Smokeless Tobacco’s Influence
on Microbial Life
Brendan Connolly
PJAS
Central Catholic High School
Grade 9
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)
Previous Studies
• Show that smokeless tobacco affects
the cardiovascular system less than
smoked tobacco.
• Increased cancer risk.
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.
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.
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.
Purpose
•To examine the effects
of smokeless tobacco on
human microbial flora.
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.
Materials
• LB plates
•
• LB Media (0.5% yeast extract,
1% tryptone, 1% sodium
•
chloride)
• Micropipettes
•
• Sterile Pipette tips
•
• Vortex
• Sterile Dilution Fluid (100mM •
•
KH2PO4, 100mM K2HPO4,
10mM MgSO4, 1mM NaCl)
•
• Sidearm Flasks (125mL)
Spreading Platform, spreader
bar, ethanol
Escherichia coli bacteria
(DH5-alpha)
Skoal Fine Cut Smokeless
Tobacco
Ethanol
Micro tubes
Matches
0.22 micron sterile syringe
filters
1.
2.
3.
4.
5.
6.
7.
8.
9.
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.
Test Tube Ingredients
Concentration 0%
0.01% 0.10%
Sterile Dilution
Fluid
9.9 mL 9.89 mL9.8 mL
E coli
Smoke-less
tobacco
Total
1%
8.9 mL
0.1 mL 0.1 mL 0.1 mL
0.1 mL
0 mL 0.01 mL0.1 mL
10 mL 10 mL 10 mL
1 mL
10 mL
Liquid Exposure
250
Number of Colonies
P-value= 1.35E-12
200
150
100
50
0
0.00%
0.01%
0.10%
1.00%
Concentration of Smokeless Tobacco
Survivorship Curve of E. coli
Number of Colonies
250
200
150
100
50
0
0.00% 0.20% 0.40% 0.60% 0.80% 1.00% 1.20%
Concentration of Smokeless Tobacco
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
 12.7105 > 2.47, significant
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.
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.)
References
• http://profiles.nlm.nih.gov/NN/B/B/F/D/_/nn
bbfd.pdf
• http://en.wikipedia.org/wiki/Dipping_tobacco
• http://en.wikipedia.org/wiki/Tobacco
• http://en.wikipedia.org/wiki/Human_flora
• http://en.wikipedia.org/wiki/Gut_flora
• http://www.textbookofbacteriology.net/norm
alflora_3.html