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

Transcription

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) Concentration Chart- Lemon Juice and GSE 0% 0.1% 1% 10% Sterile Dilution Fluid 9.9 ml 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) PH effect (GSE)

13 Liquid Pulse Procedure (Continued) 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 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 Colonies Lemon Juice (Liquid Pulse) Effect on E. coli survivorship P value= E % 0.10% 1% 10%

16 Dunnett s Test (Liquid Pulse) Lemon Juice Concentration T Value Interpretation 0.1% Insignificant 1% Significant 10% Significant

17 Colonies Lemon Juice (Infusion) Effect on E. coli survivorship P Value= % 0.10% 1% 10%

18 Dunnett s Test (Infusion) Lemon Juice Concentration T Value Interpretation 0.1% Insignificant 1% Significant 10% Significant

19 Colonies GSE (Liquid Pulse) Effect on E. coli survivorship P Value= E % 0.10% 1% 10%

20 Dunnett s Test (Liquid Pulse) GSE Concentration T Value Interpretation 0.1% Insignificant 1% Significant 10% Significant

21 Colonies GSE (Infusion) Effect on E. coli survivorship P Value= % 0.10% 1% 10%

22 Dunnett s Test (Infusion) GSE Concentration T Value Interpretation 0.1% Insignificant 1% Significant 10% Significant

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

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

28 Lemon Juice (Liquid Pulse) Data GSE (Liquid Pulse) 0% 0.1% 1% 10% Lemon Juice (Infusion) 0% 0.1% 1% 10% % 0.1% 1% 10% GSE (Infusion) 0% 0.1% 1% 10%

29 Anova (Lemon Juice Liquid Pulse) SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

30 Anova (Lemon Juice Infusion) SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

31 Anova (GSE Liquid Pulse) SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total

32 Anova (GSE Infusion) SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total