VU Lab M3: Tube to Plate 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 M3 Activities:

1. Open the Virtual Unknown software.

2. Review the procedures covered in exercises M1 and M2.  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. <--  Be sure to pick THIS subgroup or your tests will not work!!

 * 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. From the “Media” drop-down box select DNAse Agar Plate with
Methyl Green
and give the media a name you will remember (no spaces).

* On the screen, a tube of bacteria culture (yellow) and a media plate
(green) will appear.
6. In this exercise, we will learn the technique used to transfer the bacteria to the media plate
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 media plate and select “remove caps/lids”

STEP FIVE: Drag the open tube of bacteria to the burner and place the top of the tube into the
flame – a pop up box will show below the tube when it is 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 squiggle the loop along the agar surface to inoculate it with the sample until culture
lines appear (it may take a while - up to 20 seconds).
Pop up boxes below the media 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 culture tube to the burner again and flame the opening
(remember to let go of the mouse button when dragging), then replace the cap

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

STEP NINE: Drag the media plate into the 25C incubator and let it go
(make sure to use the correct temperature incubator for this media!)

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

STEP ELEVEN: Right-click on the media tube and select “Record Results”.
7. Click on View --> Lab Report and note how many organisms were eliminated by the
DNAse results.

8. Click on View --> Identification Matrix
* Along the top of the matrix, you will see the names of all the organisms that could be the
identity of your unknown.  If you count them, the number will match the number shown as
remaining after this test on your lab report.

9. To narrow down the identity of our unknown organism, we’ll do some more tests.  
When you do an unknown identification on your own, you can choose any test you wish to
do from the list.  But, for now, we’ll choose some antibiotic plate tests so we can practice
our plate inoculation technique.

10.  Note, on the Identification Matrix, that the Novobiocin susceptibility test will give positive
results for about half the organisms and negative results for the other half.  When doing
identification tests, it is always best to try to find a test that will cut your list in half as quickly
as possible.
11. Next, we will learn the technique for making a lawn on an agar for use in an antibiotic
susceptibility test.  It may help to write this procedure on an index card for easy reference in
the future.

STEP ONE: Select Nutrient Agar Plate from the Media drop-down list.

STEP TWO: From the “Tool” dropdown list, select “Swab”.

STEP THREE: Turn on the Bunsen burner

STEP FOUR: Remove the media caps and flame the mouth of the bacterial source culture tube.

STEP FIVE: Dib the swab into the culture tube (from the top) and then squiggle it along the
surface of the agar until a change in appearance of the surface occurs (this takes a while –
up to 20 seconds)

STEP SIX: Right-click on the agar plate and choose
Novobiocin from the antibiotic list
(you are shown this list because the program knows you are doing an antibiotic sensitivity
test when you choose the swab tool).
 If the option doesn't appear, make sure your lid is off.

STEP SEVEN: Replace the media lids

STEP EIGHT: Turn off the burner

STEP NINE: Place the plate into the 37C incubator and click “New Day” then remove the
plate from the incubator.

STEP TEN: Right click on the agar plate and choose “Record Results”.  If there is a circle
around the disc where bacteria did not grow, the bacteria are sensitive to the antibiotic and
the results are positive.  If the bacteria grow right up to the edge of the disc, they are not
sensitive and the results are negative.
12. Click on View --> Lab Report and note how many organisms were eliminated by the antibiotic

13. Click on View --> Identification Matrix to see how many organisms remain

14.  Now, let’s do another test to try to narrow it down further.  Repeat the procedure from
step #11, this time choosing the
Bacitracin 2U susceptibility test (be sure to select Nutrient
Agar as your media).

15. After completing the test and recording your results you should see only one organism listed
on your identification matrix – this is the identity of your unknown!  (NOTE:  If you see more than
one, that's okay!  Just guess - at this point the point is to learn how to work the program, not to
get everything exactly right!  Actual results may vary because the program randomly assigns the

16.  Click on “Identify” in the “Unknown” drop-down box and look through your lab report to note
any errors (in red).

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

17.  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.
Click here to see the questions to answer for this lab:  Lab M3 Questions to Answer