ANTIMICROBIAL EFFECTS OF

CINNAMON OILMichael 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.

• Does exposure to cinnamon oil have any effects on gram positive or gram negative bacteria survivorship?

QUESTION

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

• 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 BACTERIA STAIN CATEGORIESGram-positive (Staph) 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

• Klett spectrophotometer and sidearm flasks

• Micropipettes

• 15 mL Sterile conical tubes with Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mMMgSO4, 1mM NaCl)

• Vortex

• Labeling tape

• Ethanol, spreader bar, matches, turntable

PROCEDURELIQUID 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%.

CHART OF CONCENTRATION0%

Cinnamon Oil(control)

0.1% Cinnamon Oil

1% Cinnamon Oil

10% Cinnamon Oil

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

PROCEDURELIQUID EXPOSURE

PROCEDURELIQUID 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 SurvivorshipLiquid Exposure

0.00% 0.10% 1.00% 10.00%0

50

100

150

200

250

Concentration of Cinnamon Oil

Nu

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P-value6.14 E-23

= Average Number of E. coli Colonies

Variable Concentration T-value Interpretation

0.1% Cinnamon Oil 7.75 Significant

1% Cinnamon Oil 27.42 Significant

10% Cinnamon Oil 27.47 Significant

T-crit = 2.88 (significant difference)

Alpha = 0.05

DUNNETT’S TEST ANALYSIS(E. coli )

LIQUID EXPOSURE

Cinnamon Oil Effects on Staph SurvivorshipLiquid Exposure

0.00% 0.10% 1.00% 10.00%0

100

200

300

400

500

600

700

800

Concentration of Cinnamon Oil

Nu

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P-value9.01 E-23

= Average Number of Staph Colonies

DUNNETT’S TEST ANALYSIS(Staph)

LIQUID EXPOSURE

Variable Concentration T-value Interpretation

0.1% Cinnamon Oil 21.76 Significant

1% Cinnamon Oil 21.94 Significant

10% Cinnamon Oil 21.92 Significant

T-crit = 2.88 (significant difference)

Alpha = 0.05

PROCEDUREAGAR 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 SurvivorshipAgar Infusion Exposure

0.00% 0.10% 1.00%0

50

100

150

200

250

Concentration of Cinnamon Oil

Nu

mb

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f C

olo

nie

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P-value7.03 E-20

= Average Number of E. coli Colonies

DUNNETT’S TEST ANALYSIS(E. coli )

AGAR INFUSION EXPOSURE

Variable Concentration T-value Interpretation

0.1% Cinnamon Oil 41.06 Significant

1% Cinnamon Oil 42.73 Significant

T-crit = 2.75 (significant difference)

Alpha = 0.05

Cinnamon Oil Effects on Staph SurvivorshipAgar Infusion Exposure

0.00% 0.10% 1.00%0

100

200

300

400

500

600

700

800

Concentration of Cinnamon Oil

Nu

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P-value2.28 E-12

= Average Number of Staph Colonies

DUNNETT’S TEST ANALYSIS(Staph)

AGAR INFUSION EXPOSURE

Variable Concentration T-value Interpretation

0.1% Cinnamon Oil 15.40 Significant

1% Cinnamon Oil 15.95 Significant

T-crit = 2.75 (significant difference)

Alpha = 0.05

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 EXTENSIONSLIMITATIONS

• 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/aboutlabs/lhbp/pathogenMolecularGeneticsSection/Pages/otto.aspx

• http://www.tradekey.com/product_view/id/1595431.htm

• http://www.organicfacts.net/organic-oils/natural-essential-oils/health-benefits-of-cinnamon-oil.html

• http://antibiotics-list.com/natural-antibiotics.html

• http://www.livestrong.com/article/126030-structure-cinnamon-oil/

RESULTS0% 0.1% 1% 10%

Plate 1 195 155 0 2

Plate 2 213 150 0 2

Plate 3 204 161 2 0

Plate 4 226 131 5 3

Plate 5 237 127 1 1

Plate 6 207 210 0 0

Plate 7 238 174 0 0

Plate 8 211 137 3 0

Average 216.375 155.625 1.375 1

COLONIES PER PLATE

0% 0.1% 1%

Plate 1 195 3 0

Plate 2 213 0 0

Plate 3 204 1 0

Plate 4 226 1 0

Plate 5 237 0 0

Plate 6 207 2 0

Plate 7 238 - 0

Plate 8 211 - -

Average 216.375 1.1667 0

0% 0.1% 1%

Plate 1 828 0 0

Plate 2 713 0 0

Plate 3 471 1 0

Plate 4 586 0 0

Plate 5 551 0 0

Plate 6 759 1 0

Plate 7 795 - 0

Plate 8 800 - -

Average 687.875 0.333 0

0% 0.1% 1% 10%

Plate 1 828 9 0 0

Plate 2 713 2 0 4

Plate 3 471 3 2 2

Plate 4 586 8 0 0

Plate 5 551 15 1 0

Plate 6 759 7 0 0

Plate 7 795 3 0 1

Plate 8 800 1 0 0

Average 687.875 6 0.375 0.875

E. coli – Liquid Exposure Staph – Liquid Exposure

E. coli – Agar Infusion Exposure Staph – Agar Infusion Exposure

LD 50 for E. coli and Staph

0.00% 0.20% 0.40% 0.60% 0.80% 1.00% 1.20%0

100

200

300

400

500

600

700

800

= E. coli= Staph

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Concentration of Cinnamon Oil

LD 50E. coli = est. 0.045%Staph = 0.32%