VU Lab M8: Differential Media – TSI & Citrate
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 M8 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 2 (make sure to pick #2 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 M? button at the top of the lab page and read through the background and
procedures for the Triple Sugar Iron Agar Slant (commonly abbreviated TSI or TSIA).
7. From the Media dropdown box, select Triple Sugar Iron Agar.
8. Using the tube to tube transfer procedure, and a wire instead of a loop, inoculate the media
and place it in the 37C incubator.
9. Click “New Day” one time then remove the tube from the incubator and record your results
using the guidelines in the next step.
10. The TSI test gives more information than other tests we have looked at. If the agar turns
yellow, acid has been formed whereas if it turns magenta, alkaline products have been formed.
If the agar has cracks, gas was formed. And, if there is a black section of agar, hydrogen sulfide
was formed. If the agar has remained intact and red, the test is negative. Interpretation of the
results for a TSI test can be found at:
NOTE: The TSI test duplicates the results found by the Phenol Red Glucose Broth with Durham
Tube test. When you start to choose your own tests later in the term, it is not necessary to do
both tests. The TSI test is more efficient because it packs more information into only one test.
This is why TSI tubes are included as part of your Wet Lab kit.
11. Next, click on the M? button at the top of the lab page and read through the background and
procedures for the Simmon’s Citrate Agar Slant.
12. From the Media dropdown box, select Simmon’s Citrate Agar Slant.
13. Using the tube to tube transfer procedure, and a wire instead of a loop, inoculate the media
and place it in the 37C incubator.
14. Click “New Day” one time then remove the tube from the incubator and record your results.
15. Identify the organism as Enterobacter aerogenes 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:
16. Repeat steps #7-14 for a new unknown from the subgroup Media Tests 3 (make sure to
17. After completing the tests, identify your unknown as Salmonella typhii 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 2: ___________ and Media Tests 3: _______________
2. Research the TSI test. This test is referred to as a “Triple Sugar” test. What are the three
sugars included in the test agar? How do each of these sugars compare structurally?
(You may want to refer back to your question answers for lab M5 for help).
3. Research the molecule “citrate”. How is it related, structurally, to the sugars listed in the
4. 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)?
5. At the end of all of the tests, how many organisms were remaining for the Media Test 2
sample? _____________ for the Media Test 3 sample? ______________
6. 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 2 on your own. And, also, for
Media Test 3. Be sure to give your reasons for choosing each test, based on what you see in
the Identification Matrix.
7. Although students learning the techniques of the Micro lab often find it helpful to have an
instructor on hand to show them the procedures, the VU program does have some benefits over
the wet lab environment. Describe two things you have found beneficial or interesting about
using the VU program.