1. ANTIMICROBIAL EFFECTS OF CINNAMON OIL Michael DeSantis Grade 10 Central Catholic High School

2. 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

3. PREVIOUS STUDIES Studies have shown that cinnamon oil is effective against: HIV Herpes in vitro Melanoma Alzheimer's disease in mice

4. 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

5. 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.

6. 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

7. 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 CATEGORIES Gram-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

8. PURPOSE To assess the effects of cinnamon oil on the survivorship of E. coli and Staph bacteria colonies.

9. 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.

10. 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 KH 2 PO 4, 100mM K 2 HPO 4, 10mMMgSO 4, 1mM NaCl) Vortex Labeling tape Ethanol, spreader bar, matches, turntable

11. 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 10 8 cells/mL. 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 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%.

12. CHART OF CONCENTRATION 0% Cinnamon Oil (control) 0.1% Cinnamon Oil 1% Cinnamon Oil 10% Cinnamon Oil Microbe0.1 mL SDF9.9 mL9.89 mL9.8 mL8.9 mL Cinnamon Oil 0 mL0.01 mL0.1 mL1 mL Total10 mL PROCEDURE LIQUID EXPOSURE

13. 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 10 3 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.

14. Cinnamon Oil Effects on E. coli Survivorship Liquid Exposure P-value 6.14 E-23 = Average Number of E. coli Colonies

15. Variable Concentration T-valueInterpretation 0.1% Cinnamon Oil7.75Significant 1% Cinnamon Oil27.42Significant 10% Cinnamon Oil27.47Significant T-crit = 2.88 (significant difference) Alpha = 0.05 DUNNETT’S TEST ANALYSIS (E. coli ) LIQUID EXPOSURE

16. Cinnamon Oil Effects on Staph Survivorship Liquid Exposure P-value 9.01 E-23 = Average Number of Staph Colonies

17. DUNNETT’S TEST ANALYSIS (Staph) LIQUID EXPOSURE Variable Concentration T-valueInterpretation 0.1% Cinnamon Oil21.76Significant 1% Cinnamon Oil21.94Significant 10% Cinnamon Oil21.92Significant T-crit = 2.88 (significant difference) Alpha = 0.05

18. 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.

19. Cinnamon Oil Effects on E. coli Survivorship Agar Infusion Exposure P-value 7.03 E-20 = Average Number of E. coli Colonies

20. DUNNETT’S TEST ANALYSIS (E. coli ) AGAR INFUSION EXPOSURE Variable Concentration T-valueInterpretation 0.1% Cinnamon Oil41.06Significant 1% Cinnamon Oil42.73Significant T-crit = 2.75 (significant difference) Alpha = 0.05

21. Cinnamon Oil Effects on Staph Survivorship Agar Infusion Exposure P-value 2.28 E-12 = Average Number of Staph Colonies

22. DUNNETT’S TEST ANALYSIS (Staph) AGAR INFUSION EXPOSURE Variable Concentration T-valueInterpretation 0.1% Cinnamon Oil15.40Significant 1% Cinnamon Oil15.95Significant T-crit = 2.75 (significant difference) Alpha = 0.05

23. 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.

24. 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.

25. REFERENCES http://www.niaid.nih.gov/LabsAndResources/labs/aboutl abs/lhbp/pathogenMolecularGeneticsSection/Pages/ott o.aspxhttp://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/natural- essential-oils/health-benefits-of-cinnamon-oil.htmlhttp://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/http://www.livestrong.com/article/126030-structure- cinnamon-oil/

26. RESULTS 0%0.1%1%10% Plate 119515502 Plate 221315002 Plate 320416120 Plate 422613153 Plate 523712711 Plate 620721000 Plate 723817400 Plate 821113730 Average 216.375155.6251.3751 COLONIES PER PLATE 0%0.1%1% Plate 119530 Plate 221300 Plate 320410 Plate 422610 Plate 523700 Plate 620720 Plate 7238-0 Plate 8211-- Average 216.3751.16670 0%0.1%1% Plate 182800 Plate 271300 Plate 347110 Plate 458600 Plate 555100 Plate 675910 Plate 7795-0 Plate 8800-- Average 687.8750.3330 0%0.1%1%10% Plate 1828900 Plate 2713204 Plate 3471322 Plate 4586800 Plate 55511510 Plate 6759700 Plate 7795301 Plate 8800100 Average 687.87560.3750.875 E. coli – Liquid ExposureStaph – Liquid Exposure E. coli – Agar Infusion ExposureStaph – Agar Infusion Exposure

27. LD 50 for E. coli and Staph Number of Colonies Concentration of Cinnamon Oil LD 50 E. coli = est. 0.045% Staph = 0.32%