Vitamin C and Ibuprofen Effects on Escherichia Coli. Timothy Leisenring Grade 11 Central Catholic High School

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1 Vitamin C and Ibuprofen Effects on Escherichia Coli Timothy Leisenring Grade 11 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 Ibuprofen/Vitamin C Ibuprofen-anti-inflammatory drug that is used to reduce hormones-used for inflammation or pain in the body -reduces fever, headaches, and injuries in the body. Vitamin C-water-soluble nutrient found in foods-acts as an anti-oxidant. Improves the absorption of iron-helps the immune system from diseases. As people age, their Vitamin C levels increase.

4 Ibuprofen/Vitamin C Effects Ibuprofen-cause chest pain, urinating, nausea, and dizziness. Ibuprofen has caused hepatitis in some patients, but this is a rare effect. Vitamin C is thought of to resist cancer and heart disease-free radicals in the human body. Essential for helping the common cold, repairing tissue in all parts of the body.

5 Brands Tested Children s Motrin Active Ingredient: 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 Liquid Vitamin C Pure ascorbic acid

6 Problem Accompanied with drug side effects, combination of over the counter drugs can have dire side effects Do common daily over the counter drugs have synergistic effects and reduce survivorship of human bacterial flora?

7 Microbial Flora Present in the respiratory, urinary, and digestive tracts as well as the skin Consists of mostly bacteria and few protists and fungi Most are harmless, but some can cause diseases. Influences human anatomy, physiology, and overall health bacteria are harbored within and on humans. Do consumables such as pain relievers and/or vitamins cause a disruption in the flora of the body?

8 Large and diverse group of gram (-) bacteria Surrounded by an extra cell wall composed of lipopolysaccharides Free living, symbiont, or pathogen Common prokaryotic cell model Escherichia coli

9 Purpose: To determine if various combinations of Ibuprofen and Vitamin C have an adverse effect on human coliform populations.

10 Hypotheses Null Hypothesis: The combinations of Ibuprofen and Vitamin C will not cause a change in the survivorship of E. coli outside of chance. Alternative Hypothesis: The combinations of Ibuprofen and Vitamin C will cause a significant decrease in the survivorship of E. coli.

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

12 Procedure 1. E. coli was grown overnight in sterile LB agar. 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 (37 C) 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. Vitamin C 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 Concentration Test tube 1 Test tube 2 Test tube 3 Test tube 4 Test tube 5 Test tube 6 Test tube 7 E.coli 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml Vitamin C 0 ml 0 ml 1 ml 1 ml 0.1 ml 1 ml 0.1 ml Ibuprofen 0 ml 0.3 ml 0 ml 0.1 ml (0.1%) 0.03ml 0.3 ml 0.3 ml 0.1 ml 0.03 (0.1%) ml 0.03mL 0.3mL 0.3mL 0.03mL SDF 9.9 ml 9.6 (3 mlmg/ml) 8.9 ml (38.87 mg/ml) ml 9.5 ml (3 mg/ml) 8.6 ml ( mg/ml) ml Total 10 ml 8.87 ml 10 ml 10 ml 9.5 ml 10 ml 8.6 ml 10 ml 10 ml 9.77 ml 10 ml

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 Effects of Vitamin C and Ibuprofen on E.coli Survivorship P-value= Alpha= 0.05

16 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 t- value > t-crit

17 Dunnett s Test: E.coli Variable Concentrations T-Value Interpretation T-Crit= % vitamin C and 0.3% ibuprofen 1% vitamin C and 0.3% ibuprofen 0.1% vitamin C and 3% ibuprofen 1% vitamin C and 3% ibuprofen Not significant Not significant Not significant 3.52 SIGNIFICANT

18 Conclusions The null hypothesis was rejected for the following combinations of drugs: 0.1%Vitamin C, 0.3% Ibuprofen 1%Vitamin C, 0.3%Ibuprofen 0.1% Vitamin C, 3% Ibuprofen The only combination that did not result in significant reduction of survivorship was 1% Vitamin C and 3% Ibuprofen.

19 Limitations The plating was not exactly synchronized, which could have resulted in extra bacterial replication in the tubes. Only a few concentrations were tested Only one type of exposure tested (liquid pulse)

20 Future Changes Use a different model. Test different concentrations between Vitamin C and Ibuprofen. Test different variables. Test effects on growth rate

21 References