VU Lab M6: Amino Acid Metabolism

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 M6 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
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 so be sure you don’t click it).

4. Read through the case study and assign the
Gram Stain status.

5.  In this lab we will start to explore some of the more complex tests used for unknown
identification – tests of amino acid metabolism.

6. All of the tests we have looked at up to this point showed results after one click of the
“New Day” button.  The tests in this lab, however, are more complex and do not show full results
that quickly.  

7.  Click the blue “M?” button at the top of the lab page and read through the background and
procedures for the Arginine dihydrolase test, Lysine decarboxylase test, and Ornithine
decarboxylase test.  Notice that all tests have the same general procedure and appearance,
and all three take 2 “New Days” to fully complete.

8. From the Media dropdown box, select
Arginine Dihydrolase Broth.

9. Using the tube to tube transfer procedure learned in previous labs, inoculate the media and
place it in the 37C incubator.

10.  Click “New Day” one time then remove the tube from the incubator and observe it.  If the tube
is still purple, the results are negative.  If the tube is yellow, it needs one more day to finish the test.

11.  If the tube is yellow, place it back into the 37C incubator and click “New Day” one time.

12.  Remove it from the incubator and record your results (if it stayed yellow after two days,
the test is negative but if it returned to purple on the second day, the test is positive).

13.  Repeat the two day procedure from steps #8-12 for
Lysine Decarboxylase Broth and
Ornithine Decarboxylase Broth, recording results after the second day.

14.  Next, we will look at a procedure that involves only one day of incubation, but requires the
addition of reagents before the results can be read.

15.  Click the blue “M?” button at the top of the lab page and read through the background and
procedures for the Phenylalanine Agar Slant test.

16.  From the Media dropdown box, select
Phenylalanine Agar.

17.  Using the tube to tube transfer procedure learned in previous labs, inoculate the agar, place
it in the 37C incubator, press the “New Day” button, then remove the tube from the incubator.

18.  From the Tool dropdown box, select “Dropper”.

19.  From the Reagent dropdown box, select
10% Ferric Chloride.
20.  Remove the cap from the tube and add the reagent.

21.  Next, select
0.1N HCl from the Reagent dropdown box and add it to the tube also.

22.  A color change to green is a positive result.  No color change is a negative result.  
Record your results for the test.  (NOTE: for Media Test 2 the result should be negative, although
for some reason the program sometimes accidentally shows the picture for positive.  
Record the test for this organism as negative, even if it shows green for this one unknown only.)

23.  View your Lab Report and Identification Matrix.

24.  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:

25.  Repeat steps #8-23 above for a new unknown from the subgroup Media Tests 4
(make sure to choose #4!).

26.  After completing the tests, identify your unknown as
Proteus vulgaris.  Review and 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 4: _______________

2.  When organisms use amino acids for energy instead of carbohydrates, they must first go
through the process of “deamination”.  Explain what deamination is, and how it relates to the
urea cycle.  (This must be in your own words, credit is not given for copy and paste answers.)

3.  Examine the structure of the amino acids arginine, lysine, and phenylalanine at this webpage:
Which of these three amino acids shows a structure most different from the others?  How is it

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 4 sample? ______________

6.  Is it possible to fully identify an unknown using only amino acid metabolism tests?  
Explain your answer.