1. Antimicrobial Effects of Orange/Mango Juice
Jack O'Toole
Grade 9
Pittsburgh Central Catholic High School

2. Problem
What are the effects of orange/mango juice on two different types of bacteria?

3. Rationale
Almost all consumables are tested for their effects on human cells.
The micro flora, however, should also be considered.
Do consumables such as orange/mango juice cause a disruption in the flora of the body?

4. Micro Flora
Present in the respiratory, urinary, and digestive tracts as well as the skin.
Consists of mostly bacteria, few protists and fungi.
Most are harmless, but some can cause diseases.
Influences human anatomy, physiology, and overall health.
Over 1000 different types of bacteria are harbored within and on humans.
Ingested substances may have an effect on the population size of the flora

5. Gram(-) vs. Gram(+) Bacteria
Cell wall contains an extra
layer of lipopolysaccharides for
extra protection.
Outer membrane protects
bacteria from several antibiotics.
Most pathogenic bacteria in humans are Gram (+) organisms.
Simple cell wall.
Some antibiotics work against the formation of the cell wall.

6. Escherichia Coli (E.coli)
Large and diverse group of gram (-) bacteria.
Free living, symbionts, or pathogens.
Live in the intestinal tract of many mammals.
Most strains are not pathogenic.
Serve as a common prokaryotic cell model.

7. Staphylococcus Epidermidis (Staph)
Common surface symbiont in many mammals (Human).
Gram (+) bacteria
Most strains considered non-pathogenic.
Pathogenic strains can be life-threatening.

8. Health Benefits of Orange Juice
one of the healthiest beverage
boost immune system function
reduce signs of aging
protect against cancer
boost cellular repair and metabolism
detoxify the body
improve circulation
improves blood pressure
reduces inflammation
lowers cholesterol levels

9. Health Benefits of Mango Juice
very low in saturated fat, cholesterol and sodium
excellent source of dietary fiber, vitamin B6, vitamin A and vitamin C.
rich in minerals like potassium, magnesium and copper
one of the best sources of quercetin, betacarotene, and astragalin. These powerful antioxidants neutralize free radicals which cause Ailments like heart disease, premature aging, cancer and degenerative diseases throughout the body

10. Previous Studies
E.coli and Staph are common cell models used in many Biomedical investigations.
Other fruit juices have been tested, revealing interesting anti-microbial properties.

11. Purpose
To assess the effects of Orange/Mango Juice on the survivorship of Staphylococcus epidermidis and Escherichia coli .

12. Hypotheses
Null Hypothesis: Orange/Mango Juice will have no significant effect on the survivorship of E.coli and Staph.
Alternative Hypothesis: Orange/Mango will have significantly reduce the survivorship of E.coli and Staph.

13. Materials Incubator Gloves/goggles Klett Spectrophotometer
Micro and Macro pipettes + Sterile tips
Spreader bars
Escherichia coli
Staphylococcus epidermidis (Staph)
Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mMMgSO4, 1mM NaCl)
Bunsen Burner
Turn-table
Vortex
Incubator
Gloves/goggles
Klett Spectrophotometer
20 Sterile Test Tubes
Ethanol
Luria Broth Agar Plates (0.5% yeast extract, 1% tryptone, 1% NaCl)
Simply Orange with Mango

14. Procedure (Liquid Pulse)
1. Bacteria (E.coli and Staph) 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 10⁵ cells/mL. 5. The Orange/Mango Juice was sterile filtered 6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 1%, 5% and 10%.

15. Chart of Liquid Concentration0% 1% 5%
10%
Microbe
0.1mL
SDF
9.9mL
9.8mL
9.4mL
8.9mL
Orange/Mango Juice
0mL
0.5mL
1mL
Total
10mL

16. Procedure (Liquid Pulse)
µL of cell culture was then added to the Orange/Mango juice solutions, yielding a final volume of 10 mL and a cell density of approximately 10^3 cells/mL. 8. The solutions were vortexed. 9. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB- agar plates. 10. The plates were incubated at 37 C for 24 hours. 11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

17. Procedure (Agar Infusion)
Plates were infused as follows:
2. The plates were incubated for 1 hour (37°C) to promote infusion.
3. 100µL from the control tube (0%) was spread onto the plates.
4. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Concentration
SDF
Variable
Low
180µL
20µL
High
0µL
200µL

18. Orange/Mango Juice Effects on E.Coli Survivorship (Liquid Pulse)
P-value < 0.1^-6

19. Orange/Mango Juice Effects on E.Coli Survivorship (Agar Infusion)
P-value < 0.1^-6

20. Orange/Mango Juice Effects on Staph Survivorship (Liquid Pulse)
P-value < 0.1^-6

21. Orange/Mango Juice Effects on Staph Survivorship (Agar Infusion)
P-value < 0.1^-6

22. Anova Abbreviation for analysis of variance
Statistical test comparing variation within and between experimental groups
If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence)

23. Dunnett's Test Follow up test to the Anova
Localizes source of variation compared to the control.
T Value is greater than your T Critical value than the difference is significant.

24. Dunnett's Test Staph (Liquid Pulse)
Concentration Groups
T-Value
T-Crit Value
Results 1%
5.634
Significant 5%
18.008
10%
38.361

25. Dunnett's Test Staph (Agar Infusion)
Concentration Groups
T-Value
T-Crit Value
Results
Low
68.748
4.9646
Significant
High
83.334

26. Dunnett's Test E.Coli (Liquid Pulse)
Concentration Groups
T-Value
T-Crit Value
Results 1%
5.129
Significant 5%
12.46
10%
21.002

27. Dunnett's Test E.Coli (Agar Infusion)
Concentration Groups
T-Value
T-Crit Value
Results
Low
2.112
4.9646
Insignificant
High
15.682
Significant

28. Conclusion
Reject the null hypothesis that there will be no significant effect on the survivorship of Staph and E. coli.
Each tested concentration of Orange/Mango significantly reduced the survivorship of both E. Coli and Staph except for the Low concentration of the E.coli agar infusion.

29. Limitations Only two Bacteria tested. Only one exposure time.
Only one brand of orange/mango juice tested
Study doesn't account for other factors affecting the bacteria

30. Extensions Use other brands Use other bacteria
Use multiple exposure times
Do more trials per group
More control over human interference

31. Works Cited
benefits/beverage/orange-juice.html
benefits/fruit/mango.html

32. E.Coli Anova (Liquid Pulse)
P-value < 0.1^-6

33. E.Coli Anova (Agar Infusion)
P-value < 0.1^-6

34. Staph Anova (Liquid Pulse)
P-value < 0.1^-6

35. Staph Anova (Agar Infusion)
P-value < 0.1^-6