The Effects of Alcohol and Nicotine on Microbial Flora

Jeff Van Kooten Grade 11Pittsburgh 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

• Often made through fermentation • Fermentation is the process by which sugar is

broken down by yeast into carbon dioxide and alcohol • Used and abused by millions • 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 filters3. 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 )mLNicotine 1 (N) mL .01 (N)mL .1(N)mL 1 (N)mLSDF 8.4mL 8.89mL 8.8mL 7.9mLMicrobe .1mL .1mL .1mL .1mLTotal Volume 10mL 10mL 10mL 10mL

0.00% 0.10% 1.00% 10.00%0

20

40

60

80

100

120

140

160

180

153

134

101.589.166

Nicotine Effects on Yeast Survivorship

Nicotine

Nicotine Concentrations

Resu

lting

Num

ber o

f Col

onie

s

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

0.00% 1.00% 5.00% 10.00%0

20

40

60

80

100

120

140

160

180

153

133.33

105.6694.66

Ethyl Alcohol Effects on Yeast Survivorship

Ethyl

Concentration of Ethyl

Resu

lting

Num

ber o

f Col

onie

s

Dunnett’s TestT-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%Nicotine .1% Nicotine 1% Nicotine 10% Nicotine

0%Ethyl 0% (N) 0% (E ) .1% N 0% (E ) 1% N 0% (E ) 10% N 0% (E )

1%Ethyl 0% (N) 1% (E ) .1% (N) 1% (E ) 1% (N) 1% (E ) 10% (N) 1% (E )

5%Ethyl 0% (N) 5% ( E) .1% (N) 5% (E ) 1% (N) 5%(E ) 10% (N) 5% (E )

10%Ethyl 0% (N) 10% (E ).1% (N) 10% (E ) 1% (N) 10% (E ) 10% (N) 10% (E )

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)

0

20

40

60

80

100

120

140

160

180

153

119.5105.833

84.66

106

69.6662.5

97.5

81.5

49.83

Nicotine and Ethyl Alcohol Effects on Yeast Survivorship

Nicotine and Ethyl Concentrations

Resu

lting

Num

ber o

f Col

onie

s

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

• Extensions • Use different concentrations of nicotine and ethyl alcohol • Test carcinogenic effects• Test on Escherichia coli populations

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

Data Analysis

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 6 918 153 44.8Column 2 6 800 133.3333 23.86667Column 3 6 634 105.6667 18.66667Column 4 6 568 94.66667 17.06667

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 12617.33 3 4205.778 161.1409 3.56E-14 3.098391Within Groups 522 20 26.1

Total 13139.33 23

Data Analysis (Cont.)Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 6 918 153 44.8Column 2 6 805 134.1667 48.16667Column 3 6 609 101.5 24.7Column 4 6 535 89.16667 10.56667

ANOVASource of Variation SS df MS F P-value F critBetween Groups 15488.79 3 5162.931 161.048 3.578E-14 3.098391Within 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 Total0.1%Ethyl

Count 6 6 6 18Sum 717 636 585 1938Average 119.5 106 97.5 107.6667Variance 16.3 19.6 23.5 104.3529

0.5%Ethyl Count 6 6 6 18Sum 635 418 375 1428Average 105.8333 69.66667 62.5 79.33333Variance 18.96667 35.06667 9.5 399.5294

1%Ethyl Count 6 6 6 18Sum 508 379 299 1186Average 84.66667 63.16667 49.83333 65.88889Variance 15.06667 885.3667 54.16667 498.8105

Two Factor (Cont.)Total

Count 18 18 18Sum 1860 1433 1259Average 103.3333 79.61111 69.94444Variance 232.2353 652.6046 455.9379

ANOVASource of Variation SS df MS F P-value F crit

Sample 16373.48 2 8186.741 68.37901 2.28E-14 3.204317Columns 10626.04 2 5313.019 44.37651 2.27E-11 3.204317

Interaction 1032.074 4 258.0185 2.155076 0.089486 2.578739Within 5387.667 45 119.7259

Total 33419.26 53