1. Carbohydrate Influence on UV Stressed Yeast
Jason Beiriger
CCHS, Grade 10
2nd Year in PJAS

2. Ultraviolet Rays
Light waves that have shorter wavelengths, thus greater energy, than visible light
They range from 400nm to 10nm
Given off from the sun but most are absorbed by the ozone layer

3. Damage due to UV light
Damage includes skin burn, sun poisoning, skin irritation, redness, photo-aging, nausea, and possibly skin cancer
FDA Protection methods include sun screen, hats, and radiation-blocking clothing
Can cause DNA to form dimers, leading to replication errors, mutations

4. Oxidative stress UV light can also result in oxidation stress
Increases oxidant production in cells
Free Radical accumulation can lead to compounding stress
Results in cellular degeneration
May cause direct cell death or induce cancer
High glucose levels induce an increase in enzymatic activity leading to the synthesis of amino acids

5. Antioxidants
Antioxidant- a molecule capable of preventing the oxidation of other molecules
Oxidation- a chemical reaction that transfers electrons from a substance to an oxidizing agent
Oxidation reactions can produce free radicals, which can damage cells

6. Two structures of D-glucose
A monosaccharide
Important carbohydrate
Source of energy to many cells
Stimulates the production of enzymes which assemble amino acids
Two structures of D-glucose
Could supplementing the cell with carbohydrates influence their stress protein response?

7. Yeast Easy to grow and culture Unicellular
Most studied cell in the world
Saccharomyces cerevisiae
Similar cell cycle, biochemistry and genetics to other eukaryotic cells, like those in humans

8. Objective/Purpose
To determine if carbohydrate supplementation will be effective in protecting Saccharomyces cerevisiae from UV light stress

9. Null Hypothesis Alternate Hypothesis
Glucose supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae
Glucose supplementation will significantly aid the survival of UV stressed Saccharomyces cerevisiae

10. Materials
60 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar)
Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1mM CaCl2, 100mM NaCl)
Klett spectrophotometer
Sterile pipette tips and Micropipettors
Vortex
Sidearm flask
Spreader bar
Ethanol
Micro burner
Saccharomyces cerevisiae (yeast)
UV Hood
Rubber Gloves
Test tubes
Test Tube Rack
SDF Test Tubes
Microtubes
Glucose
Incubator
YEPD media

11. Procedure
Saccharomyces cerevisiae was grown overnight in sterile dilution YEPD media.
A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
The culture was incubated at 30 degrees Celsius until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL.
The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
The glucose was diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL. For example:
1 mL of 20% glucose solution mL of SDF = final concentration of almost 2% glucose. (the addition of 0.1 mL of cell culture will result in a total of 10 mL and a 2% concentration)

12. Chart of Test Tube Components
0% glucose
0.1% glucose
2% glucose
Microbe
0.1 mL
SDF
9.9 mL
9.4 mL
8.9 mL
Glucose stock (2g/10mL)
0 mL
0.5 mL
1 mL
Total
10 mL

13. Procedure 1. Liquid exposure
mL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL mL of the solution was transferred into each of 6 microtubes. The microtubes were exposed to UV radiation in a culture hood for the following time periods: 0, 40, 100 seconds. 8. After UV exposure, the yeast was suspended using a pipette mL aliquots were removed from the tubes and spread onto YEPD agar plates. 10. The plates were incubated at 30 degrees Celsius for 48 hours. 11. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

14. Procedure 2. Plated Exposure
0.1 mL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
The yeast was suspended using a pipette.
0.1 mL aliquots were removed from the tubes and spread onto YEPD agar plates.
The plates were exposed to UV radiation in a culture hood for the following time periods: 0, 40, 100 seconds.
The plates were incubated at 30 degrees Celsius for 48 hours.
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

15. Sample ANOVA used in experiment
Abbreviation for analysis of variance
Statistical test comparing variation within and between experimental groups
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
0% no UV 6
464
0% UV
189
31.5
91.5
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 1
2.4E-05
Within Groups 10
Total 11
If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence)
Sample ANOVA used in experiment

16. Glucose Remediation Effects on Cells in Liquid Exposure
1.307E-03
Number of colonies
2.4E-05
Concentration of Glucose

17. Glucose Remediation Effects on Plated Cells
0.0949
7.6E-08 Significant interaction
4.45E-02
Number of colonies
Concentration of Glucose

18. Key Questions Liquid Exposure Plated Exposure
Did UV hurt the cells? 2.4E-05 Significant
Did sugar hurt the cells? Insignificant
Did 5% sugar hurt the cells? Insignificant
Did 10% sugar hurt the cells? Insignificant
Did sugar help remediate UV damage? 6.02E-07 Significant interaction
Plated Exposure
Did UV hurt the cells? 6.17E-06 Significant
Did sugar hurt the cells? Insignificant
Did 5% sugar hurt the cells? 4.45E-03 Significant
Did 10% sugar hurt the cells? Insignificant
Did sugar help remediate UV damage? 7.6E-08 Significant interaction

19. Conclusion Null Hypothesis Hypothesis
Carbohydrate supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae
REJECTED by analysis
Carbohydrate supplementation will significantly aid the survival of UV stressed Saccharomyces cerevisiae
SUPPORTED by analysis

20. Limitations Further Testing
Due to slight differences in positioning in the UV hood, the cultures may have received slight differences in the amount of exposure to the ultra-violet rays
Synchronizing the exact times of plating
More replicates
Utilize various wavelengths of UV light
Different concentrations of glucose
Different carbohydrate

21. Sources www.FDA.com http://www.ncbi.nlm.nih.gov/pubmed/8097593
Fraser-Reid, Bert, "van't Hoff's Glucose", Chem. Eng. News 77 (39): 8
Fred W. Schenck “Glucose and Glucose-Containing Syrups” in Ullmann's Encyclopedia of Industrial Chemistry 2006, Wiley-VCH, Weinheim. doi: / a12_457.pub2
Solubility of D-glucose in non-aqueous solvents,