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

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

3. 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

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

5. 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

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

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

8. 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.

9. 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

10. 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.

11. 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

12. 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.

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

14. 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

15. 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%

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

18. 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

19. 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.

20. References

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

22. 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