1. Mouthwash Effectiveness in Eliminating Bacteria By: Matthew Mancuso Department of Biology Tennessee Technological University, Cookeville, TN 38505

2. Overview Introduction Introduction Background Background Hypothesis Hypothesis Materials, Methods, and Data Collection Materials, Methods, and Data Collection Timelines Timelines Expected Results Expected Results Benefits Benefits

3. Introduction This will test the effectiveness of several kinds of mouthwash which claim to be antibacterial: Scope®, Listerine®, and Cepasol®. This will test the effectiveness of several kinds of mouthwash which claim to be antibacterial: Scope®, Listerine®, and Cepasol®. Against several common strains of bacteria found in the mouth: Genus Streptococcus, Bacillus, and Pseudomonas. Against several common strains of bacteria found in the mouth: Genus Streptococcus, Bacillus, and Pseudomonas.

4. Objective To determine if commercially available mouthwashes are capable of performing the advertised elimination of common oral bacteria. To determine if commercially available mouthwashes are capable of performing the advertised elimination of common oral bacteria.

5. Experimental Tests will be run both with dilutions of the mouthwash samples in tubes, and streak plates with discs treated with mouthwash. Tests will be run both with dilutions of the mouthwash samples in tubes, and streak plates with discs treated with mouthwash. These will determine the strength of the antimicrobial agent in the mouthwash, as well as its ability to inhibit formation of new bacterial colonies. These will determine the strength of the antimicrobial agent in the mouthwash, as well as its ability to inhibit formation of new bacterial colonies.

6. Expectations All 3 brands contain alcohol, which is effective in killing bacteria in solution, the tubes should show a correlation between alcohol % and ability to eliminate bacteria. All 3 brands contain alcohol, which is effective in killing bacteria in solution, the tubes should show a correlation between alcohol % and ability to eliminate bacteria. On the plates the alcohol will evaporate leaving only the antibacterial agents to slow the growth of bacteria, these will show the strength of the other antimicrobial agents in the mouthwash. On the plates the alcohol will evaporate leaving only the antibacterial agents to slow the growth of bacteria, these will show the strength of the other antimicrobial agents in the mouthwash.

7. Background Data recorded in these tests should correlate with other studies on similar effects of mouthwashes. Data recorded in these tests should correlate with other studies on similar effects of mouthwashes. These include dilution tests which suggest the effectiveness of alcohol, as well as active ingredient tests. These include dilution tests which suggest the effectiveness of alcohol, as well as active ingredient tests.

8. Journal Sources “The Effect of Mouthwash on the Bacteria Found in the Mouth Over a Period of Time.” Dana A. Blackburn (Bulletin of the South Carolina Academy of Science, Annual 2003) “The Effect of Mouthwash on the Bacteria Found in the Mouth Over a Period of Time.” Dana A. Blackburn (Bulletin of the South Carolina Academy of Science, Annual 2003) “The Role of Therapeutic Antimicrobial Mouthrinses in Clinical Practice.” Michael L. Barnett. (Journal of the American Dental Assoc, June 2003 ) “The Role of Therapeutic Antimicrobial Mouthrinses in Clinical Practice.” Michael L. Barnett. (Journal of the American Dental Assoc, June 2003 ) “Effects of a Chlorhexidine Gluconate – Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems.” Robert G. Bartolo; Andrew J. McBain; Carl E. Catrenich; Duante Charbonneau; Ruth G. Ledder; Peter Gilbert. (Applied Environmental Microbiology Aug. 2003) “Effects of a Chlorhexidine Gluconate – Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems.” Robert G. Bartolo; Andrew J. McBain; Carl E. Catrenich; Duante Charbonneau; Ruth G. Ledder; Peter Gilbert. (Applied Environmental Microbiology Aug. 2003) “The Antimicrobial Effect of Mouth Rinses When Used for 15 Seconds.” Darcie Bell; Laura Bretsch; Michael Lents; James D Kettering. (Journal of Dental Hygiene, Fall 2000) “The Antimicrobial Effect of Mouth Rinses When Used for 15 Seconds.” Darcie Bell; Laura Bretsch; Michael Lents; James D Kettering. (Journal of Dental Hygiene, Fall 2000)

9. Hypothesis Antimicrobial mouthwashes are effective at killing bacteria but not as effective as the product labeling suggests. Antimicrobial mouthwashes are effective at killing bacteria but not as effective as the product labeling suggests.

10. Materials: Plates Bacteria: Millions of each, Streptococcus, Pseudomonas, and Bacillus, bacteria commonly found on food and in the mouth. Agar plates: 12, one for each type of bacteria/mouthwash pairing and 3 for control. Testing discs: (60)5 per plate, this will give many sets of data for each mouthwash while not wasting plates.

11. Methods: Plates Each of the 3 species of bacteria will be cultured in broth then applied in streaks to agar plates so the bacteria are evenly distributed about the surface.

12. Methods: Plates Discs soaked in each of the different mouthwashes will then be applied to the agar plates (1 type per plate), and discs soaked in water will be applied to the control.

13. Methods: Plates After the bacteria is allowed to incubate for several days the zones of inhibition (area around the discs where no bacteria is growing) will be measured.

14. Methods: Plates The size of the zone corresponds to the ability of the antimicrobial agent in the mouthwash to inhibit the growth of bacteria.

15. Data Collection: Plates Bacillus Agar Plate Results Inhibition radius in mm from disc Disc # 12345 Scope Listerine Cepasol Control

16. Materials: Tubes Test tubes:60, for each of the 3 mouthwash to be mixed at each of 5 dilutions (1, 1:1, 1:10, 1:100, 1:1000) and each to be paired with the 3 different bacteria, and 15 for the control, a dilution of common laboratory antiseptic to which the results may be compared. Broth: nutrient broth to be used in each of the test tubes as the diluting agent. No other lab equipment will be necessary, as results will be immediately visible to the naked eye.

17. Methods: Tubes The first test tube of each dilution is first filled with 110mL of the pure mouthwash. The 4 subsequent tubes are filled with 90mL of broth.

18. Methods: Tubes 10mL of liquid is then removed from the 1 st tube and placed into the 2 nd, making this mixture 1/10 as strong as the original, this is continued from the 2 nd to the 3 rd, 3 rd to the 4 th, and 4 th to the 5 th.

19. Methods: Tubes After a few days each tube will be examined to determine if bacteria are growing in it, typically indicated by sediment and cloudiness of the broth. Then the lowest concentration of mouthwash with no bacteria growing in it can be compared to the control to determine the strength of the antimicrobial agents in each type.

20. Data Collection: Tubes Bacillus Test Tube Results Bacteria present (pos. or neg.) Dilution 11/101/1001/1,0001/10,000 Scope Listerine Cepasol Control

21. Timelines Preparation of each set of tests should take less than 1 day. Preparation of each set of tests should take less than 1 day. Incubation should be run for 2 or 3 days to be sure maximum growth has occurred. Incubation should be run for 2 or 3 days to be sure maximum growth has occurred. Examination of the results should take less than 1 day as well. Examination of the results should take less than 1 day as well.

22. Expected Results It is expected to see a correlation between alcohol % and bacteria killed in solution as alcohol is a known antimicrobial. It is expected to see a correlation between alcohol % and bacteria killed in solution as alcohol is a known antimicrobial. Results from the various antiseptics in the products should vary as the effectiveness of these specific chemicals on the bacteria may be different, though it is expected that Listerine, having more antimicrobial substances should prove more effective than the other 2. Results from the various antiseptics in the products should vary as the effectiveness of these specific chemicals on the bacteria may be different, though it is expected that Listerine, having more antimicrobial substances should prove more effective than the other 2.

23. Benefits This research should help a person to decide what mouthwash to purchase based upon its contents and not just the claims listed on the bottle. This research should help a person to decide what mouthwash to purchase based upon its contents and not just the claims listed on the bottle.