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