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| Lab
Exercise Outline |
| In order to cause disease, bacteria must carry genes that encode the necessary virulence proteins. As you read in Module 2 - Home, bacteria can acquire new genes by several different processes. In this lab exercise, you will observe the transfer of antibiotic resistance genes among bacteria. Antibiotic resistance does not help Pseudomonas syringae attack plants, but it is a good system for studying gene transfer. |
Experimental
Objective
It is known
that bacteria can easily acquire resistance to new antibiotics. In this experiment
we will test if antibiotic resistance can be transferred between bacteria that
are put in close proximity to each other.
Work
in groups of 2 or 3
Materials:
Marker
(one per student group)
Sterile Water (at least 2 ml per student group)
Microfuge
tubes (four per student group)
Pipette droppers (four per student group)
Bacterial
Cultures- P. syringae, E. coli, and helper strain
Disposable
loops
Media plate with no antibiotics (one per student group)
Media plate
with antibiotic A (one per student group)
Media plate with antibiotic B (one
per student group)
Media plate with antibiotics A and B (one per student group)
Methods:
Lab Session
1
Previous studies of bacteria have shown that that P. syringae
is resistant to Antibiotic A and E. coli is resistant to antibiotic B.
Today we will test if these bacteria can transfer antibiotic resistance to each
other through the process of conjugation. First, we will confirm their antibiotic
resistance by streaking each of these bacteria on plates containing antibiotics
A or B. This experiment also includes a helper strain, which increases the efficiency
of the conjugation process, but is not resistant to any antibiotics.
Test
to Confirm Antibiotic Resistance
1. You will need to get three agar plates,
one with antibiotic A, one with antibiotic B, and one with no antibiotic. Your
instructor will also give you access to two plates with bacteria growing on them.
One plate will have a culture of P. syringae and the other will have a culture
of E. coli. Take some time to observe the bacteria and write some of the distinguishing
features of each culture in the space below.
E.
coli:
P. syringae:
2. Label one Antibiotic A plate
and one Antibiotic B plate as shown below:
3. Using a disposable loop, streak P. syringae or E. coli on the
plates in the pattern shown below:
Session 1 Analysis
1. An hypothesis is essentially
an idea or educated guess on what you think or expect the answer to a question
to be. Your hypothesis will either will or will not be supported by the results
of your experiment. What is your hypothesis in the above experiment?
2.
What information did you base your hypothesis on?
3.
What results do you expect based on your hypothesis?
4.
What other results might you see and how would they change your hypothesis?
Setting
up the conjugation
Next
we will test if antibiotic resistance can be transferred from one strain
to another.
1. Label three microfuge
tubes: P. syringae, E. coli, or Mix.
2.
Label the bottom of a media plate (no antibiotics) as shown below:
3.
Using a transfer pipette, fill each tube with approximately 500 ml sterile water
(the tubes are graduated).
4.
Using a loop, scrape a generous amount of cells off of the P. syringae plate and
shake off in the P. syringae micro-centrifuge tube.
5.
Dispose of the P. syringae loop as instructed to by your instructor.
6.
Take a clean sterile loop and repeat this for the E. coli.
7.
Using a pipette dropper, pipette the water in the tubes up and down to unclump
the cells. Before disposing of the pipette dropper, put of drop of each suspension
(P. syringae or E. coli) into the C tube and then a drop onto the
media plate where you labeled. Try to combine approximately equal amounts of each
of the three bacteria in the Mix tube. (remember to use a different dropper for
each different type of bacteria.)
8.
Gently shake the Mix tube to mix.
9.
Using a new pipette dropper, spot a drop of the cell suspension from the conjugation
mixture onto the media plate where you labeled Mix.
10.
Let dry and keep at room temperature for 48 hours.
Lab Session 2
1. Before you begin, check the plates you streaked during session 1. What do you
see? Which bacteria were able to grow on:
a. Media with antibiotic A:
b.
Media with antibiotic B:
c. Media
with no antibiotic:
2. Label
one media plate with antibiotics A and B with P. syringae, E. coli,
and Mix as shown below:
3. Streak the bacteria spot from the plate from session 1 (with no antibiotics)
in the corresponding space on the plate with both antibiotics.
4.
Incubate your plate at room temperature for at least 48 hours and answer the following
questions.
a. What is your hypothesis in the above experiment?
b. What information did you base your hypothesis on?
c.
What results do you expect based on your hypothesis?
d.
What other results might you see and how would they change your hypothesis?
Lab
Session 3
Check the plate with both antibiotics for growth. Did
you see growth of :
· E. coli
·
P. syringae
· The
mixture
Post-lab
questions:
1. Based
on your observations from session 1, which bacteria is growing on the plate with
both antibiotics that you plated on session 2? How do you account for this growth?
2. There are several ways bacteria can be resistant to an antibiotic: they can
develop a spontaneous mutation, have an enzyme that breaks down the antibiotic, or have a mechanism that pumps the antibiotic out of the bacterium.
Spontaneous mutations occur at random and are reletively rare, while genes for
anitbiotic resistance enzymes are often able to be transferred from one bacteria
to another. Which one of these did the bacteria in this module have?
3.
What problems can the over use of antibiotics cause? Explain how these problems
are caused.
