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Ethanol Effects Upon Microbial Flora Survivorship
Siddarth NarayanGrade 9
North Allegheny Intermediate High School
What effect does ethanol have upon E Coli and Staph cultures?
Problem
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
Large and diverse group of gram (-) bacteria Free living, symbiontes, or pathogens Live in the intestinal tract of many
mammals. Most strains are not pathogenic Serve as a common prokaryotic cell model.
Escherichia coli (E.Coli)
Common surface symbiont in many mammals (Human).
Gram (+) bacteria Most strains considered non-pathogenic. Pathogenic strains can be life-threatening.
Staphylococcus epidermidis (Staph)
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
Ethanol
To examine effects of ethanol upon endosymbiontes
Purpose
Null Hypothesis: Ethanol will not significantly affect microbial survivorship
Alternative Hypothesis: Ethanol will significantly reduce the survivorship of E coli and staph cells.
Hypotheses
LB agar plates (LB media + 1.5% agar)
LB media (0.5% yeast extract, 1% tyrptone, 1% NaCL)
Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading turntable
Spreader bar Ethanol (Ethyl Alcohol) Sterile capped test
tubes with Sterile distilled water.
DH5 Alpha E coli Strain
Staphylococcus epidermidis (Ward’s)
0.22 micron syringe filters + 10mL syringe
Klett
Materials
1. The microbes were grown overnight in sterile LB 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 108 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%, 1%, 10%, 25% and 50% to a total of 9.9 mL.
6. 0.1 mL of Staph/ E coli 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% 1% 10% 25% 50%
Sterile Dilution Fluid
9.9 mL 9.8 mL 8.9 mL 7.4 mL 4.9 mL
Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL
Ethanol 0 mL 0.1 mL 1 mL 2.5 mL 5 mL
Total Volume
10 mL 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 and spread on LB agar plates (3 plates per concentration)
9. The plates were then incubated at 37 degrees for 24 hours.
10. The resulting colonies were counted.
Procedure (Continued)
E Coli Survivorship
5
P Value=1E-05
50 25 10 1 00
50
100
150
200
250
300
350
400
450
Ethanol Concentration (%)
Avera
ge N
um
ber
of
Cells
Data Analysis (ANOVA)
Anova: Single Factor (E Coli)
SUMMARYGroups Count Sum Average Variance
50% 3 2 0.667 0.33325% 3 1 0.333 0.33310% 3 1152 384 11788
1% 3 889 296.3 34900% 3 773 257.7 246.3
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 4E+05 4 93988 30.27 1E-05 3.478Within Groups 31051 10 3105
Total 4E+05 14
Concentration T- Value Interpretation
1% .81 Insignificant
10% 3.3 Insignificant
25% 7.6 Significant
50% 7.6 Significant
E Coli Dunnett’s Test Analysis(Alpha= .05) (T Crit=3.48)
Staph Survivorship P Value=1.46E-11
50 25 10 1 00
50
100
150
200
250
300
350
400
450
500
Series1
Data Analysis (ANOVA)Anova: Single Factor (Staph)
SUMMARYGroups Count Sum Average Variance
50% 3 0 0 025% 3 399 133 32710% 3 1422 474 468
1% 3 1141 380.3333 44.333330% 3 1092 364 268
ANOVASource of Variation SS df MS F P-value F crit
Between Groups 462882.3 4 115720.6 522.5191 1.46E-11 3.47805
Within Groups 2214.667 10 221.4667
Total 465096.9 14
Concentration T-Value Interpretation
1% 1.7 Insignificant
10% 11.7 Significant
25% 24.5 Significant
50% 38.7 Significant
Staph Dunnett’s Test Analysis(Alpha=.05) (T Crit=3.48 )
Staph and E coli LD50
0 1 10 25 500
50
100
150
200
250
300
350
400
450
500
Ethanol Concentration s (%)
Nu
mb
er
of
Ce
lls
E coli~ 22% Staph~ 24%
The null hypothesis can be rejected for the following concentration of ethanol and species: E coli (25%, 50%), Staph (10%, 25%, 50%)
Both microbes display similar survivorship
Conclusions
Low sample sizes Only one type of exposure (Liquid Pulse
Exposure) Limited concentrations of variable Only one exposure time
Limitations
More replicates being used in same setup Other exposures (diffusion assay) Varied exposure time Synergistic Effects Can ethanol benefit survivorship in certain
quantities? Does ethanol only have an effect upon
pathogenic endosymbiontes?
Further Study
Data Results (E Coli)
Plate 1 Plate 2 Plate 3
0% Ethanol 263 270 240
1% Ethanol 364 255 270
10% Ethanol 420 262 470
25% Ethanol 0 1 0
50% Ethanol 0 1 1
Data Results (Staph)Plate 1 Plate 2 Plate 3
0% 360 350 382
1% 382 373 386
10% 480 492 450
25% 114 135 150
50% 0 0 0
E Coli ANOVA Data Analysis
Staph ANOVA Data Analysis
0 1 10 25 500%
200000%
400000%
600000%
800000%
1000000%
1200000%
1400000%
GroupsCountSumAverageVariance
References www.ncbi.nlm.nih.gov/pubmed/19609257 www.mayoclinic.com/health/staph
-infections/DS00973 www.mayoclinic.com/health/e-coli/DS01007 www.life.umd.edu/classroom/.../GramPosvs
GramNeg.htm www.ncbi.nlm.nih.gov/pubmed/15112929 http://
jb.asm.org/content/157/1/233.abstract