Name: ______

This experiment is designed to compare the effectiveness of mouthwashes containing alcohol (Listerine) to non-alcohol based mouthwashes (Crest) in reducing the populations of oral bacteria. Samples can be retrieved over time, allowing two variables to be tested:

  1. type of mouthwash (alcohol vs. non-alcohol)
  2. time oral sample is exposed to mouthwash (15 seconds vs. 30 seconds)

Materials Needed for Each Pair of Students:

  • 2 empty test tubes
  • 1 test tube with sterile water
  • 1 test tube rack
  • 2 plastic Petri dishes containing agar (Day 1)
  • 5 sterile cotton swabs

Additional Materials Needed:

  • pipettes
  • permanent marker or wax pencil for labeling
  • non-alcohol mouthwash (such as Crest)
  • alcohol-based mouthwash (such as Listerine)

Day 1

  1. You'll need a clean 50mL beaker. Set up the following at your lab table: ring stand, o-ring, gas line, bunsen burner, and wire mesh. The bottom of the o-ring needs to be 5 cm from the top of the bunsen burner. Use a metric ruler to adjust this distance and lock the o-ring. These mixing proportions make enough nutrient agar to prepare one Petri dish. Measure out exactly .5 gram of agar using the triple beam balance (Place a ½ sheet of computer paper on the balance and add agar until you get the needed amount.) Place exactly 20mL of water in the graduated cylinder. Pour the .5 gram of agar in the 50mL beaker. Place the 20 mL of water, from the graduated cyliner, into the 50mL beaker with the agar you just added. Light the bunsen burner and allow the mixture to heat up. Once this mixture starts to boil, allow it to go for one more minute to completely dissolve the agar. The mixture should be clear with no particles floating around in the solution. Allow the mixture to cool for 5 minutes (use the hot hands and set the beaker on the lab table) before moving on to the next step.
  2. Separate the Petri dish (there's a top and a bottom) and carefully fill the bottom half of the Petri dish with warm agar nutrient solution. Use the top half of the Petri dish to loosely cover the bottom portion (set the lid ajar to allow moisture to escape) and allow the solution to cool and harden until tomorrow. You will need to do this part of the lab two times so that you have two separate Petri dishes.

Day 2

Mouthwash Experiment Procedure

Labeling Petri Dish

Always label the bottom of a Petri dish (the half that contains the agar). Students can use a permanent marker to draw a line down the center bottom of each Petri dish.

The Petri dishes should be labeled with:

  • group members initials
  • type of mouthwash one alcohol one non-alcohol
  • one half of each plate should be labeled “15 seconds” the other “30 seconds.”

Labeling and Filling Test Tubes

Label one blank test tube alcohol (or Listerine) and the other non-alcohol (or Crest). Then, use two separate pipettes to add a total of 3ml of the appropriate mouthwash to each.

Taking the Bacterial Sample

This experiment is timed, so students should have all materials set up and labeled before starting. Only one student should provide the oral sample.

  1. Sweep sterile swab around mouth to obtain a good oral sample.
  2. Stir the swab containing oral sample in the test tube of sterile water. This inoculates the saline with bacteria from the mouth.
  3. Use a clean pipette to add 1 ml of the now inoculated saline to each of the test tubes of mouthwash. As soon as the inoculated saline is added, the timed experiment has begun.
  4. Gently swirl or flick the test tubes to mix in the oral sample.
  5. After 15 seconds use a new sterile swab to take a sample from the Listerine test tube and then wipe that swab over the half of the corresponding agar plate.
  6. Do the same for the Crest test tube, taking a new sterile swab, obtaining a sample from the Crest test tube and wiping it over the surface of the corresponding agar plate. (This works best if two students work together, one taking the Listerine sample, the other the Crest sample).
  7. Repeat same procedure at 30 seconds, swabbing sample on “30 second” labeled side of agar plate.

Incubating Bacterial Samples

After Petri dishes are incubated for at least 24 hours the class can examine and compare the number and variety of bacterial colonies growing on the surface of the agar. A colony is made up of million of bacteria that are visible as a dot on the agar’s surface.