VU Lab M2: Tube to Tube Transfer (Aseptic Technique)

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 M2 Activities:

1. Open the Virtual Unknown software.

2. Review the procedures covered in exercise M1.  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
Gram Positive Coccus.

  * 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.
NOTE: If you are stuck on any of the above directions, go back and review the procedures from lab M1.
5. From the “Media” drop-down box select Phenol Red Glucose
Broth with Durham Tube
and give the media a name you will
remember (no spaces).

  * On the screen, two tubes will appear.  The left (yellow) tube
contains the bacteria sample and the right (red) tube is the phenol
red glucose growth media.
6. In this exercise, we will learn the technique used to transfer
the bacteria to the media tube with a minimum of contamination.  
It may help to write this procedure on an index card for easy
reference in the future.

STEP ONE:  From the “Tool” dropdown list, select “Loop”
STEP TWO: Right-click on the Bunsen burner to turn it on

STEP THREE: Place the loop icon into the burner flame until it turns red

STEP FOUR: Right-click on the red tube and select “remove caps/lids”

STEP FIVE: Drag the open tubes to the burner and place the top of the tubes into the flame – a
pop up box will show below the tubes when they are adequately flamed
(when dragging tubes, click on the tube and then let go of the mouse button when dragging –
if you drag the tubes with the mouse button depressed, the program may not recognize that
the tubes have been flamed)

STEP SIX: Dip the loop into the source tube (the yellow one) to scoop up some bacteria and
immediately dip the loop into the media (the red tube) to inoculate it with the sample – be sure to
enter both tubes from the top through the tube opening. Pop up boxes below the tubes will
confirm you are proceeding correctly.  If you do not see the pop up boxes, let go of the mouse button.

STEP SEVEN: Drag the tubes to the burner again and flame the openings (remember to let go of
the mouse button when dragging), then replace the caps

STEP EIGHT: Flame the loop tool in the burner to remove bacterial residue

STEP NINE: Drag the red tube into the 37C incubator and let it go (it will disappear into the
incubator but you can see it is in there by right clicking on the incubator)

STEP TEN: Turn off the burner then click the “New Day” button

STEP ELEVEN: Right-click on the media tube and select “Record Results”.  Any color change or
bubble formation is a positive result.  Reference:

If you have trouble with the innoculation process watch this video (starting at minute 5:00):
7.  Next, select Phenol Red Lactose Broth from the Media drop-down box and repeat the above
procedure.  Remember, all tests will show up on your lab report so be sure to do all steps for full
credit on this lab.  (For results, all phenol red broths use the same color changes as shown in the link
above for positive or negative results – only the sugar metabolized is different).

8. Then, select
Phenol Red Fructose Broth from the Media drop-down box and repeat the above
procedure.   This time, pay particular attention to the state of the stoplights in the upper right corner as
you work.

NOTE:  If "Phenol Red Fructose Broth" is not showing in your list, it is most likely because your
unknown is not a Gram Positive Coccus.  You MUST choose the "Gram Positive Coccus" subgroup
in step #3 above or this test will not appear!  (In this case, you'll need to start over.)
9.  Next, click on View --> Lab Report.

10.  Finally, click on “Identify” in the “Unknown” drop-down box.  

* Choose an identity – if more than one is available, it is okay to guess at this point.

  NOTE:  If you get an "All microbes eliminated" error, watch this video for how to fix it:

11.  Look through your lab report and note any errors (in red).  

12. Before leaving your report.  Click File --> Save As to name it and save it to your computer.   BE
SURE TO SAVE THIS REPORT – you must upload it to the lab dropbox for credit on this lab.
Questions:  Answer the following questions in a Word document and upload the document
to the correct dropbox, along with your .pdf lab report saved from the VU program.  
Both the .pdf report 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): ___________________

2.   Fully explain (in sentences) the purpose of the Bunsen burner in the aseptic transfer procedure.
Explain the likely outcome for the media if the burner step was ignored.

3.  Did your unknown bacteria show any difference in results for the three sugars tested?  
If so, explain the differences in the sugars used.  If not, explain how the sugars are similar in structure.  
(Please be aware with these questions that each student is assigned a different unknown and may
have different question answers – your results and answers may need to be different from those of
your classmates, depending on which case study the program assigned.  
If you have not yet taken Chemistry, you may have to do some independent research for
some of the questions, but all answers must be in your own words –
copy and paste will not count for credit.)

4.  In step #8 of the lab you were asked to pay attention to the state of the stoplights as you
inoculated the sample.  Describe what you observed happen with the lights.  
Explain the reason for the changes.

5.  After identifying your unknown in step #10 your lab report will show errors in red.  Choose one
error, explain why/how the error was made, and what you can do to prevent the error in the future.

6. Open your lab report (click View --> Lab Report) and use it to answer the following questions:

  a.  How many bacteria types were eliminated by the Glucose test?  _____________  
by the Lactose test? ______________  by the Fructose test? ____________________

  b.  How many bacteria types are remaining as possible identities of the unknown after the
end of testing? _____________

  c.  Assigned unknown identity: ______________________________

  d.  Identified unknown identity: ______________________________