Does Mouthwash Kill Oral Bacteria?

The following is a slightly more complex (but more effective) experiment than the procedure previously published in the Suite101 articles 1. "Does Mouthwash Kill Bacteria? Data Collection " and 2. "Does Mouthwash Kill Bacteria? Data Analysis". See photos at the bottom of this article for additional information on setup and method.

Like the previous experiment, this one is also 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 standard test tubes
• 1 test tube with sterile physiological saline or sterile water
• test tube rack
• 2 plastic Petri dishes containing TSY agar
• 5 sterile cotton swabs

Additional Materials Needed:

• 1 ml plastic disposable pipettes
• permanent marker or wax pencil for labeling
• non-alcohol mouthwash (such as Crest)
• alcohol-based mouthwash (such as Listerine)
• biohazard bags

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 half that contains TSY).

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 saline. This inoculates the saline with bacteria from the mouth.
3. Use a new disposable 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" labeles side of agar plate.

Disposal of Materials

Used swabs should be placed in biohazard bags and all test tubes should be autoclaved prior to cleaning.

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.