1. Mouthwash Effects on Microbial Flora
Gabe Winbush
Grade 9
Pittsburgh Central Catholic High School

2. Question
Which mouthwash is more effective in killing bacteria: Tom’s or Listerine? And do higher concentrations of mouthwash have a significant effect on bacteria survivorship?

3. Mouthwash: Kills germs that cause bad breath Helps prevent gum disease
Gingivitis
Inflammation of the gums
Periodontitis
Inflammation and infection of the ligaments and bones that support teeth
Sometimes contains alcohol to help kill bacteria

4. Mouthwashes used in this experiment:
Tom’s
Natural mouthwash
Alcohol free
Gluten free
Listerine
Is the only nationally branded antimicrobial mouthwash to receive the ADA Seal of Acceptance for fighting plaque and gingivitis.
Claims to protect teeth for 24 hours
Contains alcohol

5. Gram+ vs. Gram- Bacteria
Staph: Gram Positive Bacteria
E. coli: Gram Negative Bacteria
Staph is model for Gram+
Gram+ bacteria have a thick peptidoglycan layer
Gram+ bacteria appear purple when stained
E. coli is model for Gram-
Gram- bacteria have membrane covering thin layer of peptidoglycan
Gram- appear pink when stained

6. Staph (Staphylococcus Epidermidis)
Gram positive
Round (Cocci)
Forms grape-like clusters
Symptoms
Boils
Redness
Swelling

7. E. coli (Escherichia coli)
Gram negative
A common symbiont found in animal intestines (including humans)
Most strains are harmless
Some pathogenic strains are found in food as a result of cross contamination (many times during the slaughtering process)
Symptoms
Diarrhea
Urinary tract infections
Pneumonia

8. Purpose of Experiment
To assess the effects of different concentrations of two different mouthwashes on gram+ and gram- bacterial models

9. Hypotheses
Null Hypotheses: The two mouthwashes will not significantly vary in reducing bacterial survivorship
Alternate Hypotheses: Listerine will reduce bacterial survivorship more effectively than Tom’s

10. Materials Sterile mouthwashes (Listerine and Tom’s) Vortex Ethanol
Metal wire spreader
Micro pipettes and tips
Macro pipettes
Test tube rack
Glass tubes
Incubator
Escherichia coli (dh5 alpha)
Staphylococcus epidermidis
LB agar plates
SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)

11. Procedure E. coli/Staph was grown overnight in sterile LB media.
A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
The culture was incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately cells/ml.
The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.
The mouthwashes were diluted with sterile dilution fluid to concentrations of 0%, 1%, 10%, and 50% to total 9.9 ml.
0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 cells/ml.

12. Chart of concentrations
0 %
0.1% 1% 5%
Sterile Dilution Fluid (SDF)
9.9 ml
9.8 ml
8.9 ml
4.9 ml
E. coli/Staph
0.1 ml
Mouthwash
0 ml
1 ml
5 ml
Total
10 ml

13. Procedure cont.
The solution was mixed by vortexing and placed at room temperature for 15 minutes.
After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on LB plates.
The plates were incubated at 37°C for 24 hours.
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

14. Procedure (infused plates)
1. E. coli was grown overnight in sterile LB media.
2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
3. The culture was placed in an incubator at 37°C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.
4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
µl of each mouthwash were spread evenly on LB Agar plates.

15. Procedure cont. (infused plates)
The plates were then inverted and placed in an incubator at a heat of 37°C for 15 minutes
After vortexing to evenly suspend cells, 100 µl of E. coli/Staph from control tube was spread on the plates
The plates were incubated at 37°C for 24 hours
The resulting colonies were counted. Each colony is assumed to have arisen from one cell

16. Effect of Mouthwash on Staph Survivorship
P=1.08E-08
P= 0.17

17. Effect of mouthwash on E. coli Survivorship
P=1.78E-09
P=1.24E-08
P= 0.75

18. Dunnett’s Test Staph T Crit.= 3.29 E. coli T Crit.= 3.29
Concentration of mouthwash
T-value
interpretation
1% (Listerine)
1.09
Not significant
8.2
Significant
10% (Listerine)
10.98
15.6
50% (Listerine)
13.89
19.12
1% (Tom’s)
2.76
Not Significant
5.92
10% (Tom’s)
8.02
13.75
50% (Tom’s)
10.76
14.52

19. Number of colonies on infused plates graph

20. Dunnett’s Test (infused plates)
T Crit.= 2.57
Artificial Mouthwash
Natural Mouthwash
Type of bacteria
T-value
Interpretation
Staph
1.12
Not significant
0.88
E. coli
0.49
0.37

21. Mouthwash effects on microbial survivorship

22. Conclusions Null hypothesis can be accepted
The 10% and 50% concentrations of mouthwash had a significant negative effect on the survivorship of staph
The 1%, 10%, and 50% concentrations of mouthwash had a significant negative effect on the survivorship of E. coli
The two mouthwashes did not have a significant difference in the survivorship of bacteria
The infused plates did not significantly differ from normal plates in the amount of bacteria killed

23. Limitations and Extensions
Plating was not perfectly synchronized
Only four concentrations of mouthwash were used
Extensions
Use more than two variables
Use more concentrations
Use more types of bacteria

24. References