Mouthwash Effectiveness in Mouthwash Effectiveness in Eliminating BacteriaEliminating Bacteria

By: Matthew Mancuso

Department of BiologyTennessee Technological University, Cookeville, TN 38505

OverviewOverview

IntroductionIntroductionBackgroundBackgroundHypothesisHypothesisMaterials, Methods, and Data CollectionMaterials, Methods, and Data CollectionTimelinesTimelinesExpected ResultsExpected ResultsBenefitsBenefits

IntroductionIntroduction

This will test the effectiveness of This will test the effectiveness of several kinds of mouthwash which several kinds of mouthwash which claim to be antibacterial: Scope®, claim to be antibacterial: Scope®, Listerine®, and Cepasol®.Listerine®, and Cepasol®.

Against several common strains of Against several common strains of bacteria found in the mouth: Genus bacteria found in the mouth: Genus Streptococcus, Bacillus, and Streptococcus, Bacillus, and Pseudomonas. Pseudomonas.

ObjectiveObjective

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

ExperimentalExperimental

Tests will be run both with dilutions of the Tests will be run both with dilutions of the mouthwash samples in tubes, and streak mouthwash samples in tubes, and streak plates with discs treated with mouthwash.plates with discs treated with mouthwash.

These will determine the strength of the These will determine the strength of the antimicrobial agent in the mouthwash, as antimicrobial agent in the mouthwash, as well as its ability to inhibit formation of new well as its ability to inhibit formation of new bacterial colonies.bacterial colonies.

ExpectationsExpectations

All 3 brands contain alcohol, which is All 3 brands contain alcohol, which is effective in killing bacteria in solution, the effective in killing bacteria in solution, the tubes should show a correlation between tubes should show a correlation between alcohol % and ability to eliminate bacteria.alcohol % and ability to eliminate bacteria.

On the plates the alcohol will evaporate On the plates the alcohol will evaporate leaving only the antibacterial agents to slow leaving only the antibacterial agents to slow the growth of bacteria, these will show the the growth of bacteria, these will show the strength of the other antimicrobial agents in strength of the other antimicrobial agents in the mouthwash.the mouthwash.

BackgroundBackground

Data recorded in these tests should Data recorded in these tests should correlate with other studies on similar effects correlate with other studies on similar effects of mouthwashes.of mouthwashes.

These include dilution tests which suggest These include dilution tests which suggest the effectiveness of alcohol, as well as the effectiveness of alcohol, as well as active ingredient tests.active ingredient tests.

Journal SourcesJournal Sources ““The Effect of Mouthwash on the Bacteria Found in the Mouth Over a The Effect of Mouthwash on the Bacteria Found in the Mouth Over a

Period of Time.” Dana A. Blackburn (Bulletin of the South Carolina Period of Time.” Dana A. Blackburn (Bulletin of the South Carolina Academy of Science, Annual 2003)Academy of Science, Annual 2003)

““The Role of Therapeutic Antimicrobial Mouthrinses in Clinical Practice.” The Role of Therapeutic Antimicrobial Mouthrinses in Clinical Practice.” Michael L. Barnett. (Journal of the American Dental Assoc, June 2003 )Michael L. Barnett. (Journal of the American Dental Assoc, June 2003 )

““Effects of a Chlorhexidine Gluconate – Containing Mouthwash on the Effects of a Chlorhexidine Gluconate – Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems.” Robert G. Bartolo; Andrew J. McBain; Carl E. Catrenich; Ecosystems.” Robert G. Bartolo; Andrew J. McBain; Carl E. Catrenich; Duante Charbonneau; Ruth G. Ledder; Peter Gilbert. (Applied Duante Charbonneau; Ruth G. Ledder; Peter Gilbert. (Applied Environmental Microbiology Aug. 2003)Environmental Microbiology Aug. 2003)

““The Antimicrobial Effect of Mouth Rinses When Used for 15 Seconds.” The Antimicrobial Effect of Mouth Rinses When Used for 15 Seconds.” Darcie Bell; Laura Bretsch; Michael Lents; James D Kettering. (Journal Darcie Bell; Laura Bretsch; Michael Lents; James D Kettering. (Journal of Dental Hygiene, Fall 2000)of Dental Hygiene, Fall 2000)

HypothesisHypothesis

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

Materials: PlatesMaterials: Plates

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

Methods: PlatesMethods: Plates

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

Methods: PlatesMethods: Plates

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

Methods: PlatesMethods: Plates

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

Methods: PlatesMethods: Plates

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

Data Collection: PlatesData Collection: Plates

BacillusBacillus Agar Plate ResultsAgar Plate Results

Inhibition radius in mm from discInhibition radius in mm from disc

Disc #Disc #

11 22 33 44 55

ScopeScope

ListerineListerine

CepasolCepasol

ControlControl

Materials: TubesMaterials: Tubes

Test tubes:60, for each of the 3 mouthwash to be Test tubes:60, for each of the 3 mouthwash to be mixed at each of 5 dilutions (1, 1:1, 1:10, 1:100, mixed at each of 5 dilutions (1, 1:1, 1:10, 1:100, 1:1000) and each to be paired with the 3 different 1:1000) and each to be paired with the 3 different bacteria, and 15 for the control, a dilution of bacteria, and 15 for the control, a dilution of common laboratory antiseptic to which the results common laboratory antiseptic to which the results may be compared .may be compared .

Broth: nutrient broth to be used in each of the test Broth: nutrient broth to be used in each of the test tubes as the diluting agent.tubes as the diluting agent.

No other lab equipment will be necessary, as No other lab equipment will be necessary, as results will be immediately visible to the naked eye.results will be immediately visible to the naked eye.

Methods: TubesMethods: Tubes

The first test tube of The first test tube of each dilution is first each dilution is first filled with 110mL of filled with 110mL of the pure mouthwash.the pure mouthwash.

The 4 subsequent The 4 subsequent tubes are filled with tubes are filled with 90mL of broth.90mL of broth.

Methods: TubesMethods: Tubes

10mL of liquid is then 10mL of liquid is then removed from the 1removed from the 1stst tube and placed into tube and placed into the 2the 2ndnd, making this , making this mixture 1/10 as mixture 1/10 as strong as the original, strong as the original, this is continued from this is continued from the 2the 2ndnd to the 3 to the 3rdrd, 3, 3rdrd to the 4to the 4thth, and 4, and 4thth to to the 5the 5thth..

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

Data Collection: TubesData Collection: Tubes

BacillusBacillus Test Tube ResultsTest Tube Results

Bacteria present (pos. or neg.)Bacteria present (pos. or neg.)

DilutionDilution

11 1/101/10 1/1001/100 1/1,0001/1,000 1/10,0001/10,000

ScopeScope

ListerineListerine

CepasolCepasol

ControlControl

TimelinesTimelines

Preparation of each set of tests should Preparation of each set of tests should take less than 1 day.take less than 1 day.

Incubation should be run for 2 or 3 days to Incubation should be run for 2 or 3 days to be sure maximum growth has occurred.be sure maximum growth has occurred.

Examination of the results should take less Examination of the results should take less than 1 day as well.than 1 day as well.

Expected ResultsExpected Results

It is expected to see a correlation between It is expected to see a correlation between alcohol % and bacteria killed in solution as alcohol % and bacteria killed in solution as alcohol is a known antimicrobial.alcohol is a known antimicrobial.

Results from the various antiseptics in the Results from the various antiseptics in the products should vary as the effectiveness of products should vary as the effectiveness of these specific chemicals on the bacteria may be these specific chemicals on the bacteria may be different, though it is expected that Listerine, different, though it is expected that Listerine, having more antimicrobial substances should having more antimicrobial substances should prove more effective than the other 2.prove more effective than the other 2.

BenefitsBenefits

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