1. Vitamin C Effects on Stressed Microbes Michael Limbacher Central Catholic High School February 4, 2012

2. Heat Stress on Cells  Heat stress effects the cell in many ways.  Respiration  Reproduction  Life Span  Is there a way to increase the survivorship of cells after they have been damaged by heat stress?  Vitamin C Effects is being tested.

3. Ways Cells Respond to Stress  In sudden emergencies, cells create stress proteins that are programed to repair damage that the stress induces.  Stress proteins often restore damaged proteins to normal shape, thus restoring function.  These proteins also are programed to cope against infection, autoimmune disease, and in some cases cancer.

4. Pure Vitamin C  One of the safest and most effective nutrients  Available in production in liquid and solid form  Benefits for Humans  Prevents immune system deficiencies  Prevents eye disease and skin wrinkling  Protects against cardiovascular disease  Helps prevent free radicals

5. Orange  First cultivated in China at c. 2500 B.C.  Is best grown in tropical to semitropical places  70 mg of Vitamin C per orange  Nutritional benefits for humans  Resistance against infectious agents  Helps maintain healthy skin and mucus membrane  Also possibly prevents the body from lung and oral cavity cancers

6. Escherichia coli (E. coli)  Gram-negative bacteria  Commonly found in the lower intestine of warm-blooded animals  Most E. coli strains are harmless to humans; however, some are very harmful.  Commonly used in biological experiments  Easy manipulation  Easy adaption to cultivation in a laboratory environment

7. Staphylococcus epidermidis (Staph e.)  Gram-positive bacteria  Typically harmless skin-dwelling microorganisms  Commonly used in microbiological experiments, due to variety of strain types within species  Each strain has different features and toxins

8. Purpose  Determine if 1% and 10% of Vitamin C and Orange Extract can increase the survivorship of stressed and unstressed microbes.

9. Hypotheses  Null: Addition of Pure Vitamin C and Orange Extract will not have an effect on the survivorship of stressed or unstressed microbes, E. coli and Staph e.  Alternate: Addition of Vitamin C and Orange Extract will increase survivorship of heat stressed or unstressed microbes, E. coli and Staph e.

10. Materials  Sterile test tubes  Test tube rack  Micropipettes  Pipette tips  Klett Spectrophotometer and sidearm flasks  Escherichia coli  Staphylococcus epidermidis  LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Sterile Dilution Fluid [SDF] (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  California Naval Orange Extract  Pure Liquid Vitamin C (Ingredients: Vitamin C (300 mg) Purified Water, Sucrose, Glycerin, Methylparaben, Propylparaben  Filter Paper  55°C Water  Spreader Bar  Vortex  Beaker  Thermometer  Incubator

11. Procedures (1) 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 sterile sidearm flasks. 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 10 8 cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

12. Procedures (2) 5. Vitamin C/Orange Extract, SDF, and Microbes were added to sterile tubes to create the desired experimental concentrations.

13. Procedures (3) 6. The solutions were vortexed and allowed to sit at room temperature (Unstressed) or bathed at 55ºCelsius (Stressed) for 15 minutes. 7. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates. 8. Plates (144 total) were incubated at room temperature for 48 hours. 9. Colonies were counted. Each colony was assumed to have arisen from one cell.

14. Supplement Effects on Unstressed E coli Orange P Value=.405278Vitamin C P Value=.364967

15. Supplement Effects on Unstressed Staph e Orange P Value=.000127Vitamin C P Value=.342471 Staph e. T-crit= 2.86 Concentration T Value Interpretation Orange 1%5.594Significant Orange 10% 4.376Significant T>T crit

16. Supplement Effects on Stressed E coli

17. Supplement Effects on Stressed Staph e Orange P Value= 2.16 E-05 Vitamin C P Value= 7.27 E-11

18. Dunnett’s Stat Test E coli T crit= 2.86Staph e T crit= 2.86 ConcentrationT ValueInterpretationConcentrationT ValueInterperetation Orange 1%4.119SignificantOrange 1%2.11Not Significant Orange 10%18.697SignificantOrange 10%6.763Significant Vitamin C 1%3.472SignificantVitamin C 1%3.849Significant Vitamin C 10%19.563SignificantVitamin C 10%17.108Significant STRESSED

19. Conclusions  Vitamin C and Orange Extract did not have a significant effect on survivorship of E. coli in unstressed conditions. (Accept Null)  Orange Extract appeared to adversely effect Staph e. survivorship in unstressed conditions. (Reject Null) Vitamin C, however, did not have a significant effect. (Accept Null)  Vitamin C and Orange Extract appeared to significantly increase the survivorship of stressed E. coli and Staph e. (Reject Null) (Insufficient evidence for stressed protection for 1% Orange Extract in Staph e.)

20. Limitations/Extensions  Limitations  Lag time in plating  Only 3 concentrations were used.  Only one temperature for stress was used.  Extensions  Increase Sample Size  Use different types of antioxidants (Lycopene, Vitamin A, Vitamin E)  Expose to a different type stress (UV)  Increase exposure time to the Vitamin C

21. References  “Lysogeny Broth” Protocols Online. Web. 24 Dec 2011..  “What Is E. Coli? (Escherichia Coli).” Medical News Today. Web. 24 Dec 2011.  “Staph Infection (Staphylococcus aureus) MedicineNet. Web. 24 Dec 2011.  “Orange Fruit Nutrition Facts.” Power Your Diet. Web. 24 Dec 2011  “The Benefits of Vitamin C.” WebMD. Web. 24 Dec 2011. http://www.webmd.com/diet/guide/the-benefits-of-vitamin-c

22. COLONY COUNT (1)

23. COLONY COUNT (2)

24. ANOVA Sample