Alcohol Effects On a Eukaryotic Cell Model

Austin BruggerGrade 9

Pittsburgh Central Catholic High School

What are the actual effects of alcohol on a Eukaryotic Cell Model?

Problem

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

Saccharomyces cerevisiae (Yeast)

More than 10% of your body mass is due to symbionts and pathogens

Mostly Prokaryotic cells Some may cause diseases Also protect the body from foreign invaders Reinforce immune system, prevent

allergies, and produce vitamins Lie within skin, saliva, and gastrointestinal

tracts Break down abnormally large nutrients

Human Symbionts, Pathogens, and Flora

Often made through the process of fermentation

Fermentation is the process by which yeast breaks down sugar into carbon dioxide and alcohol.

Common uses include: Household chemicals, alcoholic beverages, and medical chemicals and disinfectants

Very toxic substance with numerous effects on body

Ethyl Alcohol

To assess the effects of alcohol on the survivorship of Saccharomyces cerevisiae

Purpose

Null Hypothesis: Ethyl alcohol will not affect the survivorship of Saccharomyces cerevisiae.

Alternative Hypothesis: Ethyl alcohol will significantly reduce the survivorship of Saccharomyces cerevisiae.

Hypotheses

YEPD agar plates (YEPD media + 1.5% agar)

YEPD media (1% yeast extract, 2% peptone, 2% glucose)

Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading turntable

Spreader bar Ethanol (Ethyl Alcohol) Sterile capped test

tubes with Sterile distilled water.

Saccharomyces cerevisiae (Yeast) (Obtained from Jones Lab, CMU)

0.22 micron syringe filters + 10mL syringe

Klett Spectrophotometer

Materials

1. Yeast was grown overnight in sterile YEPD media.2. A sample of the overnight culture was added to fresh

media in a sterile sidearm flask.3. 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.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 0.1%, 1%, and 10% to a total of 9.9 mL.

6. 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

Concentration Chart0% 0.1% 1% 10%

Sterile Dilution Fluid

9.9 mL 9.89 mL 9.8 mL 8.9 mL

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Ethyl Alcohol

0 mL 0.01 mL 0.1 mL 1 mL

Total Volume

10 mL 10 mL 10 mL 10 mL

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 at time 10 minutes and 30 minutes 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.

Procedure (Continued)

Amount of Colonies per plate (10 min Exposure)Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Averag

e

0% Alcohol

294 255 220 278 285 302 272

0.1% Alcohol

196 180 209 210 204 192 198

1% Alcohol

163 148 158 184 190 197 173

10% Alcohol

142 140 138 154 140 136 141

Data Results

Amount of Colonies per plate (30 min Exposure)Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Averag

e

0% Alcohol

205 213 167 186 198 200 202

0.1% Alcohol

159 167 158 164 120 148 153

1% Alcohol

120 143 140 146 138 123 135

10% Alcohol

110 116 101 112 107 99 107

Data Results (Continued)

0.00% 0.10% 1.00% 10.00%0

25

50

75

100

125

150

175

200

225

250

275

300

10 Min Exposure30 Min Exposure

Concentration of Alcohol

Re

su

ltin

g N

um

be

r of

Co

lon

ies

P-Value: 0.000306

P-Value: 0.000293

P-Value: 0.001888 P-Value:

.000000481

Alcohol Effects on Yeast Colonies

P-Value: 0.00000000166

P-Value: 0.000000781

10 Min Exposure

Variable Concentration

T-Value Interpretation

0.1% Alcohol 6.65 Significant

1% Alcohol 8.92 Significant

10% Alcohol 11.77 Significant

Dunnett’s Test

30 Min Exposure

Variable Concentration

T-Value Interpretation

0.1% Alcohol 3.51 Significant

1% Alcohol 5.37 Significant

10% Alcohol 8.26 Significant

T-Critical = 3.1Alpha= 0.05

Survivorship of Yeast

0 1 2 3 4 5 6 7 8 9 10 11 120

50

100

150

200

250

300

10 Min Ex-posure

Concentration of Alcohol

Resu

ltin

g N

um

ber

of

Colo

nie

s

Ratio of Survivorship of Variables to Control (10 Min Exposure)

0.1% Alcohol 1% Alcohol 10% Alcohol

Control (0% Alcohol)

72% 63% 51%

Analysis of Exposure Time

Ratios of Survivorship of Variables to Control (30 Min Exposure)

0.1% Alcohol 1% Alcohol 10% Alcohol

Control (0% Alcohol)

74% 65% 52%

Did the duration of exposure have a significant effect?

Data Analysis (ANOVA)Anova: Single Factor10 Min ExposureSUMMARY

Groups Count Sum Average Variance

0% Alcohol 6 1634 272.3333 916.2667

0.1% Alcohol 6 1191 198.5 132.7

1% Alcohol 6 1040 173.3333 387.0667

10% Alcohol 6 850 141.6667 40.66667

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

Between Groups 55788.46 3 18596.15 50.37219 1.66E-09 3.098391

Within Groups 7383.5 20 369.175

Total 63171.96 23

Data Analysis (Dunnett’s Test)

10 min Exposure

Mv Mc MSE n T

0.10% 198.5 272.3333 369.175 6 73.833311.093166

066.6557463

91

1% 173.3333 272.3333 369.175 6 9911.093166

068.9244134

11

10% 141.6667 272.3333 369.175 6130.666

611.093166

0611.779017

75

30 Min Exposure

T-Crit for both was 3.1

Mv Mc

0.10% 152.6667 186 270.4417 6 33.33339.4945896

873.5107678

26

1% 135 186 270.4417 6 519.4945896

875.3714801

46

10% 107.5 186 270.4417 6 78.59.4945896

878.2678664

99

Increased concentrations of alcohol lowered the amount of yeast cells that grew.

Increased exposure did not have a significant effect on the amount of surviving colonies.

Higher concentrations of alcohol, since they are more potent, could also be effective in reducing the amount of yeast colonies that could survive.

Conclusions

Incorrect concentrations of alcohol could have been produced.

Spreader bars couldn’t have been sterilized properly

During the testing, plating could have possibly been unsynchronized.

Only one type of alcohol was used.

Limitations

Different types of alcohol More alcoholic concentrations Experiment on Staphylococcus epidermidis

and Escherichia coli populations Study effects on neurological effects on

flora of the body Perform a Try pan blue exclusion assay

to test for dead cells.

Further Studies

http://pubs.niaaa.nih.gov/publications/aa63/aa63.htm

http://www.alcohol.vt.edu/Students/alcoholEffects/brainBody.htm

http://biology.clc.uc.edu/courses/bio104/cellresp.htm

http://www.anaerobicrespiration.net/general/anaerobic-respiration-fermentation/#more-32

http://www.healthy.net/scr/article.aspx?Id=772

Sources