Effects of Lemon Juice On E. Coli Survivorship Chris Seekings Pittsburgh Central Catholic Grade 9 1st Year participating in pjas

Problem How do lemon juice concentrate and naturally squeezed lemons effect the survivorship of E. coli?

Lemon A sour yellow fruit native to a tree in Asia No specific formula, it is a liquid mixture not a compound. Has a pH level of below 7, making it acidic

Giant Eagle Brand Lemon Juice Concentrate Contains two different preservatives, sodium bisulfite and sodium benzoate and lemon oil along with filtered water.

Microbial Flora •Not much is known about the association between humans and their flora •Mutualistic, parasitic, pathogenic, and commensal •Normal Flora provide nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes

Escherichia Coli (E. Coli) Group of gram (-) bacteria Live in intestinal tract of many mammals Serve as a common prokaryotic cell model

Purpose To test the effects of two types of lemon juice, Giant Eagle Brand Concentrate and freshly squeezed, on E. coli survivorship.

Hypotheses •Null Hypothesis: There will be no effect of lemon juice concentrate and squeezed lemons in the survivorship of E.coli. •Alternative Hypothesis: Both lemon juice concentrate and freshly squeezed lemons will significantly reduce the survivorship of the bacteria.

Materials •Spreader bars •YEPD Agar Plates (1% Yeast Extract, 2% Peptone, 2% Glucose) •Escherichia coli C600 •Burner Filter Paper •Sidearm Flask •Turn-table •Vortex •Incubator •Cone/Beaker •Lemon, Giant Eagle Brand Lemon Juice Concentrate •SDF (Sterile Dilution Fluid)- 100mm, KH2Po4, 10mm,MgSO4, 1mm NaCl •20 Sterile Test Tubes •Ethanol

Procedures (Liquid Exposure) 1. Bacteria (E.coli) were 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°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL. 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^8 cells/mL. 5.The lemons were then juiced and sterilized along with the lemon juice concentrate. 6. The following ingredients were combined to create the desired concentrations.

Procedures (Concentration Chart) 0% 0.10% 1% 10% Sterile Fluid 9.9mL 9.89mL 9.8mL 8.9mL Microbe 0.1mL Lemon Juice (Freshly Squeezed, Concentrate) 0mL 0.01mL 1mL Total Volume 10mL

Procedure (Continued) 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.

P-Value- 2.38E-28 + 2.13E-24 For every concentration, the pH level was between 6-7

Lemon Juice Concentrate Dunnett's Test Results Squeezed Lemon Juice Concentration T-Value T-Crit Results 0.10% 2.9 3 Not Significant 1% 9.6875 Significant 10% 34.285 Lemon Juice Concentrate Concentration T-Value T-Crit Results 0.1% 3.195 2.88 Significant 1% 36.171 10% 42.457

LD50 of Lemon Juice Concentrate is at a concentration of approximately 0.65% LD50 of Squeezed Lemon Juice is at a concentration of Approximately 4%

Limitations •Plating could have been unsynchronized •Only 3 concentrations of variable tested •Only one exposure time utilized Only liquid pulse exposure used

Extensions Test more than one exposure time Test other concentrations of the variable Do a larger number of replicates for more accurate results Use agar infusion for a prolonged exposure

Conclusion The null hypothesis was rejected for both lemon juice concentrate and squeezed lemon juice The alternative hypothesis was accepted due to both lemon juice concentrate and squeezed lemon juice having a negative effect on E. coli. The lemon juice concentrate had a greater negative effect on the E. coli compared to the squeezed lemon juice.

References http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGramNeg.htm http://textbookofbacteriology.net/normalflora.html https://www.reference.com/science/chemical-formula-lemon-juice-228bec6157fd4490

A-Nova Test Results Freshly Squeezed Lemon Juice Results Lemon Juice Concentrate Results Freshly Squeezed Lemon Juice Results

Freshly Squeezed Lemon Juice (Number of Colonies Per Plate) 0% 121 107 103 101 105 97 102 0.1% 96 93 99 98 86 1% 79 72 76 81 66 83 84 10% 7 9 11 8 14 6 P-Value- 2.13E-24 Lemon Juice Concentrate (Number of Colonies Per Plate) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 0% 121 107 103 101 105 97 102 0.1% 100 86 99 98 95 1% 16 17 19 23 13 15 10% 4 1 7 P-Value- 2.38E-28