Media Tests 1: ___________ and Media Tests 2: _______________
2. Tryptophan is an amino acid broken down by a complex pathway (how complex it is:
http://www.genome.jp/kegg-bin/show_pathway?map00380 ) where indole is one of the
products formed. The point of the breakdown process is to form Acetyl CoA for Glycolysis.
Is glycolysis necessary for aerobic respiration, fermentation, or both?
Explain. (In your own words).
3. Given the complexity of the pathway shown in the link above, would it make more sense for
bacteria to prefer to use glucose or tryptophan for energy? Explain your opinion in full sentences
and your own words.
4. The MR and VP tests are both fermentation tests. Explain two specific differences between
energy production by aerobic respiration versus by fermentation.
5. Open your two lab reports from this lab and compare them side by side. Which test results
were the same? Which test results were different? Were any of the test results not helpful
(did not eliminate any organisms)? If so, which one(s)?
6. At the end of all of the tests, how many organisms were remaining for the Media Test 1
sample? _____________ for the Media Test 2 sample? ______________
7. Check the Identification Matrix for each sample. Explain which test you would do next if you
were to continue trying to narrow down the identity of Media Test 1 on your own. And, also, for
Media Test 2. Be sure to give your reasons for choosing each test, based on what you see in
the Identification Matrix.