1. Synergistic Effect of Ibuprofen and Alcohol on Flora
Ryan McNelis
Grade 11
Pittsburgh Central Catholic High School

2. Rationale for Experiment
Commonly, ingested materials are investigated for effects on human cells.
However, effects on the human flora may also be important.

3. 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

4. Escherichia Coli (E. Coli)
Large and diverse group of gram (-) bacteria
Free living, symbiont, or pathogen
Live in the intestinal tract of many mammals
Most strains are not pathogenic
Serve as a common prokaryotic cell model

5. Ibuprofen and Ethanol Ibuprofen: Anti-inflammatory drug
Used to reduce hormones-used for inflammation or pain in the body-reduces fever, headaches, and injuries in the body.
Ethanol:
Primary alcohol found in beverages.
Created by the fermentation of sugars by yeast.

6. Ibuprofen and Ethanol: Negative Effects
Ibuprofen: associated with cardiovascular risks, skin blistering, nausea, headache, and dizziness
Ethanol: causes lack of balance, gastrointestinal disease; over long term may act as a carcinogen and contribute to birth defects.

7. Brand Tested Children’s Dye Free Ibuprofen 100mg of Ibuprofen per 5 mL
Inactive Ingredients: acesulfame potassium, anhydrous citric acid, D&C yellow no.10, FD&C red no.40, flavors, glycerin, polysorbate 80, pregelatinized starch, purified water, sodium benzoate, sucrose, xanthan gum

8. Problem
Accompanied with drug side effects, combination of over the counter drugs and alcohol can have dire side effects
Do a common daily over the counter drug (Ibuprofen) and ethanol have synergistic effects and reduce survivorship of human bacterial flora?

9. Purpose
To investigate whether various concentrations of Ibuprofen and Ethanol have an adverse synergistic effect on microbial flora populations.

10. Hypotheses
Null: The concentrations of Ibuprofen and ethanol will have no significant effect on E. Coli survivorship
Alternative: The concentrations of Ibuprofen and ethanol will cause a significant decrease in the survivorship of E. Coli.

11. Materials
E. coli
LB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl)
Latex Gloves
2 syringe Sterile Filters
Micropipettes
BunsenBurners
Micro tubes
Spread Bar
Children’s Ibuprofen
Incubator
ethanol
Ethanol
Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Matches
Vortex
Klett Spectrophotometer
30 LB agar plates

12. Procedure E. coli was grown overnight in sterile LB agar.
A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL.
The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
Ibuprofen and ethanol was mixed with the appropriate amount of Sterile Dilution Fluid to create a 20% stock. Ibuprofen was used directly from the bottle, after sterile filtration. 100 μL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 104 cells/mL. The following ingredients were mixed to create the desired experimental exposures:

13. Chart of Concentrations
0% Alcohol
0.1 % Alcohol
1% Alcohol
0% Ibuprofen
0, 0
0, Low
0, High
0.5% Ibuprofen
Low, 0
Low, Low
Low, High
5% Ibuprofen
High, 0
High, Low
High, High

14. Procedure
6. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
μL aliquots were removed from the tubes and spread on LB plates.
8. The plates were incubated at 37 degrees for 24 hours.
9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
10. The appropriate statistical analyses were performed to adequately assess the hypothesis.

15. Results 0% Alcohol 0.1 % Alcohol 1% Alcohol 0% Ibuprofen
0.5% Ibuprofen
151
138
5% Ibuprofen
158
204
219

16. Results P (Ethanol)= 0.0000265 P (Ibuprofen)= 0.345
P (interaction)=

17. Statistical Analyses
ANOVA: Single Factor Analysis of Variation Statistical test that compares the means of multiple groups P-value from ANOVA testing identifies significance if P-value is <0.05 Dunnett’s Test Statistical test that compares an experimental group directly to the control group Identifies that the experimental group is significantly different than the control if the t-value > t-crit Two Factor ANOVA Can reveal synergistic effects of two variables

18. Dunnett’s Test T-Crit=2.15 Concentration T-Value Interpretation
L IBU, L Ethanol
1.59
Not Significant
H IBU, L Ethanol
0.14
L IBU, H Ethanol
1.72
H IBU, H Ethanol
2.69
Significant

19. Conclusions
The null hypothesis was rejected; the data supports a synergistic effect
The null hypothesis was rejected for the following concentration of ibuprofen and ethanol:
5% Ibuprofen, 1% Ethanol (H, H)
Ethanol appeared to have a significant effect, Ibuprofen alone did not

20. Limitations and Extensions
The plating was not precisely synchronized, which could have resulted in extra bacterial reproduction in the tubes
Only one type of exposure tested (liquid pulse)
Only a few concentrations were tested
Extensions
Test a larger range of concentrations
Test on other models (yeast)
Other exposure methods
Varying exposure times

21. References www.drugs.com/ibuprofen.html

22. 2 Factor ANOVA Count 18 Sum 3023 2811 2973 Average 167.9444 156.1667
Total
Count 18
Sum
3023
2811
2973
Average
Variance
618.5
ANOVA
Source of Variation SS df MS F
P-value
F crit
Sample 2
2.65E-05
Columns
Interaction 4
Within 45 53

23. Single Factor ANOVA Anova: Single Factor SUMMARY Groups Count Sum
Average
Variance
Column 1 6
1030
Column 2
900
150
1335.6
Column 3
830
Column 4
891
148.5
969.5
Column 5
894
149
57.2
Column 6
880
Column 7
1102
Column 8
1017
169.5
389.5
Column 9
1263
210.5
280.3
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 8
5.086
Within Groups 45
Total 53