Download Attack of the Superbugs Lab Introduction

Attack of the Superbugs
Lab Introduction
Mrs. Stewart
Medical Interventions
Superbugs
• What is a
“superbug”?
– Bacteria that
have become
stronger and
less responsive
to antibiotic
treatment
How can bacteria share resistance?
Genetic Transfer
Method
Conjugation
Description
The one-way transfer of DNA
(plasmid) between bacteria in close
cellular contact
Transformation
The genetic modification of a
bacterium by incorporation of free
DNA from the surrounding
environment (usually caused by
another ruptured bacterial cell)
Transduction
The transfer of genetic material from
one bacterium to another by a genetic
vector (bacteriophage virus)
This lab uses 2 types of Bacteria:
• E. coli I
– contains a gene found on the chromosomal DNA
coding for streptomycin resistance
• E. coli II
– contains a gene found on the plasmid DNA coding
for ampicillin resistance.
4 types growth plates
LB Agar
LB = Luria Broth - growth
medium for bacterial cells
LB + Amp
*Amp = Ampicillin
LB + Str
*Str = Streptomycin
LB /Str /Amp
*Str/Amp = Streptomycin &
Ampicillin
Lab Day 1: Confirmation Plates
• Obtain 1 of each type of growth plate
– 4 plates total
• Streak E. coli I on 4 plates
• Streak E. coli II on 4 plates
• Incubate overnight
How to label the plates
I
II
Group Name
I
II
Group Name
Evaluate
• Today’s lab (step #1) is called
“preparing the confirmation
plates”. What are we
confirming?
These plates will
confirm that our E.
coli strains are
resistant to the
antibiotic we said
they were
Lab Day 2:
• Observe confirmation plate results
• Prepare “mix” plate
– You will literally be mixing the two types of
bacteria together onto one plate
• Incubate overnight
Predicted Results
Observed Results
E. coli I
E. coli I
E. coli II
E. coli I
LB agar
LB + Str
LB +
Amp
LB + Str
+ Amp
+ = Growth
-- = No Growth
Prediction
• Predict the results from your confirmation
plates that you will expect to record
Predict
• What will happen when the E. coli I
strain is mixed with the E. coli II strain?
Evaluate
• Look at the genes each strain of E. coli carries.
If conjugation occurs, specifically describe
which strain will transfer what DNA.
Lab Day 3:
• Use new “mixed” bacterial cultures and
spread onto the 3 types of antibiotic plates
• Incubate overnight
Predict
• What would you expect to see on
these growth plates?
• Why?
Lab Day 4
• Observe and record your results
• Remember: Pictures of lab results will be
required in your lab reports
*Growth plates will be discarded after today.
Any results not recorded will be lost.
Conclusion Questions
1. Why did the E. coli I strain grow on both the LB agar
plate and the LB agar plate with streptomycin but
not grow on the LB agar plate with ampicillin?
2. Explain what the results indicate about the new
strain of bacteria produced when both strains of E.
coli were mixed together.
3. Based on the results of the experiment as well as
what you learned about the mechanism of gene
transfer between bacterial cells, was the
streptomycin resistant gene transferred from Strain I
to Strain II, or was the ampicillin resistant gene
transferred from Strain II to Strain I? How do you
know?
Day:
Lab
Day 1
Time
Needed:
~ 30
minutes
Lab
Day 2
~ 30
minutes
Lab
Day 3
~ 30
minutes
Lab
Day 4
~ 10
minutes
Student Lab Schedule
Activity:
How to Store:
Students prepare
confirmation
plates.
Students observe
confirmation plates
and prepare “Mix
Plate.”
Students streak
cultures from “Mix
Plate” onto
antibiotic plates.
Students observe
& record results
Invert the plates,
label, and incubate
at 37˚C for 24 hours.
Invert the plates,
label, and incubate
at 37˚C for 24 hours.
Invert the plates,
label, and incubate
at 37˚C for 24 hours.
Clean-up all
materials.