VU Lab M7: Differential Media – Blood & Salt

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 M7 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 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).

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

5.  In this lab we will start to explore media known as “selective” and “differential” media.  
Media that are selective contain an inhibitor that allows some bacteria to grow, but inhibits others.  
Media that are differential allow all bacteria to grow, but show a different appearance in the media
after growth depending on how the bacteria use the media components.  More information on
common selective and differential media can be found here:
http://amrita.vlab.co.in/?sub=3&brch=73&sim=720&cnt=1

6.  Click on the M? button at the top of the lab page and read through the background and
procedures for the 6.5% NaCl Broth test.

7.  From the Media dropdown box, select
Nutrient Broth with 6.5% NaCl.  Salt media is
selective – it only allows bacteria to grow if they are a species that can tolerate salty conditions.

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

9.  Click “New Day” one time then remove the tube from the incubator and observe it.  If the
tube is clear, no growth occurred and the results are negative.  If the tube is cloudy (turbid),
growth occurred and the results are positive.

10.  Click on the M? button at the top of the lab page and read through the background and
procedures for the Blood Agar test.  

11.  Next, from the Media dropdown box, select
5% Sheep Blood Agar Plate.  Blood agar is
differential.  Most bacteria will grow on blood agar, but different species will use the blood to
different degrees resulting in results that look different.

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

13.  Click “New Day” one time then remove the plate from the incubator and observe it.

14.  Record the results using the following website for reference:
http://www.wisc-online.com/Objects/ViewObject.aspx?ID=MBY4307

15.  Identify the organism as Staphylococcus aureus 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

16.  Repeat steps #7-14 for a new unknown from the subgroup Media Tests 6 (make sure to
choose #6!).

17.  After completing the tests, identify your unknown as
Staphylococcus saprophyticus 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 5:  ___________  and Media Tests 6: _______________

2.  Salt is such a vital resource for food preservation that salaries to ancient workers were
sometimes paid in salt.  In fact, the word “salary” comes from the Roman word for “salt”.  
Research and explain the basic principle behind the way salt works to inhibit bacterial growth
in food. (Answer must be in your own words, copy and paste is not accepted for credit).

3.  Research blood hemolysis and answer the following questions (Answers must be in your
own words, copy and paste is not accepted for credit):

a.  Why does blood agar have a green color when alpha hemolytic bacteria grow on it?
b.  Why does blood agar become clear when beta hemolytic bacteria grow on it?  
c.  Why does blood agar not change in appearance when gamma hemolytic growth occurs?

4.  At the end of all of the tests, how many organisms were remaining for the Media Test 5
sample? _____________ for the Media Test 6 sample? ______________

5.  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 5 on your own.  And, also, for
Media Test 6.  Be sure to give your reasons for choosing each test, based on what you see in
the Identification Matrix.  If you are having trouble with the function of the Identification Matrix,
watch this video for help:
http://www.virtualunknown.com/vumie/help/index.htm#VUMICRO/Bacterial_Identification.htm