Post on 27-Apr-2020
The Effects of Alcohol and Nicotine on Microbial Flora
Jeff Van Kooten
Grade 11
Pittsburgh Central Catholic High School
Microbial Flora
• The internal and external flora has eukaryotic fungi, protists, and bacteria
• Not much is known about the association between humans and their flora
• Effects can be mutualistic, parasitic, pathogenic, and commensal
• Provides nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes
• Substance intakes by humans might have unintended effects on these important functions of the flora
Ethyl Alcohol
• Fermentation is the process by which sugar is broken down by yeast into carbon dioxide and alcohol
• Used and abused by millions
• Alcohol is a depressant
• Alter the processes in the mind
• Can be used as an antiseptic
Nicotine
• derived from the tobacco plant
• considered a stimulant
• effects many areas in the body including the central nervous system and the endocrine (hormone) system
• Chewing tobacco
• Carcinogen
• Contains nicotine
•
Saccharomyces cerevisiae (Yeast) • Used in many cell/biochemical investigations
• Easy to manipulate and rapidly grows
• As a eukaryote, it shares similar biochemistry, cell cycle, and genetics with more advanced organisms
• Utilized in this study as a human cell model and as a model of human Eukaryotic symbionts or pathogens
Purpose
• Test the effects of alcohol and nicotine individually and synergistically on Saccharomyces cerevisiae survivorship
Hypotheses
Null: Nicotine and Ethyl Alcohol will not significantly effect yeast survivorship
Alterative: Nicotine and Ethyl Alcohol will significantly reduce yeast survivorship individually
Alternative: Nicotine and Ethyl Alcohol will interact synergistically to reduce yeast survivorship
Materials • YEPD agar plates (YEPD
media + 1.5% agar) • YEPD media (1% yeast
extract, 2% peptone, 2% glucose)
• Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1mM CaCl2, 100mM NaCl)
• Sterile pipette tips • Micropipettes • Vortex • Incubator • Sidearm flask • Spreading turntable
• Spreader bar
• Ethanol (Ethyl Alcohol)
• Sterile capped test tubes with Sterile distilled water.
• Hot plate
• Coffee filters
• Saccharomyces cerevisiae (Yeast)
• Flavorless chewing tobacco
Procedure
1. 25 grams of chewing tobacco was mixed with 150mL of water 2. After thoroughly mixing the solution the tobacco leaves were drained
out and the solution was filtered through coffee filters 3. The solution was then put on a hot plate and allowed to boil for 10
minutes (yielding a high [nicotine]) 4. Yeast was grown overnight in sterile YEPD media. 5. A sample of the overnight culture was added to fresh media in a
sterile sidearm flask. 6. The culture was placed in an incubator until a density of 50 Klett
spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL.
7. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
8. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 0.1% 1.0% 5.0%, and 10% to a total of 9.9 mL.
9. 0.1 mL of yeast was then added to the test tubes, yielding a final volume of 10 mL and a cell density of 103 cells/mL per tube.
Procedure (Continued)
Concentrations 0.0% .10% Nicotine 1.0% (N) 10.0% (N) 1.0% Ethyl 5.0% ( E ) 10.0% ( E )
Ethyl 0mL 0mL 0mL 0mL .1mL .5mL 1mL
Nicotine 0mL .01 mL .1mL 1mL 0mL 0mL 0mL
Sterile Dilution Fluid 9.9 mL 9.89mL 9.8mL 8.9mL 9.8mL 9.4mL 8.9mL
Microbe .1mL .1mL .1mL .1mL .1mL .1mL .1mL
Total Volume 10mL 10mL 10mL 10mL 10mL 10mL 10mL
Concentrations 1.0% ( E ) .10% (N) 1.0% ( E ) 1.0% (N) 1.0% ( E ) 10.0% (N) 5.0% ( E ) .10% (N) 5.0% ( E ) 1.0% (N)
Ethyl .1 ( E )mL .1 ( E )mL 0.1 ( E )mL .5 ( E )mL .5 ( E )mL
Nicotine .01 (N)mL .1 (N)mL 1 (N)mL .01 (N)mL .1(N) mL
SDF 9.79mL 9.7mL 8.8mL 9.39mL 9.3mL
Microbe .1mL .1mL .1mL .1mL .1mL
Total Volume 10mL 10mL 10mL 10mL 10mL
Procedure (Continued)
7. The solutions were mixed by vortexing and allowed to sit at room temperature
8. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on YEPD agar plates (6 plates per concentration)
9. The plates were then incubated at 30 degrees for 48 hours.
10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
Concentration 5.0% ( E ) 10.0% (N) 10.0% ( E ) .10% (N) 10.0% ( E ) 1.0% (N) 10.0% (E) 10.0% (N)
Ethyl .5 ( E )mL 1 ( E )mL 1 ( E )mL 1( E )mL
Nicotine 1 (N) mL .01 (N)mL .1(N)mL 1 (N)mL
SDF 8.4mL 8.89mL 8.8mL 7.9mL
Microbe .1mL .1mL .1mL .1mL
Total Volume 10mL 10mL 10mL 10mL
153
134
101.5
89.166
0
20
40
60
80
100
120
140
160
180
0% 0.10% 1.00% 10%
Res
ult
ing
Nu
mb
er o
f C
olo
nie
s
Nicotine Concentrations
Nicotine Effects on Yeast Survivorship
Nicotine
Dunnett’s Test
Concentration T-value Interpretation
.01% Nicotine 5.761243 Significant
.1% Nicotine 15.75422 Significant
1% Nicotine 19.52709 Significant
T-Crit
3.1
153
133.33
105.66
94.66
0
20
40
60
80
100
120
140
160
180
0.00% 1.00% 5.00% 10%
Res
ult
ing
Nu
mb
er o
f C
olo
nie
s
Concentration of Ethyl Alcohol
Ethyl Alcohol Effects on Yeast Survivorship
Ethyl Alcohol
Dunnett’s Test T-Crit
3.1
Concentration T-value Interpretation
.1%Ethyl 6.667737 Significant
.5%Ethyl 16.27352 Significant
1%Ethyl 19.77707 Significant
Chart of concentrations
0.0%Nicotine 0.1% Nicotine 1.0% Nicotine 10% Nicotine
0.0%Ethyl 0.0% (N) 0% (E ) 0.1% N 0% (E ) 1.0% N 0% (E ) 10% N 0% (E )
1.0%Ethyl 0.0% (N) 1% (E ) 0.1% (N) 1% (E ) 1.0% (N) 1% (E ) 10% (N) 1.0% (E )
5.0%Ethyl 0.0% (N) 5% ( E) 0.1% (N) 5% (E ) 1.0% (N) 5%(E ) 10% (N) 5.0% (E )
10%Ethyl 0.0% (N) 10% (E ) 0.1% (N) 10% (E ) 1.0% (N) 10% (E ) 10% (N) 10% (E )
153
119.5
105.833
84.66
106
69.66 62.5
97.5
81.5
49.83
0
20
40
60
80
100
120
140
160
180
Control .1%(N)1%(E)
.1%(N)5%(E)
.1%(N)10%(E)
1%(N)1% (E)
1%(N)5% (E)
1%(N)10% (E)
10%(N)1% (E)
10%(N)5% (E)
10%(N)10%(E)
Re
sult
ing
Nu
mb
er
of
Co
lon
ies
Nicotine and Ethyl Concentrations
Nicotine and Ethyl Alcohol Effects on Yeast Survivorship
Conclusion
• Increased concentrations of Nicotine appeared to significantly effect yeast survivorship
• Increased concentrations of Alcohol also appeared to significantly effect yeast survivorship
• Accept the first alternative hypothesis
• No evidence of synergic effects of nicotine and alcohol on yeast survivorship
Limitations and Extensions
• Limits • Only yeast was used as a model
• Plating was not completely synchronized
• Pure nicotine was not used
• Only one kind of alcohol was used
• Limited amount of concentration
• Only survivorship accessed
• Cell health?
• Extensions • Use different concentrations of nicotine and ethyl alcohol
• Test carcinogenic effects
• Test on Escherichia coli populations
• Growth curve
Sources
• http://www.encyclopedia.com/topic/tobacco.aspx
• http://en.wikipedia.org/wiki/Human_flora
• http://en.wikipedia.org/wiki/Gut_flora
• http://www.alcohol.vt.edu/Students/alcoholEffects/brainBody.htm
• http://www.knowswhy.com/reasons-why-alcohol-is-bad-for-your-health/
Data Analysis
Anova: Single Factor
SUMMARY Groups Count Sum Average Variance
Column 1 6 918 153 44.8 Column 2 6 800 133.3333 23.86667 Column 3 6 634 105.6667 18.66667
Column 4 6 568 94.66667 17.06667
ANOVA Source of Variation SS df MS F P-value F crit
Between Groups 12617.33 3 4205.778 161.1409 3.56E-14 3.098391 Within Groups 522 20 26.1
Total 13139.33 23
Data Analysis (Cont.) Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Column 1 6 918 153 44.8
Column 2 6 805 134.1667 48.16667
Column 3 6 609 101.5 24.7
Column 4 6 535 89.16667 10.56667
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 15488.79 3 5162.931 161.048 3.578E-14 3.098391
Within Groups 641.1667 20 32.05833
Total 16129.96 23
Data Analysis ( Two Factor) Anova: Two-Factor With Replication
SUMMARY 0.01%Nicotine 0.1%Nicotine 1%Nicotine Total
0.1%Ethyl
Count 6 6 6 18
Sum 717 636 585 1938
Average 119.5 106 97.5 107.6667
Variance 16.3 19.6 23.5 104.3529
0.5%Ethyl
Count 6 6 6 18
Sum 635 418 375 1428
Average 105.8333 69.66667 62.5 79.33333
Variance 18.96667 35.06667 9.5 399.5294
1%Ethyl
Count 6 6 6 18
Sum 508 379 299 1186
Average 84.66667 63.16667 49.83333 65.88889
Variance 15.06667 885.3667 54.16667 498.8105
Two Factor (Cont.) Total
Count 18 18 18 Sum 1860 1433 1259 Average 103.3333 79.61111 69.94444 Variance 232.2353 652.6046 455.9379
ANOVA Source of Variation SS df MS F P-value F crit
Sample 16373.48 2 8186.741 68.37901 2.28E-14 3.204317 Columns 10626.04 2 5313.019 44.37651 2.27E-11 3.204317
Interaction 1032.074 4 258.0185 2.155076 0.089486 2.578739 Within 5387.667 45 119.7259
Total 33419.26 53