Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?

This science fair project involves the use of the bacteria E. coli. While E. coli is not considered a biohazardous or dangerous bacteria, it is important to always properly clean and dispose of bacteria and supplies that come in contact with it. See the Bacterial Safety guidelines for more details on how to handle bacterial cleanup and waste.

Preparing Plates for Disk Diffusion Test

For this experiment, it is important to innoculate the plate with a uniform distribution of bacterial colonies, and to use the exact same procedure for each plate. Here are the steps for innoculating the control and test plates.

1. Note: if you want to use triclosan, here is a suggested formulation: 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 500 μg/mL (Bittell and Hughes, 2003).
2. Prepare serial two-fold dilutions of each disinfectant.
   1. For each disinfectant, label 3 microcentrifuge tubes with disinfectant name and tube number:
      
      

      Tube #	Conc. (%)
      1	50%
      2	25%
      3	12.5%

      
      
      
   2. Pipette 500 μL of water into each tube.
   3. Pipette 500 μL of full-strength disinfectant into the first tube. Mix thoroughly
   4. Using a fresh tip, pipette 500 μL of solution from the first tube into the second tube. Mix thoroughly.
   5. Use a fresh tip, pipette 500 μL of solution from the second tube into the third tube. Mix thoroughly.
3. You can use a pencil or permanent marker to label each sterile disk with a code for the disinfectant concentration for that disk (e.g., the tube number from the serial dilution). You will need four disks for each concentration. If you label the disks, keep track of the codes in your lab notebook, wrap the disks in aluminum foil, and sterilize in a 300° oven for 30 minutes.
4. Use a permanent marker to divide the bottom of the three test plates into four equal sections. Label each plate with the disinfectant and dilution to be tested.
5. Use a permanent marker to divide the control plate into four equal sections. Label the plate "no disinfectant."
6. Using proper sterile technique, innoculate each plate uniformly. Dip a sterile cotton-tipped applicator swab into the K-12 E. coli bacterial solution and then swab it gently across the plate. Swab in three directions (120° apart) to insure complete coverage of the plate. Cover the plate and wait at least five minutes for the plate to dry.
7. For the test plates, use sterile forceps to hold a single disk by the edge with forceps and dip it into the disinfectant solution to be tested (a different concentration for each test plate). Touch the disk against the side of the tube to drain off excess liquid. Place a single disinfectant disk in the center of each of the marked sections on your test plates. Use the forceps to gently press each disk against the agar surface to insure good contact. Remember to use the exact same technique for each disk—consistency is very important for this experiment.
8. For the control plate, apply sterile disks dipped in sterile water to each of the marked sections.
9. Incubate all of the plates, inverted (agar on top), overnight at 37°C (or longer if necessary, for example, at lower temperature).

Measuring Zones of Inhibition

1. After overnight incubation, examine your plates (keep them covered at all times).
   1. The control plates should show uniform colonies over the entire surface of the plate. If the distribution is highly uneven, you will need to improve your innoculation technique and repeat the experiment. The filter disks should not impede bacterial growth, since they contained only water.
   2. If your disinfectants are effective at the concentrations you tested, you should see zones of inhibition around the disinfectant disks. The clear zones around each disk should have a uniform width, since diffusion of the compounds through the agar should be uniform in every direction. If this is not the case, suspect either your impregnation technique, or poor contact of the filter paper with the agar.
2. Measure the diameter of the zone of inhibition for each disk. Keeping the lid of the plate in place, use a ruler to measure the diameter of the disk plus the surrounding clear area in millimeters (mm).
   1. Include the diameter of the disk in your measurements. For example, if your disk has a diameter of 6 mm, and the clear area has a width of 3 mm beyond the disk, the diameter of the zone of inhibition that you should measure and record would be 12 mm (6 mm + 3 mm + 3 mm). This is the standard way that zones of inhibition are measured.
   2. You will get four separate measurements for each dilution of each disinfectant—one from each quarter section of the test plate.
3. Use the values from Table 1 to evaluate the bacterial response to each compound (Johnson and Case, 1995).
   
   

   	Diameter of zone of inhibition (mm)
   Resistant	10 or less
   Intermediate	11–15
   Susceptible	16 or more

   
   
   
4. Are the diameters consistent across all four sections? Calculate the average and the standard deviation of the diameter of the zone of inhibition for each disinfectant.
5. Do the diameters of the zones of inhibition decrease as the concentration of disinfectant decreases?

Selecting for Resistant Bacteria

Now you will select the most resistant bacteria from each plate, and repeat the exposure to diluted disinfectant. You will repeat this selection process 4 times.

1. For each plate, use a sterile swab to pick up bacterial colonies growing closest to the disinfectant-impregnated disk.
2. Swirl the swab in a tube of sterile water (10 ml) to dilute the bacteria. Cover the tube and agitate it.
3. Repeat steps 4–9, in the Preparing Plates for Disk Diffusion Test section.

Bacterial Safety

Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders.

• Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
• Make sure to wash your hands thoroughly after handling bacteria.
• Proper Disposal of Bacterial Cultures
  • Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
  • Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
  • After bleach treatment is completed, these items can be placed in your normal household garbage.
• Cleaning Your Work Area
  • At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
  • Be aware of the possible hazards of disinfectants and use them carefully.