Oil of Oregano Antibacterial Assessment 1 Alex Senchak Grade 10 Central Catholic High School

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 4 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.

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 9 Hypothesis Oil of Oregano exposure will significantly reduce the survivorship of E. coli and Staphylococcus epidermidis. Oil of Oregano exposure will not significantly reduce the survivorship of E. coli and Staphylococcus epidermidis.

Materials Beaker Micro and macro pipettes + tips Spreader bars 76 LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) Escherichia coli bacteria Staphylococcus epidermidis bacteria Oil of Oregano Burner Turn-table Tube racks Vortex Incubator Gloves\goggles Klett Spectrophotometer SDF (sterile dilution fluid). 20 sterile test tubes Ethanol 10

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

0% Stock 0.01% Stock 0.1% Stock 1% Stock Microbe 0.1 SDF Oil of Oregano Total Chart of Concentration (mL)

Procedure Cont 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 10 3 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

14 P Value 1.07 E-12

Dunnett’s Test Analysis (E. coli) T Critical = 3.26 (significant) Alpha = 0.05 Oil of Oregano Concentration T ValueInterpretation 0.01% 5.75 Significant 0.1% Significant 1% Significant 15

16 P Value 2.4 E-16

Dunnett’s Test Analysis (Staph) T Critical = 3.26 (significant) Alpha =.005 Oil of Oregano Concentration T ValueInterpretation 0.01% 7.21 Significant 0.1% Significant 1% Significant 17

18

19 LD 50- E. Coli- est. 1% Staph- est. 0.45%

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

21 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

Limitations 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. There was a lag time when plating the cells. Exposure times to the Oil of Oregano varied slightly. 22

Works Cited Contrast, By. "Oil of Oregano: All Anecdotes, No Science « Science-Based Pharmacy." Science-Based Pharmacy. Web. 26 Dec "Deseret Biologicals | Introduction to Phenolics." Deseret Biologicals | Deseret Biologicals. Web. 26 Dec "Oil of Oregano." Healing Naturally by Bee - Home Page. Web. 26 Dec "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 "Oregano Cures - FAQ (Frequently Asked Questions)." Oregano Cures - The Definitive Medicinal Spice Oil Site. Web. 26 Dec "Oregano Oil Health Benefits When It Is the Real Deal." Free Articles Directory | Submit Articles - ArticlesBase.com. Web. 26 Dec "Uses of Oil of Oregano." Source for Nutritional Supplements, Vitamins, Minerals and Herbs for Men and Women. Web. 26 Dec "The Wolfe Clinic - Oil of Oregano." The Wolfe Clinic - Advanced Healing for Degenerative and Chronic Disease. Web. 26 Dec

E. Coli Results 24 Staph Results