VU Lab M4: Gram Positive vs. Gram Negative Bacteria

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 M4 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 3 (make sure to pick #3 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 tests used to determine the identification of
unknown organisms.  

6.  In wet labs, one of the first tests completed is often the
Oxidase test.  This test is a little different
than other tests in the lab because it is completed by watching a video clip. (Remember, it may
help to make index cards of all test procedures for future reference).

STEP ONE: In the View menu, click “
Oxidase Video” and watch the video.

STEP TWO: If the test strip turns purple, the result is positive.  If the test strip remains white,
the result is negative.

STEP THREE: Record your results.

7.  Another test that is useful to perform early in the identification process is the
OF Glucose
Broth
test.

STEP ONE: Select
OF Glucose Broth from the media drop-down list.

STEP TWO: Complete an aseptic tube-to-tube transfer using the wire tool instead of the loop
tool (refer to lab M2 if you do not recall all of the steps for this procedure).

STEP THREE: Incubate the sample at 37C for one “New Day” after inoculation.

STEP FOUR: Read and record the results.  A positive result on this test indicates glucose was
used and the sample will turn yellow.  In a negative result, the sample remains green.  
Reference:
http://superfarmer.wordpress.com/category/lab/lab-manual/of-glucose-test/

8.  The above sample was exposed to oxygen in the air.  Another version of this test is carried out
with a layer of oil covering the media so that oxygen cannot reach the sample.

9.  Repeat the procedure from step #7 using the media
OF Glucose Broth with Oil and
record your results.

10.  View your lab report.  If you have done the tests correctly, none of the above tests will have
eliminated any organisms.  Don’t be discouraged, we’re just learning the steps at this point!

11.  As we saw in the Gram Stain for the Media Tests 3 sample, this organism is Gram Negative.  
The tests we have done so far are more useful for Gram Positive organisms.  So, now we’ll move
on to a Gram Positive one.  But first, go ahead and identify the unknown for the Media Tests 3
sample (the unknown in this case is
Salmonella typhii).  

12.  View and save your lab report – you’ll be uploading two .pdf reports for this lab.

13. Select a new unknown (click on the “New” button with the orange biohazard symbol) with the
subgroup
Media Tests 5 (make sure to pick #5 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).

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

15.  Repeat the steps #6-10 above for this new unknown (tests for
Oxidase, OF Glucose, and
OF Glucose with Oil).

16.  Open and view the lab report and notice the differences in eliminated organisms for this
organism when compared to the previous one.  

17.  Open and view the Identification Matrix.  Notice that many of the tests listed have
inconclusive results, noted as [+] or d.  We can’t reliably use those results to determine the
identity of the unknown, but we will use a couple of them to practice.

18.  In the View menu, choose “
Catalase Video”.  

19.  A positive result on a Catalase test shows bubbling, a negative test does not.  
Record your results after viewing the video.

20.  Next, in the Media drop-down box, select the
5% Sheep Blood Agar Plate.

21.  Inoculate the plate using the tube to plate transfer method (with loop) from
exercise M3 (be sure to review the steps from that lab if you do not remember them).

22.  Incubate blood agar in the 37C incubator for one “New Day” after inoculation.

23.  Review the following site for blood agar results:
http://www.wisc-online.com/Objects/ViewObject.aspx?ID=MBY4307  
Then, record the results for your sample.

24.  Review the Lab Report and Identification Matrix.

25.  The identity of the Media Test 5 unknown is
Staphylococcus aureus.  If this organism
is not showing on your matrix, it’s because we used the [+] and d test results.  We did this so
you could see what happens if you don’t choose tests that only have clear + and – options.  
(It may show up on your matrix, it depends on what the % was and what you chose as your
answer for each test.)

26.  Go ahead and choose one of the remaining options and identify the unknown.  Note that
Staphylococcus aureus is given as the Assigned Unknown on the lab report.  When choosing
tests to complete to identify an unknown organism it is very important to choose only
clear + vs – tests, and to avoid tests that give ambiguous results.  

NOTE:  If you get an "All microbes eliminated" error, watch this video for corrections:
https://www.youtube.com/watch?v=Pu3h8cZgOhU

27.  Save your lab report.  You will need to upload both lab reports (Media Tests 3 and
Media Tests 5) and the question answers to the dropbox for this lab for full credit.

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 3:  _______  
and Media Tests 5: ________

2. Open both lab reports side by side and compare them to answer the following question:  
Some tests, even when done correctly, do not eliminate any organisms.  Explain why this occurs.

3.  Use the resource buttons (the B?, M?, and T?) to research the Oxidase test.  
Explain (in your own words, not copy and paste) what the Oxidase test measures.  
Based on the results shown on your lab reports, is this test more useful in distinguishing among
Gram Positive or Gram Negative organisms?

4.  Use the resource buttons to research the OF Glucose Broth and OF Glucose Broth with Oil
tests.  Explain (in your own words, not copy and paste) what these two tests measure.  
Explain why the use of oil is significant.  Based on the results shown on your lab reports,
are these tests more useful in distinguishing among Gram Positive or Gram Negative organisms?

5.  Explain the difference in microscopic appearance of Gram Positive versus Gram Negative
organisms.  Explain one reason (there are several to choose from) why these organisms appear
different under a microscope.  (Always use full sentences for full credit, some questions may
require a bit of book research).