Mouthwash Effects on Microbial Flora

Gabe WinbushGrade 9

Pittsburgh Central Catholic High School

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?

Mouthwash:

• Kills germs that cause bad breath• Helps prevent gum disease

GingivitisInflammation of the gums

PeriodontitisInflammation and infection of the ligaments and bones

that support teeth

• Sometimes contains alcohol to help kill bacteria

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

Gram+ vs. Gram- BacteriaStaph: Gram Positive Bacteria• Staph is model for Gram+• Gram+ bacteria have a

thick peptidoglycan layer• Gram+ bacteria appear

purple when stained

E. coli: Gram Negative Bacteria

• E. coli is model for Gram-• Gram- bacteria have

membrane covering thin layer of peptidoglycan

• Gram- appear pink when stained

Staph (Staphylococcus Epidermidis)

• Gram positive• Round (Cocci)• Forms grape-like clusters• Symptoms– Boils– Redness– Swelling

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

Purpose of Experiment

• To assess the effects of different concentrations of two different mouthwashes on gram+ and gram- bacterial models

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

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)

Procedure1. E. coli/Staph 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 incubated until a density of 50 Klett

spectrophotometer units was reached. This represents a cell density of approximately 108-109 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The mouthwashes were diluted with sterile dilution fluid to concentrations of 0%, 1%, 10%, and 50% to total 9.9 ml.

6. 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.

Chart of concentrationsConcentration 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 0.1 ml 0.1 ml 0.1 ml

Mouthwash 0 ml 0.1 ml 1 ml 5 ml

Total 10 ml 10 ml 10 ml 10 ml

Procedure cont.

7. The solution was mixed by vortexing and placed at room temperature for 15 minutes.

8. After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on LB plates.

9. The plates were incubated at 37°C for 24 hours.10. The resulting colonies were counted. Each colony is

assumed to have arisen from one cell.

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.5. 200 µl of each mouthwash were spread evenly on LB Agar plates.

Procedure cont. (infused plates)

6. The plates were then inverted and placed in an incubator at a heat of 37°C for 15 minutes

7. After vortexing to evenly suspend cells, 100 µl of E. coli/Staph from control tube was spread on the plates

8. The plates were incubated at 37°C for 24 hours9. The resulting colonies were counted. Each colony is

assumed to have arisen from one cell

Effect of Mouthwash on Staph Survivorship

0% 1% 10% 50%0

20

40

60

80

100

120

140

160

180

200

ListerineTom's

Concentration of Mouthwash

Num

ber o

f Col

onie

s

P=5.99E-07

P= 0.12

P= 0.25

P=1.08E-08

P= 0.17

Effect of mouthwash on E. coli Survivorship

0% 1% 10% 50%0

20

40

60

80

100

120

140

160

180

ListerineTom's

Concentration of Mouthwash

Num

ber o

f Col

onie

s

P= 0.0002

P= 0.39

P=1.78E-09 P=1.24E-08

P= 0.75

Dunnett’s Test

Staph T Crit.= 3.29 E. coli T Crit.= 3.29

Concentration of mouthwash

T-value interpretation Concentration of mouthwash

T-value interpretation

1% (Listerine) 1.09 Not significant 1% (Listerine) 8.2 Significant

10% (Listerine) 10.98 Significant 10% (Listerine) 15.6 Significant

50% (Listerine) 13.89 Significant 50% (Listerine) 19.12 Significant

1% (Tom’s) 2.76 Not Significant 1% (Tom’s) 5.92 Significant

10% (Tom’s) 8.02 Significant 10% (Tom’s) 13.75 Significant

50% (Tom’s) 10.76 Significant 50% (Tom’s) 14.52 Significant

Number of colonies on infused plates graph

Staph E. Coli0

20

40

60

80

100

120

140

160

180

200

ControlListerineTom's

Type of Bacteria

Num

ber o

f Col

onie

s

Dunnett’s Test (infused plates)

Artificial Mouthwash Natural Mouthwash

Type of bacteria

T-value Interpretation Type of bacteria

T-value Interpretation

Staph 1.12 Not significant Staph 0.88 Not significant

E. coli 0.49 Not significant E. coli 0.37 Not significant

T Crit.= 2.57

Mouthwash effects on microbial survivorship

0% 1% 10% 50%0

20406080

100120140160180200 Listerine/staph

Listerine/E. coliTom's/staphTom's/E. coli

Concentration of Mouthwash

Num

ber o

f Col

onie

s

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

Limitations and Extensions

• Limitations– 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

References

• http://www.cdc.gov/nczved/divisions/dfbmd/diseases/ecoli_o157h7/

• http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab12/diseases/ecoli/ecoli.html

• http://www.medicinenet.com/staph_infection/article.htm

• http://www.ebi.ac.uk/2can/genomes/bacteria/Staphylococcus_epidermidis.html

• http://www.patient.co.uk/health/Dental-Plaque-and-Gum-Disease.htm