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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
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