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 antioxidant.
• Improves the absorption of ironhelps 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
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?
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.
Vitamin C And Ibuprofen Synergistic Effects On E-Coli
P-value
90
5.34E-14
Colonies Of E-Coli
80
Control
70
1%V
10mg/mLI
60
50
2%V
10mg/mLI
40
1%V
20mg/mLI
30
2%V
20mg/mLI
20
10
0
Control
1%V
2%V
1%V
2%V
10mg/mLI 10mg/mLI 20mg/mLI 20mg/mLI
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-cfor-common-cold