Shane Fogarty Grade 12 Central Catholic High School

Vitamin C And Ibuprofen Effects On Escherichia Coli

Rationale for Experiment

• Commonly, ingested materials are investigated for effects on human cells.

• However, effects on the human flora may also be important.

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.

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.

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

• 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?

Problem

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 • 1014 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?

Escherichia coli

Escherichia coli is a large and diverse group of gram (-) bacteria.

It is found in the intestinal tract of most animals, including humans.

Estimated to cause infection in more than 70,000 patients a year in the

United States.

E. coli has been tested in conjunction with Vitamin C/Ibuprofen before in

previous studies.

Purpose:

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

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.

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

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:

Chart of Concentration

Test tube

#1

Test tube #2

Test tube #3

Test tube #4

Test tube #5

E-Coli 0.1mL 0.1mL 0.1mL 0.1mL 0.1mL

Vitamin C 0mL 1mL (1%)

2mL (2%)

1mL (1%)

2mL (2%)

Ibuprofen 0mL 1mL (10mg/mL)

1mL (10mg/mL)

2mL (20mg/mL)

2mL (20mg/mL)

SDF 9.9mL 7.9mL 6.9mL 6.9mL 5.9mL

Total 10mL 10mL 10mL 10mL 10mL

Procedure

5. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

6. 100 µL aliquots were removed from the tubes and spread on LB plates. 7. The plates were incubated at 37 degrees for 24 hours. 8. The resulting colonies were counted visually. Each colony was assumed to

have arisen from one cell. 9. The appropriate statistical analyses were performed to adequately assess

the hypothesis.

0

10

20

30

40

50

60

70

80

90

Control 1%V

10mg/mLI

2%V

10mg/mLI

1%V

20mg/mLI

2%V

20mg/mLI

Vitamin C And Ibuprofen Synergistic Effects On E-Coli

Control

1%V

10mg/mLI

2%V

10mg/mLI

1%V

20mg/mLI

2%V

20mg/mLI

Co

lon

ies

Of

E-C

oli

P-value

5.34E-14

Dunnett’s Test Analysis T Critical = 3.26 (Significant) If T Value is greater than T Critical, it is significant.

Variable Concentration

T Value

Interpretation

1% Vitamin C 10mg/mL Ibuprofen

1.58 Not Significant

1% Vitamin C 20mgmL Ibuprofen

10.80 Significant

2% Vitamin C 10mg/mL Ibuprofen

10.69 Significant

2% Vitamin C 20mg/mL Ibuprofen

11.10 Significant

Conclusions

• The null hypothesis was rejected for the following combinations of drugs:

2%Vitamin C, 10mg/mL Ibuprofen 1%Vitamin C, 20mg/mL Ibuprofen 2%Vitamin C, 20mg/mL Ibuprofen

• The only combination that did not result in significant reduction of survivorship was 1% Vitamin C and 10mg/mL Ibuprofen.

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)

Future Changes

• Use a different model. • Test different concentrations between

Vitamin C and Ibuprofen. • Test different variables. • Test effects on growth rate

References

• www.drugs.com/ibuprofen.html • www.ncbi.nlm.nih.gov/pubmed/18461099 • www.cdc.gov/ecoli/ • www.mayoclinic.com/health/e-coli/DS01007 • www.webmd.com/cold-and-flu/cold.../vitamin-c-

for-common-cold