1. Attack of the Superbugs Lab Introduction
Mrs. Stewart
Medical Interventions

2. Superbugs What is a “superbug”?
Bacteria that have become stronger and less responsive to antibiotic treatment

3. How can bacteria share resistance?
Genetic Transfer Method
Description
Conjugation
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)

4. 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.

5. 4 types growth plates LB Agar LB + Str
LB = Luria Broth - growth medium for bacterial cells
*Str = Streptomycin
LB /Str /Amp
LB + Amp
*Str/Amp = Streptomycin & Ampicillin
*Amp = Ampicillin

6. 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

7. How to label the plates
I II
I II
Group Name
Group Name

8. 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

9. 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

10. LB agar LB + Str LB + Amp LB + Str + Amp Predicted Results
Observed Results
E. coli I
E. coli II
LB agar
LB + Str
LB + Amp
LB + Str + Amp
+ = Growth = No Growth

11. Prediction
Predict the results from your confirmation plates that you will expect to record

12. Predict
What will happen when the E. coli I strain is mixed with the E. coli II strain?

13. Evaluate
Look at the genes each strain of E. coli carries. If conjugation occurs, specifically describe which strain will transfer what DNA.

14. Lab Day 3:
Use new “mixed” bacterial cultures and spread onto the 3 types of antibiotic plates
Incubate overnight

15. Predict
What would you expect to see on these growth plates?
Why?

16. 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.

17. Conclusion Questions
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?
Explain what the results indicate about the new strain of bacteria produced when both strains of E. coli were mixed together.
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?

18. Student Lab Schedule
Day:
Time Needed:
Activity:
How to Store:
Lab Day 1
~ 30 minutes
Students prepare confirmation plates.
Invert the plates, label, and incubate at 37˚C for 24 hours.
Lab Day 2
Students observe confirmation plates and prepare “Mix Plate.”
Lab Day 3
Students streak cultures from “Mix Plate” onto antibiotic plates.
Lab Day 4
~ 10 minutes
Students observe & record results
Clean-up all materials.