The Effects of Chewing Tobacco on Microbial Flora

Marco Augello Central Catholic HS

Grade 10 Second 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 LB

Media.

2. Samples of the overnight cultures were added to fresh media

in a sterile sidearm flask.

3. The cultures were placed in an incubator (37°C) until a

density of 50 Klett spectrophotometer units was reached. This

represents a cell density of approximately 107 cells/mL.

4. The cultures were diluted in sterile dilution fluid to a

concentration 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 Concentrations

Liquid 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

100

200

300

400

500

600

700

0% 0.01% 0.10% 1%

Staph

E. coli

Chewing Tobacco Concentrations

Ave

rage

Co

lon

ies

Co

un

ted

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

100

200

300

400

500

600

700

800

900

1000

0% 1% 10%

Staph

E. coli

Chewing Tobacco Concentrations

Ave

rage

Co

lon

ies

Co

un

ted

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 Factor

Staph LP

SUMMARY

Groups Count Sum Average Variance

0% 4 2406 601.5 1243

0.01% 4 2186 546.5 349.6667

0.10% 4 1948 487 1415.333

1% 4 1664 416 1215.333

ANOVA

Source of VariationSS df MS F P-value F crit

Between Groups76157 3 25385.67 24.04325 2.31E-05 3.490295

Within Groups12670 12 1055.833

Total 88827 15

Anova: Single Factor

E. coli LP

SUMMARY

Groups Count Sum Average Variance

0% 4 2325 581.25 1418.917

0.01% 4 2083 520.75 2674.917

0.10% 4 1632 408 3060.667

1% 4 1490 372.5 1469.667

ANOVA

Source of VariationSS df MS F P-value F crit

Between Groups113203.3 3 37734.42 17.50171 0.000111 3.490295

Within Groups25872.5 12 2156.042

Total 139075.8 15

Anova: Single Factor

Staph Inf

SUMMARY

Groups Count Sum Average Variance

0% 4 3772 943 518

1% 4 3260 815 528.6667

10% 4 2747 686.75 998.9167

ANOVA

Source of VariationSS df MS F P-value F crit

Between Groups131328.2 2 65664.08 96.30126 8.39E-07 4.256495

Within Groups6136.75 9 681.8611

Total 137464.9 11

Anova: Single Factor

E. coli Inf

SUMMARY

Groups Count Sum Average Variance

0% 4 3426 856.5 377.6667

1% 4 2965 741.25 424.9167

10% 4 2521 630.25 552.25

ANOVA

Source of VariationSS df MS F P-value F crit

Between Groups102390.2 2 51195.08 113.361 4.15E-07 4.256495

Within Groups4064.5 9 451.6111

Total 106454.7 11