Citrus Fruit Antimicrobial Effects By John Seabrooke Central Catholic High School Grade 9
Antimicrobials Natural Tea tree oil Onion Lemon juice Grapefruit seed extract Cinnamon Artificial Antibiotics Bleach Hydrogen peroxide Triclosan Benzethonium Chloride
Lemon Juice cooking and medicine ph level of 2 (acidic) Home remedy for killing bacteria Acid is corrosive
Lemon Juice Chemical Composition Water Citric acid Phosphoric acid Melic acid Citronellol Citral Pectin Hesperidin D- Limonene Sesquiterpen Phellandrene
Grapefruit Seed Extract (GSE) All-natural antimicrobial found to contain synthetic preservatives benzethonium chloride ph level of 2
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
Escherichia coli (E. coli) Gram negative bacteria Lab prokaryotic cell model Many different types Many are not harmful Ubiquitous Mammalian symbiote
Purpose To determine the effect of lemon juice and grapefruit seed extract on E. coli survivorship
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.
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
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%.
Liquid Pulse Procedure (Continued) Concentration Chart- Lemon Juice and GSE 0% 0.1% 1% 10% Sterile Dilution Fluid 9.9 ml 9. 89 ml 9.8 ml 8.9 ml Microbe 0.1 ml 0.1 ml 0.1 ml 0.1 ml Variable 0 ml 0.01 ml 0.1 ml 1 ml Total Volume 10 ml 10 ml 10 ml 10 ml PH effect (Lemon Juice) 7 7 7 5 PH effect (GSE) 7 7 7 6
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.
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.
Colonies Lemon Juice (Liquid Pulse) Effect on E. coli survivorship 700 600 P value= 2.94055E-07 500 400 300 200 100 0 0% 0.10% 1% 10%
Dunnett s Test (Liquid Pulse) Lemon Juice Concentration T Value Interpretation 0.1% 2.613 Insignificant 1% 6.941 Significant 10% 9.284 Significant
Colonies Lemon Juice (Infusion) Effect on E. coli survivorship 700 600 P Value=0.000505063 500 400 300 200 100 0 0% 0.10% 1% 10%
Dunnett s Test (Infusion) Lemon Juice Concentration T Value Interpretation 0.1% 0.198 Insignificant 1% 3.431 Significant 10% 8.900 Significant
Colonies GSE (Liquid Pulse) Effect on E. coli survivorship 600 500 P Value=2.21499E-09 400 300 200 100 0 0% 0.10% 1% 10%
Dunnett s Test (Liquid Pulse) GSE Concentration T Value Interpretation 0.1% 3.121 Insignificant 1% 5.114 Significant 10% 10.207 Significant
Colonies GSE (Infusion) Effect on E. coli survivorship 600 500 P Value= 0.0050967 400 300 200 100 0 0% 0.10% 1% 10%
Dunnett s Test (Infusion) GSE Concentration T Value Interpretation 0.1% 1.935 Insignificant 1% 8.319 Significant 10% 10.529 Significant
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.
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.
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.
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
Works Cited http://www.biology-online.org/ http://www.nutribiotic.com/ http://www.biologycorner.com/ http://www.cdc.gov/ecoli/
Lemon Juice (Liquid Pulse) Data GSE (Liquid Pulse) 0% 0.1% 1% 10% 621 419 306 293 592 518 344 243 597 562 444 335 578 522 372 325 497 468 367 282 Lemon Juice (Infusion) 0% 0.1% 1% 10% 702 886 542 452 669 620 546 302 587 568 555 384 641 502 651 418 693 686 478 387 0% 0.1% 1% 10% 576 486 380 227 516 479 458 272 627 427 403 279 549 506 399 302 568 465 421 209 GSE (Infusion) 0% 0.1% 1% 10% 702 487 284 365 669 465 402 284 587 629 387 218 641 762 345 274 693 597 361 236
Anova (Lemon Juice Liquid Pulse) SUMMARY Groups Count Sum Average Variance Column 1 5 2885 577 2240.5 Column 2 5 2489 497.8 3053.2 Column 3 5 1833 366.6 2550.8 Column 4 5 1478 295.6 1343.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 241082.6 3 80360.85 34.98399 2.94E-07 3.238872 Within Groups 36753.2 16 2297.075 Total 277835.8 19
Anova (Lemon Juice Infusion) SUMMARY Groups Count Sum Average Variance Column 1 5 2836 567.2 1651.7 Column 2 5 2363 472.6 868.3 Column 3 5 2061 412.2 867.7 Column 4 5 1289 257.8 1483.7 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 252911.4 3 84303.78 69.22345 2.21E-09 3.238872 Within Groups 19485.6 16 1217.85 Total 272397 19
Anova (GSE Liquid Pulse) SUMMARY Groups Count Sum Average Variance Column 1 5 3292 658.4 2152.8 Column 2 5 3262 652.4 21622.8 Column 3 5 2772 554.4 3848.3 Column 4 5 1943 388.6 3101.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 237910.2 3 79303.38 10.32405 0.000505 3.238872 Within Groups 122902.8 16 7681.425 Total 360813 19
Anova (GSE Infusion) SUMMARY Groups Count Sum Average Variance Column 1 6 3292 548.6667 73970.67 Column 2 6 2940.001 490.0002 69097.4 Column 3 6 1779.01 296.5017 22779.11 Column 4 6 1377.1 229.5167 15221.92 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 417999 3 139333 3.078007 0.0050967 3.098391 Within Groups 905345.5 20 45267.28 Total 1323345 23