Bacteria resistance to antibiotics
The materials required for this science fair project are:
- 15 agar Petri dishes
- 15 disinfected swabs
- 1 bottle of disinfected water
- 1 piece of filter paper
- 1 hole puncher
- 4 test tubes
- 1 measuring cylinder
- 100mg of antibiotic ampicillin
- 1 digital weighing scale
- 1 beaker of water
- E Coil bacteria culture
- 1 forceps
- 1 marker pen
1. The independent variable for this science project is the concentration of the antibiotic used. The dependent variable is the size of inhibition zone (which is the area free frombacteria). This is determined by measuring the diameter of the area using a ruler. The constants (control variables) are the number of times the tests are carried out, room temperature and the ingredients in the Petri dish agar.
2. Prepare 15 Petri dishes using the agar are and store in a refrigerator.
3. Bring 3 of the Petri dishes to room temperature before the start of experiment Label the Petri dishes 1mg, 3mg and 5mg.
4. Prepare and label the antibiotic mixture as follows :
a. Labeled 1mg - 1mg of ampicillin is mixed with 10ml water
b. Labeled 3mg - 3mg of ampicillin is mixed with 10ml water
c. Labeled Marked 5mg - 5mg of ampicillin is mixed with 10ml water
5. Mix 10ml of E Coli bacterium culture with 40ml water in the 4th test tube. Rinse the swab using the disinfected water. Dip it into the test tube containing the bacteria culture and swipe it gently over the agar surface of the 3 Petri dishes. Ensure that you cover the entire surface area of the dish, by rubbing in at least 3 different directions.
6. Punch out circular plates of filter paper using a hole puncher. Label the circular plates of filter paper 1mg, 3mg and 5mg. Pick up the filter paper plates using the forceps and dip them into the ampicillin solution according to the labelled concentrations. Place the filter paper plates at the center of the Petri dish according to their labels and cover them. Keep the dishes in a cool dry place for the bacteria to incubate for 4 days.
7. After 4 days, there will be a circular area around the filter paper plate called the inhibition zone where there will be no bacterial growth. Measure the diameter of this area record it in the table provided below
The concept of the inhibition zone is attributed to the Kirby-Bauer disk diffusion method. This method is used by microbiologists to assess the effectiveness of antimicrobial agents. As the ampicillin diffuses out from the filter paper into the agar, there will be a higher concentration of ampicillin nearer the disk. This concentration decreases gradually, as the distance from the edge of the disc increases. As long as the concentration of ampicillin at any given distance is high enough to properly act as an antibacterial agent, no bacteria will grow at that location. Hence, if the bacteria has increased resistance, a smaller inhibition zone will be seen, and vice versa.
8. Collect the surviving bacteria along the border of the inhibition zone are using the disinfected swab and use it to prepare the bacteria culture needed for subsequent experiments
Repeat steps 3-8 another 4 times. Measure the diameter of the inhibition zone for the repeated science fair projects and record the results in the table provided below.
Always follow laboratory safety guidelines and always practice sterile technique when handling microbes. Never have any food or drink at your workstation and always thoroughly wash your hands with disinfectant soap or alcohol before leaving your workstation. Always dispose of used material in a biohazard bag. If none are available, the bacteria should be destroyed with bleach before being disposed of.