1. Oil of Oregano Effects on Microbial Flora Oil of Oregano Effects on Microbial Flora Cameron Herbst Pittsburgh Central Catholic High School

2. The Claim  Hailed as a highly potent purifier, antiviral, antibacterial, antifungal, antiparasitic.  Benefits respiratory health, joint/muscle flexibility, destroy organisms that contribute to skin infections/digestive problems, strengthen immune system

3. The Claim  Countless studies have evaluated the affect of ingested material on human cells and physiology.  A key component of the body is the natural flora - especially those within the alimentary canal. These belong to the families: Clostridium, Fusobacterium, Escherichia, Bacteroides, Eubacterium, and Lactobacillus, among others.

4. Oil of Oregano  Derived from wild oregano: Origanum vulgare  Two key compounds: carvacrol, thymol (studies suggest may combat microorganisms which cause illness)  Ingredients: 60% carvacrol (3.2 mg), olive oil

5. Production and Uses of Oil of Oregano  Marketed as “supports general well-being”  Obtained from crushing the leaves of the wild oregano plant  Dosage is 2 drops  Can be ingested or applied topically

6. Risks of Oil of Oregano Use  Reduces the body’s ability to absorb iron  Not advisable for pregnant women, stimulates blood flow in the uterus, weakening lining of fetus in the womb  Can cause skin irritation, rashes, or vomiting if you have allergies to thyme, basil, mint, or sage

7. E. coli E. coli  One of the most common forms of bacteria; Free living, symbiotic or pathogenic  Has been utilized as the most studied prokaryote  There are many of different strains of E. coli, most of which are non- pathogenic. However, there are strains which can produce fatal diseases

8. Staphylococcus epidermidis  Part of our normal/skin flora, gram-positive  Anaerobic, but grows best in aerobic conditions  Opportunistic pathogen, requires major breach in hosts defenses  Non-pathogenic strain utilized

9. Purpose  To determine whether oil of oregano in various concentrations will affect the survivorship of intestinal flora (E. coli) and Staphylococcus epidermidis.

10. Hypothesis  Null – Survivorship of E. coli and Staphylococcus epidermidis in varying concentrations of oil of oregano would not vary significantly from the control.  Alternative – Survivorship of E. coli and Staphylococcus epidermidis in varying concentrations of oil of oregano would vary significantly from the control.

11. Materials  Ethanol (sterilization of instruments)  Latex gloves  E. coli DH5 alpha  Micropipette  Tube rack  8 Tubes  SDF (per 1 liter) (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  Turn table  LB agar plates  LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Bunsen burner  Spreader bars  Matches  Sterile pipette tips  Incubator  Vortex  Klett spectrophotometer  Staphylococcus epidermidis

12. Procedure 1)E. coli and Staphylococcus epidermidis 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 cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4)The culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/mL. 5) The tubes were prepared as follows:

13. Procedure Tube #Oil of Oregano (mL) SDF (mL)E. coli/Staph (mL) Final Volume (mL) Final Concentration (%) 1 (E. coli)09.90.1100 2 (E. coli)0.019.890.1100.1 3 (E. coli)0.19.80.1101 4 (E. coli)0.59.40.1105 1 (Staph)09.90.1100 2 (Staph)0.019.890.1100.1 3 (Staph)0.19.80.1101 4 (Staph)0.59.40.1105

14. Procedure 6) The E. coli and Staphylococcus epidermidis was allowed to remain exposed to the oil of oregano for 15 minutes. 7)100 µL aliquots were removed from the tubes and spread on LB plates.(6 replicates) 8)The plates were incubated at 37 degrees Celsius for 24 hours. 9)The resulting colonies were quantified. Each colony is assumed to have arisen from one cell.

15. p= 9.63E-8

16. p= 6.19E-4

17. E. coli Dunnett’s Test Variable Comparisont-valueInterpretation 0.1% vs control67.75Significant 1% vs control53.41Significant 5% vs control 23.75Significant α = 0.01 t-crit = 4.21

18. Staph Dunnett’s Test α = 0.01 t-crit = 4.21 Variable Comparisont-valueInterpretation 0.1% vs control133.70Significant 1% vs control122.70Significant 5% vs control 95.20Significant

19. Interpretation  There appeared to be a correlation between the percent concentration of oil of oregano and cell survivorship. Higher concentrations of oil of oregano resulted in fewer surviving colonies in both Staphylococcus epidermidis and E. coli.

20. Conclusion  5% oil of oregano appeared to greatly reduce E. coli survivorship  The null hypothesis can be rejected for all concentrations of oil of oregano for E. coli and Staphylococcus epidermidis.

21. Limitations  This experiment was limited in that pure oil of oregano was not able to be used.  Plating was not exactly synchronized, which could have resulted in extra time for bacterial replication.

22. Extensions  Varying exposure time of E. coli and Staphylococcus epidermidis in the tubes before plating will be tested.  To allow for longer exposure, oil of oregano will be infused directly into the LB agar.  A different symbiotic organism other than E. coli and Staphylococcus epidermidis will be tested.

23. Cited Sources  http://e-colibasics.com/ http://e-colibasics.com/  www.oiloforegano.com/ www.oiloforegano.com/  http://randi.org/site/index.php/swift- blog/1099-oil-of-oregano-miracle- cure.html http://randi.org/site/index.php/swift- blog/1099-oil-of-oregano-miracle- cure.html  www.fda.gov www.fda.gov  http://web.uconn.edu/mcbstaff/graf/Stu dent%20presentations/S%20epidermid is/sepidermidis.html http://web.uconn.edu/mcbstaff/graf/Stu dent%20presentations/S%20epidermid is/sepidermidis.html