1. Cranberry Juice Antimicrobial Properties Zane Stiles Central Catholic High School Grade 9

2. Antimicrobial effects of berries o Types of berries Raspberry Cranberry Blueberry o Previous Studies Cancer cells antioxidant activity Staphylococcus aureus and Escherichia coli

3. Cranberry Health Benefits o Cancer and disorders Cardiovascular Immune Systems o Dental Plaque o Kidney Stones

4. Urinary Tract Infections (UTIs) o On the Rise o Frequency o Cause o Common Cures

5. Cranberry Juice and UTIs o Shown to prevent or minimize pathology in vitro o Inhibit adhesion of E. coli o Results variable and moderate

6. Cranberry Juice and UTIs o Previous Results Inconclusive o High Drop-out Rate o Adverse Effects gastrointestinal intolerance weight gain drug-cranberry interactions

7. Escherichia coli o Inhabit intestinal tract o Many different types o Many are not harmful o Different types of infections o Serves as a common prokaryotic cell model

8. E. coli and Cranberry Juice o E. coli and other bacteria cause many infections o Cranberry Juice is claimed to prevent these o Does cranberry juice affect E. coli populations?

9. Purpose To determine if cranberry juice has an affect on E. coli survivorship in vitro.

10. Hypotheses o Null hypothesis: Cranberry juice does not affect E. coli survivorship in vitro. o Alternative hypothesis: Cranberry juice does affect E. coli survivorship in vitro.

11. o Labeling tape o Ethanol o Latex gloves o E. coli DH5 Alpha o Micropipettes o Micro rack o Micro tubes o LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride). o Luria Broth agar plates (to be infused with cranberry juice) Materials o Cranberry Juice (natural, unsweetened) o Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) o Turntable o Luria Broth agar plates o Bunsen burner o Spread bar o Incubator o Klett Spectrophotometer o Matches

12. Pulse Liquid Exposure Procedure 1. E. coli 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. Sterilized Cranberry juice was mixed with the appropriate amount of SDF to create cranberry juice concentrations of 10%, 1%, and 0.1%.

13. Chart of Liquid Concentration 0% Concentration 0.1% Concentration 1% Concentration 10% Concentration Microbe0.1mL SDF9.9mL9.89mL9.8mL8.9mL Cranberry Juice 0mL0.01mL0.1mL1mL Total10mL

14. Pulse Liquid Exposure Procedure 6.100 µL of cell culture was then added to the cranberry juice 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 a set of regular LB 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.

15. Agar Infusion Procedure 1. Sterilized Cranberry Juice was infused into the LB agar media in two concentrations, 10 % (approximately 100mL/L Cranberry Juice) and low (approximately 10mL/L Cranberry Juice), and used to create the LB agar plates. 2. E. coli was grown overnight in sterile LB media. 3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 4. 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. 5. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

16. Agar Infusion Procedure 6.100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. 8.The plates were incubated at 37 C for 24 hours. 9.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

17. 10 2 cells 10 3 cells/mL with cranberry juice 10 8 cells/mL (E. coli) 100 uL 10 5 cells/mL (E. coli) 100 uL

18. P-value 1.35E-08 P-value 1.35E-08 Colony Count Cranberry Juice Pulse Liquid Exposure Effects on E. coli Survivorship Concentration of Cranberry Juice P-value 1.35E-08 P<0.05 P>0.05

19. Dunnett’s Test Analysis T Critical = 3.10 (significant) Alpha = 0.05 Pulse Liquid Exposure Variable Concentration T valueInterpretation 0.1% Cranberry Juice 3.00Not Significant 1% Cranberry Juice6.18Significant 10% Cranberry Juice 10.38Significant

20. Cranberry Juice Pulse Liquid Exposure Effects on E. coli Survivorship Colony Count Concentration of Cranberry Juice

21. P-value 1.35E-08 P-value 1.35E-08 Colony Count Cranberry Juice Agar Infusion Effects on E. coli Survivorship Concentration of Cranberry Juice P<0.05 P-value 2.73E-13

22. Dunnett’s Test Analysis T Critical = 2.67 (significant) Alpha = 0.05 Agar Infusion Exposure Variable Concentration T valueInterpretation 1%11.39Significant 10%17.30Significant

23. Conclusions o The null hypothesis that cranberry juice does not affect E. coli survivorship in vitro was rejected for the concentration of 1% and 10% cranberry juice in both types of exposure. o The null hypothesis could not be rejected for the lowest concentration of 0.1% cranberry juice. o The results indicate that 1% and 10% cranberry juice did negatively influence E. coli survivorship.

24. Limitations, Extensions, and Future Studies o The E. coli plating was not exactly synchronized, possibly contributing to varying colony counts. o Infused amount of cranberry juice in the LB agar was not exact. o Does not account for dilution in the body o Larger sample sizes will be employed in the next round of testing. o Higher and lower concentrations of cranberry juice will be tested. o More species of bacteria will be tested. Limitations Extensions and Future Studies

25. References o Kessman, Scott. The Health Benefits of Cranberry Juice. July 2007 http://www.associatedcontent.com/article/34119/the_health_benefits_of_cranberry_juice.html?cat=5 o Author: unknown. Cranberry History. 2010 http://www.oceanspray.com/about/cranberry_history2.aspx o Author: unknown. E. coli bacteria: what are they, where did they come from, and why are some so dangerous? 2005 http://www.about-ecoli.com/ o Author: unknown. The History of Cranberries November 14, 2006 http://www.kitchenproject.com/history/cranberries/index.htm o Author: unknown. Growing Cranberries. 2008 http://cranberryjuice.com/about.html o Author: unknown. SuperFruit: A Rundown on Cranberry Juice. 2008 http://cranberryjuice.com/benefits.html o Author: unknown. Urinary Tract Infections in Adults. 2005 http://kidney.niddk.nih.gov/kudiseases/pubs/utiadult/ o Heather M.A. Cavanagh, Michael Hipwell, Jenny M. Wilkinson. Journal of Medicinal Food. March 2003,. March 2003, 6(1): 57- 61. o Guay, David. Cranberry and Urinary Tract Infections, May 2009 http://adisonline.com/drugs/pages/articleviewer.aspx?year=2009&issue=69070&article=00002&type=abstract