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Oil of Oregano
Antibacterial Assessment
Alex Senchak
Grade 10
Central Catholic High School
1
Problem
 Bacteria that cause infections are
becoming increasingly resistant to
antibiotics and other antimicrobial
agents.
2
Escherichia coli (E. coli)
 One of the most common forms of
bacteria found in many environments.
 Symbiont in intestinal tracts of many
mammals.
 Most non-pathogenic.
 Pathogenic strains can lead to life
threatening infections.
3
E. coli:
Gram Negative Bacteria
Cell wall is thin
extra layer of
lipopolysaccharide
which adds extra
level of protection.
If the toxin enters
the circulatory
system it causes a
toxic reaction.
This outer
membrane protects
the bacteria from
several antibiotics.
4
Staphylococcus epidermidis
 Common surface symbiont in many
mammals (human).
 Most forms considered non-pathogenic.
 Pathogenic forms can be life
threatening.
 Forms biofilms.
5
Staph:
Gram Positive Bacteria
Most pathogenic
bacteria in humans
are gram-positive
organisms.
Simple cell wall.
Antibiotics such as
penicillin work
against the
formation of the
cell wall.
6
Oil of Oregano
 Naturally occurring antibiotic.
 Carvacrol.
 Disruption of biofilms.
7
Purpose
 To determine if Oil of Oregano will
affect the survivorship of E. coli and
Staphylococcus epidermidis.
8
Null Hypothesis
 Oil of Oregano exposure will not
significantly reduce the survivorship of
E. coli and Staphylococcus epidermidis.
Hypothesis
 Oil of Oregano exposure will significantly
reduce the survivorship of E. coli and
Staphylococcus epidermidis.
9
Materials
 Beaker
 Burner
 Micro and macro pipettes + tips
 Turn-table
 Spreader bars
 Tube racks
 76 LB agar plates (1% Tryptone,
 Vortex
0.5% Yeast Extract, 1% NaCl)
 Escherichia coli bacteria
 Staphylococcus epidermidis
bacteria
 Oil of Oregano
 Incubator
 Gloves\goggles
 Klett Spectrophotometer
 SDF (sterile dilution fluid).
 20 sterile test tubes
 Ethanol
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Procedure for Testing
Oregano on Bacteria
1. Bacteria (E. coli and Staph) was 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 (37°C) 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. An oil of oregano suspension was mixed with the appropriate
amount of SDF to create oil of oregano concentrations of 1%,
0.1%, 0.01%, and 0%
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Chart of Concentration (mL)
0%
Stock
0.01%
Stock
0.1%
Stock
1%
Stock
Microbe
0.1
0.1
0.1
0.1
SDF
9.9
9.899
9.890
9.8
Oil of
Oregano
0
0.001
0.01
0.1
Total
10
10
10
10
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Procedure Cont.
6. 100 µL of cell culture was then added to the oil of oregano
solutions, yielding a final volume of 10 mL and a cell density of
approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room
temperature for 15 minutes.
8. After vortexing to evenly suspend the cells, 100 µL aliquots were
removed from the tubes and spread on LB agar plates.
9. The plates were incubated at 37°C for 24 hours.
10. The resulting colonies were counted visually. Each colony was
assumed to have arisen from one cell.
13
Effect of Oregano on E. coli
250.00
P Value
1.07 E-12
217.33
181.67
Colonies
200.00
148.17
109.33
150.00
100.00
50.00
0.00
0%
0.01%
0.10%
% of Oregano in Solution
1%
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Dunnett’s Test Analysis (E. coli)
T Critical = 3.26
(significant)
Alpha = 0.05
Oil of Oregano
Concentration
T Value
Interpretation
0.01%
5.75
Significant
0.1%
11.16
Significant
1%
25.57
Significant
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Effect of Oregano on Staph
250.00
P Value
2.4 E-16
201.17
164.17
200.00
Colonies
138.00
150.00
58.83
100.00
50.00
0.00
0%
0.01%
0.10%
% of Oregano in Solution
1%
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Dunnett’s Test Analysis (Staph)
T Critical = 3.26
(significant)
Alpha = .005
Oil of Oregano
Concentration
T Value
Interpretation
0.01%
7.21
Significant
0.1%
12.31
Significant
1%
17.90
Significant
17
Survivorship Percentage
Compared to the Control
% of Suriving Colonies
90%
80%
70%
60%
50%
40%
E. Coli
30%
Staph
20%
10%
0%
.01%
.1%
% of Oregano in Solution
1%
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% of Surviving Colonies
LD 50 for E. coli and Staph
90%
LD 50E. Coli- est. 1%
Staph- est. 0.45%
80%
70%
60%
50%
40%
E. coli
30%
20%
Staph
10%
0%
0.00%
0.20%
0.40%
0.60%
0.80%
1.00%
1.20%
% of Oregano in Solution
19
Observation
 There appeared to be a dosage effect of
the Oil of Oregano on the bacteria.
 Increasing amounts of Oil of Oregano
led to less bacteria surviving.
20
Conclusion
Null Hypothesis
 Oil of Oregano exposure will not
significantly reduce the survivorship
of E. coli or Staphylococcus
epidermidis.
 REJECTED
Hypothesis
 Oil of Oregano exposure will
significantly reduce the survivorship
of E. coli and Staphylococcus
epidermidis.
 ACCEPTED
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Limitations
• There was a lag time
when plating the
cells. Exposure times
to the Oil of Oregano
varied slightly.
Extensions
• Test a higher
concentration of Oil
of Oregano on
bacteria.
• Test different types of
bacteria.
• Infuse the agar with
oil of oregano to
provide a prolonged
surface exposure.
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Works Cited
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Contrast, By. "Oil of Oregano: All Anecdotes, No Science « Science-Based Pharmacy."
Science-Based Pharmacy. Web. 26 Dec. 2010.
<http://sciencebasedpharmacy.wordpress.com/2009/03/27/oil-of-oregano/>.
"Deseret Biologicals | Introduction to Phenolics." Deseret Biologicals | Deseret
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"Oil of Oregano." Healing Naturally by Bee - Home Page. Web. 26 Dec. 2010.
<http://www.healingnaturallybybee.com/articles/anti6.php>.
"Oil of Wild Oregano Is One of the Strongest and Most Effective Antibiotics Known to
Man." CuringHerbs.com Herbal Remedies for Your Health. Web. 26 Dec. 2010.
<http://curingherbs.com/wild_oregano_oil.htm>.
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Medicinal Spice Oil Site. Web. 26 Dec. 2010.
<http://www.oreganocures.com/faq.html#3>.
"Oregano Oil Health Benefits When It Is the Real Deal." Free Articles Directory |
Submit Articles - ArticlesBase.com. Web. 26 Dec. 2010.
<http://www.articlesbase.com/alternative-medicine-articles/oregano-oil-healthbenefits-when-it-is-the-real-deal-2840298.html>.
"Uses of Oil of Oregano." Source for Nutritional Supplements, Vitamins, Minerals and
Herbs for Men and Women. Web. 26 Dec. 2010. <http://www.healthyhealth.com/site/844294/page/541843>.
"The Wolfe Clinic - Oil of Oregano." The Wolfe Clinic - Advanced Healing for Degenerative
and Chronic Disease. Web. 26 Dec. 2010. <http://www.thewolfeclinic.com/oregano.html>.
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E. Coli Results
Staph Results
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