1. Citrus Fruit Antimicrobial Effects By John Seabrooke Central Catholic High School Grade 9

2. Antimicrobials Natural Tea tree oil Onion Lemon juice Grapefruit seed extract Cinnamon Artificial Antibiotics Bleach Hydrogen peroxide Triclosan Benzethonium Chloride

3. Lemon Juice cooking and medicine pH level of 2 (acidic) Home remedy for killing bacteria Acid is corrosive

4. Lemon Juice Chemical Composition Water Citric acid Phosphoric acid Melic acid Citronellol Citral Pectin Hesperidin D- Limonene Sesquiterpen Phellandrene

5. Grapefruit Seed Extract (GSE) All-natural antimicrobial found to contain synthetic preservatives – benzethonium chloride pH level of 2

6. GSE Ground grapefruit seeds and pulp Polyphenolics are added and chemically converted into quaternary compounds quaternary compounds used industrially – microbials but are toxic to animal life Natural form of these industrial compounds

7. Escherichia coli (E. coli) Gram negative bacteria Lab prokaryotic cell model Many different types Many are not harmful Ubiquitous Mammalian symbiote

8. Purpose To determine the effect of lemon juice and grapefruit seed extract on E. coli survivorship

9. Hypotheses Null Hypothesis- The lemon juice and grapefruit seed extract will not have a significant effect on E. coli survivorship. Alternate Hypothesis- The lemon juice and grapefruit seed extract will significantly reduce E. coli survivorship.

10. Materials E. coli (DH5 Alpha) Ethanol Latex gloves Sicilia Lemon Juice Nutribiotic Grapefruit Seed Extract Micropipettes Microtubes Sterile Dilution Fluid (per 1 liter)(100mMKH2PO4,100mMK 2HPO4,10mMMgSO4,1mMNaC l) LB Agar Plates(LB Media) (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Matches Incubator Spread bar Turntable Bunsen Burner Vortex

11. Liquid Pulse Procedure 1.E. coli was 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 degrees Celsius) until a density of 50 Klett spectrophotometer units was reached. 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.The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, 10%.

12. Liquid Pulse Procedure (Continued) 0%0.1%1%10% Sterile Dilution Fluid 9.9 mL9. 89 mL9.8 mL8.9 mL Microbe0.1 mL Variable0 mL0.01 mL0.1 mL1 mL Total Volume10 mL PH effect (Lemon Juice) 7775 PH effect (GSE)7776 Concentration Chart- Lemon Juice and GSE

13. Liquid Pulse Procedure (Continued) 6. 0.1 mL of cell culture was then added to the 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 microliters aliquots were removed from the tubes and spread on LB-agar plates. 9. The plates were incubated at 37 degrees Celsius for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

14. Agar Infusion Procedure 1.Sterilized lemon juice and grapefruit seed extract were infused into the LB agar media in three concentrations, 0.1%, 1%, and 10% (with volumes of 2, 20, and 200 microliters of variable) and used to create the LB agar plates. 2. 0.1 mL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10^3 cells/mL. 3. After vortexing to evenly suspend the cells, 100 microliters aliquots were removed from the control tube and spread onto the infused agar plates. 4. The plates were incubated at 37 degrees Celsius for 24 hours. 5. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

15. Lemon Juice (Liquid Pulse) Effect on E. coli survivorship Colonies

16. Dunnett’s Test (Liquid Pulse) Lemon Juice ConcentrationT ValueInterpretation 0.1%2.613Insignificant 1%6.941Significant 10%9.284Significant

17. Lemon Juice (Infusion) Effect on E. coli survivorship Colonies

18. Dunnett’s Test (Infusion) Lemon Juice ConcentrationT ValueInterpretation 0.1%0.198Insignificant 1%3.431Significant 10%8.900Significant

19. GSE (Liquid Pulse) Effect on E. coli survivorship Colonies P Value=2.21499E-09

20. Dunnett’s Test (Liquid Pulse) GSE ConcentrationT ValueInterpretation 0.1%3.121Insignificant 1%5.114Significant 10%10.207Significant

21. GSE (Infusion) Effect on E. coli survivorship Colonies P Value= 0.0050967

22. Dunnett’s Test (Infusion) GSE ConcentrationT ValueInterpretation 0.1%1.935Insignificant 1%8.319Significant 10%10.529Significant

23. Key Questions Did lemon juice have a significant effect on E. coli survivorship? Yes, the p values were 2.94E-07 and 0.000505. Did grapefruit seed extract have a significant effect on E. coli survivorship? Yes, the p values were 2.21E-09 and 0.005096.

24. Key Questions At which concentrations did lemon juice significantly reduce E. coli survivorship? For both the liquid pulse and infusion tests, the 1% and 10% concentrations significantly reduced E. coli survivorship. At which concentrations of GSE significantly reduce E. coli survivorship? For both the liquid pulse and infusion tests, the 1% and 10% concentrations significantly reduced E. coli survivorship.

25. Conclusion For both experiments, the null hypothesis can be rejected and the alternate hypothesis can be accepted. Lemon juice had a significant negative impact on E. coli survivorship. Grapefruit Seed Extract had a significant negative impact on E. coli survivorship.

26. Limitations and Future Studies Limitations Only one microorganism was tested Slight variations in plating time Only one exposure time Unknown active ingredient Future Studies More concentrations Different times of exposure Synergistic effects Narrow concentration range Attempt to find LD50 Try to identify the antimicrobial agent

27. Works Cited http://www.biology-online.org/ http://www.nutribiotic.com/ http://www.biologycorner.com/ http://www.cdc.gov/ecoli/

28. Data 0%0.1%1%10% 621419306293 592518344243 597562444335 578522372325 497468367282 Lemon Juice (Liquid Pulse) 0%0.1%1%10% 702886542452 669620546302 587568555384 641502651418 693686478387 GSE (Liquid Pulse) GSE (Infusion) Lemon Juice (Infusion) 0%0.1%1%10% 576486380227 516479458272 627427403279 549506399302 568465421209 0%0.1%1%10% 702487284365 669465402284 587629387218 641762345274 693597361236

29. Anova (Lemon Juice Liquid Pulse) SUMMARY GroupsCountSumAverageVariance Column 1528855772240.5 Column 252489497.83053.2 Column 351833366.62550.8 Column 451478295.61343.8 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups241082.6380360.8534.983992.94E-073.238872 Within Groups36753.2162297.075 Total277835.819

30. Anova (Lemon Juice Infusion) SUMMARY GroupsCountSumAverageVariance Column 152836567.21651.7 Column 252363472.6868.3 Column 352061412.2867.7 Column 451289257.81483.7 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups252911.4384303.7869.223452.21E-093.238872 Within Groups19485.6161217.85 Total27239719

31. Anova (GSE Liquid Pulse) SUMMARY GroupsCountSumAverageVariance Column 153292658.42152.8 Column 253262652.421622.8 Column 352772554.43848.3 Column 451943388.63101.8 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups237910.2379303.3810.324050.0005053.238872 Within Groups122902.8167681.425 Total36081319

32. Anova (GSE Infusion) SUMMARY GroupsCountSumAverageVariance Column 163292548.666773970.67 Column 262940.001490.000269097.4 Column 361779.01296.501722779.11 Column 461377.1229.516715221.92 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups41799931393333.0780070.00509673.098391 Within Groups905345.52045267.28 Total132334523