Transcript Antimicrobial Effects of Cinnamon Oilx

ANTIMICROBIAL
EFFECTS OF
CINNAMON OIL
Michael DeSantis
Grade 10
Central Catholic High School
PROBLEM
• Many infectious bacteria have or are becoming
antibiotic resistant.
• Cinnamon oil is thought to be a natural
antibiotic agent.
QUESTION
• Does exposure to cinnamon oil have any
effects on gram positive or gram negative
bacteria survivorship?
PREVIOUS STUDIES
• Studies have shown that cinnamon oil is effective
against:
•
•
•
•
HIV
Herpes in vitro
Melanoma
Alzheimer's disease in mice
CINNAMON OIL
• Bark of Cinnamomum genus
• Used by ancient Egyptians, Chinese and Koreans
as natural antibiotic
• Flavor and smell from cinnamaldehyde toxin
• Eugenol and estragole toxins
Escherichia coli (E. coli)
•
Part of the human flora; found in the human colon and digestive tract.
•
Cells are rod shaped and usually about 2 micrometers in length.
•
Gram (-)
•
One of the most studied and widely understood types of bacteria.
•
Reproduces rapidly, often within thirty minutes.
•
Commonly used as a prokaryote cell model.
•
Many strains, most non-pathogenic.
Staphylococcus epidermidis (Staph)
•
Common symbiont in mammals; part of the human skin flora
•
Gram (+)
•
Most types are non-pathogenic
•
Pathogenic forms can cause deadly infections
•
Common cause of hospital infection
•
Causes formation of biofilms
GRAM BACTERIA STAIN CATEGORIES
Gram-positive (Staph)
• Simple, thick cell wall
• Most pathogenic bacteria in
humans are gram-positive
• Antibiotics such as penicillin
prevent linking of peptidoglycan
and formation of cell wall
Gram-negative (E. coli )
• Thin cell wall of peplidoglycan
and lipid membrane
• Outer membrane is a thin extra
layer of lipopolysaccharide
which adds extra protection for
cell
• Outer membrane protects the
bacteria from several antibiotics
PURPOSE
• To assess the effects of cinnamon oil
on the survivorship of E. coli and
Staph bacteria colonies.
HYPOTHESES
• Null Hypothesis: Cinnamon oil will not have a
significant effect on E. coli and Staph
survivorship.
• Alternative Hypothesis: Cinnamon oil will
have a significant negative effect on E. coli and
Staph survivorship.
MATERIALS
• Cinnamon oil
• E. coli (DH5-alpha)
• Staph
• LB media and agar
plates (0.5% yeast
extract, 1% tryptone,
1% sodium chloride)
• Incubator
•
15 mL Sterile conical
tubes with Sterile
Dilution Fluid (100mM
KH2PO4, 100mM
K2HPO4, 10mMMgSO4,
1mM NaCl)
• Vortex
• Labeling tape
• Ethanol, spreader bar,
• Klett spectrophotometer matches, turntable
and sidearm flasks
• Micropipettes
PROCEDURE
LIQUID EXPOSURE
1. Bacteria (E. coli and Staph) were grown overnight in
sterile LB media.
2. Samples of the overnight cultures were added to fresh
media in a sterile sidearm flask.
3. The cultures were placed in an incubator (37°C) until a
density of 50 Klett spectrophotometer units was reached
for each. This represents a cell density of approximately
108 cells/mL.
4. The cultures were diluted in sterile dilution fluid to a
concentration of approximately 105 cells/mL.
5. Cinnamon oil was mixed with the appropriate amount of
SDF to create cinnamon oil concentrations of 10%, 1%,
0.1%, and 0%.
PROCEDURE
LIQUID EXPOSURE
CHART OF CONCENTRATION
0%
0.1%
1%
10%
Cinnamon Oil
Cinnamon Oil Cinnamon Oil Cinnamon Oil
(control)
Microbe
0.1 mL
0.1 mL
0.1 mL
0.1 mL
SDF
9.9 mL
9.89 mL
9.8 mL
8.9 mL
Cinnamon
Oil
0 mL
0.01 mL
0.1 mL
1 mL
Total
10 mL
10 mL
10 mL
10 mL
PROCEDURE
LIQUID EXPOSURE
6. 100 µL of cell cultures were then added to the cinnamon
oil solutions, yielding a final volume of 10 mL and a cell
density of approximately 103 cells/mL for each.
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.
Cinnamon Oil Effects on E. coli Survivorship
Liquid Exposure
Number of Colonies
250
P-value
6.14 E-23
200
150
= Average Number
of E. coli Colonies
100
50
0
0%
0.10%
1%
Concentration of Cinnamon Oil
10%
DUNNETT’S TEST ANALYSIS
(E. coli )
LIQUID EXPOSURE
T-crit = 2.88 (significant difference)
Alpha = 0.05
Variable
Concentration
T-value
Interpretation
0.1% Cinnamon Oil
7.75
Significant
1% Cinnamon Oil
27.42
Significant
10% Cinnamon Oil
27.47
Significant
Cinnamon Oil Effects on Staph Survivorship
Liquid Exposure
800
P-value
9.01 E-23
Number of Colonies
700
600
500
= Average Number
of Staph Colonies
400
300
200
100
0
0%
0.10%
1%
Concentration of Cinnamon Oil
10%
DUNNETT’S TEST ANALYSIS
(Staph)
LIQUID EXPOSURE
T-crit = 2.88 (significant difference)
Alpha = 0.05
Variable
Concentration
T-value
Interpretation
0.1% Cinnamon Oil
21.76
Significant
1% Cinnamon Oil
21.94
Significant
10% Cinnamon Oil
21.92
Significant
PROCEDURE
AGAR INFUSION EXPOSURE
1. Cinnamon oil was infused into the LB agar media in two
concentrations, 1% (approximately 10 mL/L cinnamon
oil) and 0.1% (approximately 1 mL/L cinnamon), and
used to create the LB agar plates.
2. The bacteria were grown overnight in sterile LB media.
3. After vortexing to evenly suspend the cells, 100 µL
aliquots of the control tube bacterial suspension were
added to the infused plates.
4. The plates were incubated at 37 C for 24 hours.
5. The resulting colonies were counted visually. Each
colony was assumed to have arisen from one cell.
Cinnamon Oil Effects on E. coli Survivorship
Agar Infusion Exposure
Number of Colonies
250
P-value
7.03 E-20
200
150
= Average Number
of E. coli Colonies
100
50
0
0%
0.10%
Concentration of Cinnamon Oil
1%
DUNNETT’S TEST ANALYSIS
(E. coli )
AGAR INFUSION EXPOSURE
T-crit = 2.75 (significant difference)
Alpha = 0.05
Variable
Concentration
T-value
Interpretation
0.1% Cinnamon Oil
41.06
Significant
1% Cinnamon Oil
42.73
Significant
Cinnamon Oil Effects on Staph Survivorship
Agar Infusion Exposure
800
P-value
2.28 E-12
Number of Colonies
700
600
500
= Average Number
of Staph Colonies
400
300
200
100
0
0%
0.10%
Concentration of Cinnamon Oil
1%
DUNNETT’S TEST ANALYSIS
(Staph)
AGAR INFUSION EXPOSURE
T-crit = 2.75 (significant difference)
Alpha = 0.05
Variable
Concentration
T-value
Interpretation
0.1% Cinnamon Oil
15.40
Significant
1% Cinnamon Oil
15.95
Significant
CONCLUSIONS
• The null hypothesis that cinnamon does not
effect bacteria survivorship was rejected for the
0.1%, 1%, and 10% liquid exposure
concentrations for Staph and E.coli.
• The null hypothesis was rejected for the 0.1%
and 1% agar infusion exposure concentrations
for both species.
LIMITATIONS AND EXTENSIONS
LIMITATIONS
• The cinnamon oil was somewhat insoluble and difficult
to mix into the SDF.
• The plating was not perfectly synchronized.
• Only one exposure time was utilized.
EXTENSIONS
• Test different and lower concentrations of cinnamon oil.
• Test other essential oils.
• Test other supposedly naturally antibiotic substances.
• Test cinnamon oil on other microbial models, especially
yeast and fungal models.
REFERENCES
• http://www.niaid.nih.gov/LabsAndResources/labs/aboutl
abs/lhbp/pathogenMolecularGeneticsSection/Pages/ott
o.aspx
• http://www.tradekey.com/product_view/id/1595431.htm
• http://www.organicfacts.net/organic-oils/naturalessential-oils/health-benefits-of-cinnamon-oil.html
• http://antibiotics-list.com/natural-antibiotics.html
• http://www.livestrong.com/article/126030-structurecinnamon-oil/
RESULTS
COLONIES PER PLATE
E. coli – Liquid Exposure
Staph – Liquid Exposure
0%
0.1%
1%
10%
0%
0.1%
1%
10%
Plate 1
195
155
0
2
Plate 1
828
9
0
0
Plate 2
213
150
0
2
Plate 2
713
2
0
4
Plate 3
204
161
2
0
Plate 3
471
3
2
2
Plate 4
226
131
5
3
Plate 4
586
8
0
0
Plate 5
237
127
1
1
Plate 5
551
15
1
0
Plate 6
207
210
0
0
Plate 6
759
7
0
0
Plate 7
238
174
0
0
Plate 7
795
3
0
1
Plate 8
211
137
3
0
Plate 8
800
1
0
0
Average
216.375
155.625
1.375
1
Average
687.875
6
0.375
0.875
E. coli – Agar Infusion Exposure
0%
0.1%
1%
Plate 1
195
3
0
Plate 2
213
0
Plate 3
204
Plate 4
Staph – Agar Infusion Exposure
0%
0.1%
1%
Plate 1
828
0
0
0
Plate 2
713
0
0
1
0
Plate 3
471
1
0
226
1
0
Plate 4
586
0
0
Plate 5
237
0
0
Plate 5
551
0
0
Plate 6
207
2
0
Plate 6
759
1
0
Plate 7
238
-
0
Plate 7
795
-
0
Plate 8
211
-
-
Plate 8
800
-
-
Average
216.375
1.1667
0
Average
687.875
0.333
0
LD 50 for E. coli and Staph
800
Number of Colonies
700
600
LD 50
E. coli = est. 0.045%
Staph = 0.32%
= E. coli
= Staph
500
400
300
200
100
0
0.00%
0.20%
0.40%
0.60%
0.80%
Concentration of Cinnamon Oil
1.00%
1.20%