Bacterial antibiotic resistance is an important concept in the process of understanding the nature and adaptability of bacterial species. By mating a kanamycin resistant strain with a non-resistant strain, students will observe the reproductive process and transfer of antibiotic resistance and therefore generate understanding of bacterial life cycles and evolution.
1) Become familiar with microbiology laboratory techniques.
2) Observe the bacterial resistance resulting from the mating process.
3) Develop questions and theories based on the scientific conclusions determined in the experiment.
Learning Resources and Materials
Per group of four students:
1) pOX38-Km bacteria strains and ED24 bacteria strains
2) 2 TSB plates containing Km and Spc antibiotics (1 for control, 1 for growing transformed ED24)
3) 1 TSB plate containing Km Sm antibiotics (for growing pOX38 donors)
4) Eppendorf tubes
5) transfer loop
6) Bunsen burner or alcohol lamp
7) 1xSSC stock solution
8) water bath (37 deg C)
9) micropipettor and tips
2) discard container
Development of Lesson
Students will work in a group of four to complete the experiment after listening to a lecture on bacterial resistance. This lecture will focus on the mechanics of gene recombination i.e. bacterial mating and the development and spread of bacterial resistance.
First 20 minutes of class:
1) Discuss with the class different types of bacterial infections and the many antibiotics used to treat them.
3) Lecture about the process of bacterial reproduction and the process in which bacteria are able to develop resistance and then transfer resistance through conjugation.
4) Encourage student questions about the subject and then demonstrate the lab procedure.
Next 30 minutes of class:
1) Students will obtain a test tube containing 800ml TSB (broth for bacteria) and label it with group name and "ED24 + pOX38".
2) Using a micropopettor and fresh tip, add 100 ml ED24 to the test tube.
3) Attach a fresh tip and then add 100 ml pOX38 to the test tube.
4) Place the tube in the water bath for 30 minutes.
5) While conjugation occurs, set --up the conjugation plate as follows:
6) Label the KmSpc plate with the group name, five points to spot dilution samples, and type of bacteria that should grow ("recipients--ED24"). Repeat this step for a second "donor" plate.
7) Using the micropipettor and fresh tip, fill each of five Eppendorf tubes with 180ml 1xSSC to dilute bacteria.
8) After 10 minutes, remove test tube with the conjugating bacteria from the water bath and gently tap the tube to interrupt the mating process.
9) Using a micropipettor and fresh tip, fill the first Eppendorf tube with 20ml of your sample, close and tap to mix well.
10) Draw 20 ml from the first Eppendorf tube and add to the second tube. Mix well.
11) Repeat this procedure for tubes 3-5.
12) Starting with Eppendorf tube #5, draw 10 ml of the sample and spot the 5 spot on the KmSpc "recipient" plate. Repeat with 10 ml samples from tubes 4-1.
13) Cover the plate and set aside, do not disturb until samples are dry.
14) Place in the incubator for 24 hours.
15) After 24 hours, remove your plates and draw the results of each plate. Write a summary
for each picture explaining your results.
1) For students with disabilities, allow them to participate in the experiment with additional teacher supervision or aid.
2) Encourage group involvement if possible.
1) Ensure student comprehension by evaluating summaries and diagrams.
2) Asses group participation as the experiment is carried out.
Once the students bring back their summaries and diagrams a discussion of the expected results will ensue and explanations to student questions will be provided.
Depending on the accuracy and nature of the results of this experiment, it will be evaluated for future use. This evaluation will also take into account student feedback and the possibility of more effective derivatives.
I feel that the benchmarks of this lesson were well represented by this experiment. The ability of bacteria to pass on antibiotic resistance through conjugation was demonstrated. Accommodations were made when necessary. I learned how to present this experiment and how best to explain the procedure to students who have not encountered these lab techniques before. In the future this lab will be used for this concept, however a pre-lab explanation of techniques may be helpful because of the time restraint.