Superbugs and Antibiotic Resistance

Based upon “Evolution in the Lab: Biocide Resistance in E. Coli” by Charles W. Welden and Rex. A. Hossler, published in the January 2003 Volume of the American Biology Teacher

Modified by Kirstin Bittel and Rachel Hughes

Time: 1 Full class period plus up to 6 additional partial periods
Preparation Time: 1 hour – Prep time will vary with number of agar plates prepared
NOTE - This activity requires set up several days in advance of the lesson and some materials will need to be ordered weeks in advance.
Materials: See Pre-Class Preparation 
Superbugs and Antibiotic Resistance Protocol Sheet

Through laboratory experimentation, students test antibiotics and/or antimicrobials to determine how quickly a given bacterium develops resistance to antibiotics and/or antimicrobials. Students will use their understanding of the immune system, bacteria, and natural selection to test antibiotic resistance.

Students will be able:
1. Explain how antibiotics affect the evolution of microorganisms.
2. Explain how misuse of antibiotics can lead to the evolution of antibiotic resistant bacteria.

National Science Education Standard:
Content Standard C Life Science
- Species evolve over time. Evolution is the consequence of the interactions of (1) the potential for a species to increase its numbers, (2) the genetic variability of offspring due to mutation and recombination of genes, (3) a finite supply of the resources required for life and (4) the ensuing selection by the environment of those offspring better able to survive and leave future offspring
- Natural selection and its evolutionary consequences provide a scientific explanation for the fossil record of ancient life forms, as well as for the striking molecular similarities observed among the diverse species of living organisms.

Teacher Background
Wanting to provide the cleanest and, hopefully, healthiest environment for your family is an understandable and desirable goal. To this end we are besieged by rows of antibacterial soaps at our local supermarkets and drug stores that promise to help protect our families from harmful bacteria. But are we really compromising our health with the ubiquitous use of antibacterial soaps?

There has been debate about whether the use of antibacterial soaps stimulates the development of resistant bacteria. Of the two types of antibacterial soap used, the non-residue/ rapid killing antibacterial is not considered to stimulate the development of resistant bacteria. However, those that leave a residue, such as triclosan, are thought to be more problematic as regards allowing the growth of bacteria. Low level concentrations of anti-bacterial agents are associated with resistance of bacteria to antibacterial agents. While there is no evidence that bacterial resistance to triclosan has yet to impact clinical situations, the development of bacteria that are resistant to antibacterial soaps has great implications for clinical situations.

This exercise allows students to explore bacterial resistance to triclosan. The exercise informs students about natural selection. If the selective pressure is antibacterial treatments, the population of bacteria evolves in such a manner to be resistant to these treatments.

(Information from: http://www.tufts.edu/med/apua/Q&A/Q&A_antibacterials.html - 10/10/05)

Related and Resource Websites

For more information on E.Coli K-12 and safety considerations visit:

Current Articles on Antibacterial Resistance

Pre-Lab Preparations

  • Several days before the exercise, prepare transferred E. coli (strain K-12) from stock cultures to tryptic soy agar plates, making a bacterial lawn (a more-or-less uniform sheet of bacteria covering the agar surface). Prepare at least one plate per section. It is advisable to make extras in case of contamination.
  • Prepare 20 sterile Petri dishes of tryptic soy agar and 20 tubes of sterile tryptic soy broth per laboratory group.
  • Prepare a working solution of triclosan by dissolving the powder in a solution of 17.5% ethanol and 82.5% distilled water to a final triclosan concentration of 500µg mL ^(-1). A stock of 500 mL each of triclosan and tryptics soy broth should suffice for 150 students.
  • Set up the workstations with the following items
    • a screw top jar with cotton swabs
    • a covered dish containing sterile disks of filter paper (any diameter between 5 and 10 mm will suffice)
    • Forceps labeled “Water,” “Ethanol,” and “Triclosan” sterilized by soaking in ethanol, and air-dried before use. (Lay these on paper towels and cover to avoid contamination).
    • Waste disposal buckets for used swabs, Petri dishes, and culture tubes. (It is advisable to fill these with enough disinfectant to cover the ends of the swabs as a safety precaution).
    • A rack for culture tubes and a tray for Petri dishes
    • A box or Parafilm® and scissors (or tape to seal Petri dishes with)
    • Indelible pens for labeling tubes and dishes, Tricolsan
    • Label containers containing the following: Distilled Water, Ethanol 17.5%, and Triclosan [500µg mL ^(-1)]
    • A capped tube containing 10 mL of sterile water, labeled “E. coli.”
  • Each room should also have an incubator cabinet set at 35-37°C.
  • Just before the first lab meeting, rub a sterile swab across the surface of one of the prepared dishes with a lawn of E. coli and swirl the swab in the tube of distilled water labeled “E. coli”. This provides students with a dilute, spreadable suspension of E. coli.
  • Copy articles for classroom discussion




Note A: If large quantities of plates are out of budgetary considerations for you, you might consider running this lesson as a whole class demonstration.

Note B: Although E. coli K12 is used in this lesson because of its safety and accessibility, other forms of bacteria can be used. If you decide to use something other than E.coli K12 be sure to check safety information and follow appropriate safety precautions.

Note C: Contamination can be a problem in this exercise. To reduce contamination do the following:

  • Have students use cotton swabs once and only to touch the bacteria cultures and agar plates. Instruct students not to touch the agar plates or culture broth with anything other than the swabs.
  • Have students keep the Petri dishes closed at all times except during transfer of bacteria. Even then, students should keep the lids opened briefly and narrowly. When the lid is open, it should help to be as close to the agar surface as possible.
  • When swirling the cotton swab over the agar to make a bacterial lawn, have students rub gently to avoid puncturing the agar and then dispose of the swabs in a designated waste container. All liquid cultures and plates should be placed in the same container and sterilized before disposal and labeled as biological waste.
  • Have students wash their hands thoroughly with soap (preferably triclosan-free) and hot water after handling bacterial cultures.

Note D: The use of paper disks in this lab helps students get more accurate results. Paper disks allow the treatment to diffuse through the agar in a predictable manner and give students a clear visual about the treatment gradient caused by diffusion.

Using cotton swabs gives students the ability to transfer bacteria without faculty supervision and make spreading bacteria onto the agar plates very easy.

Day 1:
1. As students enter the room, have the following question on the board, “How do new diseases come into being?” Allow students a few minutes to record and share answers. Hopefully students will being up the idea of bacteria and diseases evolving into stronger, better adapted strains. If not, you will want to ask students higher-level thinking questions to get them to that idea

2. Tell students that today they will read a brief article about bacteria and diseases. [If you want to provide more background, you may want to copy several articles so that each student in each lab group has a different piece of information to bring to the discussion.]

3. Distribute the article to the students and have them read over the report. Then ask them “What have scientists found? What do students think abut the research? How might they conduct an experiment to test the scientists’ work?”

4. Divide students into groups and tell them they will be following a protocol to test for antibacterial resistance in E. Coli K12. Be sure to tell students that while this is a relatively harmless strain of bacteria, it is still important that they follow standard safety protocol and wear lab coats, gloves, and protective eyewear.

5. Distribute protocol sheets to students and have them read over the material. What questions (if any) do they have?

6. Have students follow steps 1-3 and then return to the front of the class for a group discussion.

7. Discuss steps 4-7, which will be conducted tomorrow. What is the zone of inhibition? [This is the area where growth of the bacteria is inhibited by the treatment on the disk.] What does it mean if the zone is “large” or “small?” If the zone decreased what does that tell them? What if the zone has increased? What if it has stayed the same? [Change in the zone is related to resistance. As the zone decreases in size, bacterial resistance is increasing.] Where on the plate will students be able to find bacteria likely to be resistant to the treatment they are applying? [The bacteria nearest the zone of inhibition are more likely to be resistant to the treatment. They have been put under selective pressure by the treatment and have survived. If the reason for survival is a genetic mutation, they will pass that on to their offspring as they are reproducing.] What is the purpose of taking bacteria and transferring it to a liquid culture? [This mixes the bacteria and allows for a more even growth of bacteria on the next plate.]

Days 2-7:

  • Have students continue to collect bacteria and re-plate them following the protocol sheet. Over the weekends, have students leave bacteria sealed on a counter. Bacteria will grow slower at room temperature. If possible, a better solution would be to place the plates in a refrigerator either before or after a 24-hour incubation period.
  • When students are not collecting data or preparing the plates for the next culture, this is an appropriate time for students to begin conducting research for the final project.

What did students discover? How did bacteria respond to the different treatments? What does that tell us about resistance? How do the different treatments affect the natural evolution of bacteria? How does that affect humans, who ultimately act as “hosts” to bacteria as they reproduce? What are the benefits and drawbacks of the use of antibacterial, antimicrobials, and antibiotics?

In their science notebooks, have students write a reflective conclusion. What did they learn? What new questions do they have? How does the lab connect to “real life?”

Embedded Assessment

Can students explain how antibiotics affect the evolution of microorganisms?

Can they explain how misuse of antibiotics can lead to the evolution of antibiotic resistant bacteria?



PULSE is a project of the Community Outreach and Education Program of the Southwest Environmental Health Sciences Center and is funded by:

NIH/NCRR award #16260-01A1
The Community Outreach and Education Program is part of the Southwest Environmental Health Sciences Center: an NIEHS Award


Supported by NIEHS grant # ES06694

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Last update: November 10, 2009
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