The Effects of Chewing Tobacco on Microbial Flora

Marco AugelloCentral Catholic HS

Grade 10Second Year in PJAS

Rationale

• Chewing tobacco is widespread in society, especially among young people

• Smokeless tobacco used regularly by 6.4% of high school students and 3% of adults

Chewing Tobacco

• Grizzly fine cut tobacco• Small tobacco leaves consumed orally• Causes tooth decay, gum disease, and

other oral diseases• Carcinogen – causes pancreatic,

mouth, esophageal cancer• Contains nicotine (highly addictive)

Staphylococcus epidermidis

• Bacteria found on healthy human skin

• Gram (+)• After incubation in agar, forms

1/2 - 2 mm colonies • Used as a model for microbial

flora found in/on humans

Escherichia coli

• Large, diverse group of bacteria• Bacteria found in intestines of most mammals• Most strains are not pathogenic• Gram (-)• Used as a model for microbial flora found in the

human body

Purpose

To test the effects of chewing tobacco on Staph and E. coli populations.

Hypotheses

• Null Hypothesis: Chewing tobacco WILL NOT HAVE a significant effect on Staph and E. coli survivorship.

• Alternate Hypothesis: Chewing tobacco WILL HAVE a significant effect on Staph and E. coli survivorship.

Materials

• Escherichia coli (E. coli)• Staphylococcus epidermidis

(Staph)• Grizzly fine cut tobacco• Micro pipettes• Sterile pipette tips• Spreader bars• Ethanol• Bunsen burner• Vortex • Incubator• Test tubes

• Filter paper• Funnel• Sterile filter• Sterile Dilution Fluid

(100mM KH2PO4, 100mM KH2PO4, 10mM MgSO4, 1mM NaCl)

• LB plates + media (1% tryptone, 0.5% yeast extract, 1% NaCl)

Liquid Pulse Procedure

1. Bacteria (E.coli and Staph) were grown overnight in sterile LBMedia.2. Samples of the overnight cultures were added to fresh mediain a sterile sidearm flask.3. The cultures were placed in an incubator (37°C) until adensity of 50 Klett spectrophotometer units was reached. Thisrepresents a cell density of approximately 107 cells/mL.4. The cultures were diluted in sterile dilution fluid to aconcentration of approximately 105 cells/mL.5. A 10% stock solution of chewing tobacco and sterile water was made.6. The following components were added to sterile test tubes.

Table of ConcentrationsLiquid Pulse 0% 0.01% 0.1% 1%

CT Solution (10%) 0mL 0.01mL 0.1mL 1mL

SDF 9mL 8.99mL 8.9mL 8mL

E. Coli/Staph 1mL 1mL 1mL 1mL

Total Volume 10mL 10mL 10mL 10mL

Liquid Pulse Procedure cont’d

7. These solutions were then vortexed and allowed to sit at room temperature for 15 minutes.

8. After vortexing to allow even suspension, 100 μL aliquots were removed from the tubes and spread on LB-agar plates.

9. The plates were incubated at 37°C for 24 hours.10. The resulting colonies were counted visually, each colony

being assumed to have arisen from one cell.

Infusion Procedure

1. 0.2ml of chewing tobacco concentrations (0%, 1%, and 10%)were spread on LB agar plates and incubated for 1 hour, allowing the bacteria to absorb the chemicals. 2. 100uL aliquots of bacterial suspensions from the original control tube were spread onto the infused plates.3. The plates were incubated at 37 C for 24 hours.4. The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

Chewing Tobacco Liquid Pulse Effects

0.00% 0.01% 0.10% 1.00%0

100

200

300

400

500

600

700

StaphE. coli

Chewing Tobacco Concentrations

Aver

age

Colo

nies

Cou

nted

Staph p-value=2.31x10 -5

E. coli p-value=0.000111

Dunnett’s Tests

Staph t value t critical Significance

0% to 0.01% 2.393757056 3.5 NOT SIGNIFICANT

0% to 0.1% 4.983366962 3.5 SIGNIFICANT

0% to 1% 8.073489707 3.5 SIGNIFICANT

E. coli t value t critical Significance

0% to 0.01% 1.842645386 3.5 NOT SIGNIFICANT

0% to 0.1% 5.276666334 3.5 SIGNIFICANT

0% to 1% 6.357888007 3.5 SIGNIFICANT

Chewing Tobacco Infusion Effects

0% 1% 10%0

100

200

300

400

500

600

700

800

900

1000

StaphE. coli

Chewing Tobacco Concentrations

Aver

age

Colo

nies

Cou

nted

Staph p-value=8.39x10 -7

E. coli p-value=4.15x10 -7

Dunnett’s Tests

Staph t value t critical Significance

0% to 1% 6.932294593 4.25 SIGNIFICANT

0% to 10% 13.87812882 4.25 SIGNIFICANT

E. coli t value t critical Significance

0% to 1% 7.669616135 4.25 SIGNIFICANT

0% to 10% 15.05640478 4.25 SIGNIFICANT

Conclusions

• The null hypothesis was REJECTED for all concentrations (liquid pulse and infusion) except for the liquid pulse 0.01%

• Experiment shows that chewing tobacco does have a significant and harmful effect on Staph and E. coli

Limitations

• Only survivorship tested• Vitality of Staph/E. coli varied• Spread plating may not have been

synchronized • One exposure period• Limited replicates

Extensions

• More assessments• Better plating synchronization • Different brands/types of chewing tobacco• More exposure times for liquid pulse

Resources

• http://kidshealth.org/teen/drug_alcohol/tobacco/smokeless.html

• http://betobaccofree.hhs.gov/health-effects/smokeless-health/

• http://www.cdc.gov/tobacco/data_statistics/fact_sheets/youth_data/tobacco_use/index.htm#estimates

• http://www.ctri.wisc.edu/Smokers/smokeless_facts.htm

Anova: Single FactorStaph LPSUMMARY

Groups Count Sum Average Variance0% 4 2406 601.5 1243

0.01% 4 2186 546.5 349.66670.10% 4 1948 487 1415.333

1% 4 1664 416 1215.333

ANOVASource of VariationSS df MS F P-value F crit

Between Groups76157 3 25385.67 24.04325 2.31E-05 3.490295Within Groups12670 12 1055.833

Total 88827 15

Anova: Single FactorE. coli LPSUMMARY

Groups Count Sum Average Variance0% 4 2325 581.25 1418.917

0.01% 4 2083 520.75 2674.9170.10% 4 1632 408 3060.667

1% 4 1490 372.5 1469.667

ANOVASource of VariationSS df MS F P-value F crit

Between Groups113203.3 3 37734.42 17.50171 0.000111 3.490295Within Groups25872.5 12 2156.042

Total 139075.8 15

Anova: Single FactorStaph InfSUMMARY

Groups Count Sum Average Variance0% 4 3772 943 5181% 4 3260 815 528.6667

10% 4 2747 686.75 998.9167

ANOVASource of VariationSS df MS F P-value F crit

Between Groups131328.2 2 65664.08 96.30126 8.39E-07 4.256495Within Groups6136.75 9 681.8611

Total 137464.9 11

Anova: Single FactorE. coli InfSUMMARY

Groups Count Sum Average Variance0% 4 3426 856.5 377.66671% 4 2965 741.25 424.9167

10% 4 2521 630.25 552.25

ANOVASource of VariationSS df MS F P-value F crit

Between Groups102390.2 2 51195.08 113.361 4.15E-07 4.256495Within Groups4064.5 9 451.6111

Total 106454.7 11