1. Smokeless Tobacco’s Influence on Microbial Life
Brendan Connolly
PJAS
Central Catholic High School
Grade 9

2. Smokeless Tobacco
There are many different kinds of smokeless tobacco. (ex chewing, dipping, etc.)
Subject: Dipping tobacco
Carcinogen
Contains nicotine, which causes addiction.
Banned in several countries. (Not the United States)

3. Previous Studies
Show that smokeless tobacco affects the cardiovascular system less than smoked tobacco.
Increased cancer risk.

4. Human Microbial Flora
The microorganisms that reside in or on the surface of the body. (ex. gut, skin, oral, etc.)
Includes bacteria, fungi, and archea.
Subject bacteria: Can cause disease if growing in uncommon areas or have numbers that exceed their typical range.
Most studies-ingested material effects on human cells are done of ingested materials on human cells, BUT humans are a collection of human cells and symbionts.
An estimated 10% of body mass is symbionts.

5. Gut Flora
Consists of microorganisms that live in the digestive tracts of humans and many animals.
Have many useful functions including preventing growth of harmful pathogenic bacteria.
Studied more than any other type of flora in the body.
Includes Bacteroides, Escherichia, and Clostridium.

6. Escherichia coli
One of the most common forms of bacteria found in many environments including the intestinal tracts of mammals.
Historically utilized as the most studied prokaryote in biological research.
Most strains are non-pathogenic.
However, pathogenic forms can produce fatal illness in humans and other mammals.

7. Purpose
To examine the effects of smokeless tobacco on human microbial flora.

8. Hypotheses
Null: The smokeless tobacco will NOT have a significant effect on the survivorship of E. coli.
Alternative: The smokeless tobacco WILL have a significant effect on the survivorship of E coli.

9. Materials LB plates Spreading Platform, spreader bar, ethanol
LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Escherichia coli bacteria (DH5-alpha)
Micropipettes
Skoal Fine Cut Smokeless Tobacco
Sterile Pipette tips
Ethanol
Vortex
Micro tubes
Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, mM MgSO4, 1mM NaCl)
Matches
0.22 micron sterile syringe filters
Sidearm Flasks (125mL)

10. Procedure
Chewing tobacco extract was sterilized by means of 0.22 micron syringe filters.
Escherichia Coli B 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 diluted in sterile dilution fluid to a concentration of approxiamately 105 cells/mL.
The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9mL.
0.1 mL of cell culture was then added to the test tubes, yielding a final volume of 10mL and a cell density of approximately 103 cells/mL.
The cells were allowed to sit at room temperature for 15 minutes.
After vortexing to evenly suspend the cells, 0.1mL aliquots were removed from the tubes and spread on LB plates.
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

11. Test Tube Ingredients Concentration 0% 0.01% 0.10% 1%
Sterile Dilution Fluid
9.9 mL
9.89 mL
9.8 mL
8.9 mL
E coli
0.1 mL
Smoke-less tobacco
0 mL
0.01 mL
1 mL
Total
10 mL

12. Liquid Exposure

13. Survivorship Curve of E. coli

14. Dunnett’s Test Interpretation
t-crit = 2.47
Alpha = .05
0.01% tobacco solution
1.85 < 2.47, not significant
0.1% tobacco solution
5.78 > 2.47, significant
1% tobacco solution
> 2.47, significant

15. Conclusions
The smokeless tobacco appears to have an effect on E. Coli survivorship.
The 0.1% and 1% solutions had a significant negative effect on E. coli survivorship.
Reject the null hypothesis.
Accept the alternative hypothesis.

16. Limits and Extensions Limits Only E. coli was used as a model.
Only 4 different concentrations were used.
Plating could not be completely synchronized.
Extensions
Using different concentrations of tobacco.
A mutagenesis experiment on the host cells.
Use a Trypan Blue Exclusion Assay.
Using various prokaryotic models (gram positive etc.)

17. References http://profiles.nlm.nih.gov/NN/B/B/F/D/_/nnbbfd.pdf