1. Ascorbic Acid Remediation of UV Stressed E. Coli
Adam Sipe
Central Catholic High School
9th Grade

2. Ultraviolet Light
Electromagnetic radiation with a wavelength ranging from 100nm to 400nm
Effects
sunburn
lead to skin cancer
harm eyes
damage DNA
Sources
the sun
tanning beds
fluorescent lamps

3. Stress
Stress: anything that disrupts regular cellular processes and reduces survivorship.
Mechanical
Chemical
Oxidative
Ischemic
Heat
Photo

4. Stressed Cells UV light Interferes with basic cellular functions
Oxidative stress
Free radicals
Thymine dimers
Apoptosis

5. Response to stress In emergencies cells produce stress proteins to:
Repair damage
Cope with infection, autoimmune disease, and cancer
Activation of survival pathways
Programmed cell death

6. Antioxidants Prevent oxidation Prevent cancer, coronary heart disease
Body produces antioxidants
Can be obtained through diet

7. Vitamin C Ascorbic acid Enzyme cofactor Liquid and solid form
Benefits for humans

Effects immune deficiencies

Effects eye disease, skin wrinkling
Protects against cardiovascular disease
Neutralizes free radicals
Adults require 75 to 125 mg per day

8. Model Bacteria (E. coli)
Extremely common bacteria
Frequently used for scientific research
Rod-shaped microflora
Hundreds of species
Measure of water quality
Some strains can be extremely harmful

9. Purpose
To determine the effect of ascorbic acid on the remediation of UV stressed E. coli
To determine the effect of ascorbic acid on the survivorship of E.coli

10. Hypothesis Null Hypothesis
Ascorbic acid will have no significant effect on the survivorship of UV stressed E. coli at any UV exposure time.
Alternate Hypothesis
Ascorbic acid will have a significant impact on UV stressed E. coli survivorship.

11. Materials
E. coli
YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose)
LB media (0.5% yeast extract, 1% tryptone, 1% odium chloride)
Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO4, 10 mMMgSO4, 1mM NaCl)
Ascorbic acid
Sterile pipette tips
Micropipettes
Vortex
Incubator (37 degrees C)
Sterile spreader bars
Ethanol
Sterile capped test tubes with sterile distilled water
Lab Conoco UVC hood (254nm UVC, mW cm2 at working surface)

12. Procedures 1) A culture of E. coli was grown overnight
2) The culture of cells was diluted to a concentration of 10^5 cells/ml
3) Tubes were prepared according to the chart below, vortexed, and incubated for five minutes at room temperature
0% concentration
0.1% concentration
1% concentration
Sterile Fluid
9.9ml
9.7ml
7.9ml
Ascorbic acid stock (5%)
0ml
0.2ml
2ml
Diluted E. coli culture
0.1ml
Total
10ml

13. Procedures
4) 0.1ml aliquots of the samples from each tube and plated onto YEPD agar plates. 5 replicates of each of 5 exposure times were performed of each tube.
5) The plates were placed under a UVC hood and exposed for certain times (2, 4, 8, and 10 seconds).
6) The plates were incubated for 24 hours at 37 degrees celsius.
7) The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

14. Variation between concentrations of vitamin c
P= 4.62E-1 3
Variation between UV Exposure Times
P= 2.21E-07
Interaction Variation
P= 2E-06

15. Variation between concentrations of vitamin c
P= 4.62E-1 3
Variation between UV exposure Times
P= 2.21E-07
Variable interaction
P= 2E-06

16. Dunnett's Test Analysis
0.1% Concentration
1% Concentration
Zero Seconds Exposure
T =~ 5.199
Significant effect
T =~11.9
Variation Between Bacteria Counts of Each Variable Concentration
T-crit= 3.03
2 Seconds
4 Seconds
8 Seconds
10 Seconds
0% Concentration
T=~12.1
Significant Effect
T=~15.356
T=~15.959
T =~18.01
Variation Between Bacteria Counts of UV Exposure Times
T- crit = 3.48

17. Conclusions The null hypothesis was rejected
The alternate hypothesis was supported
E. coli showed a greater resistance to UV when exposed to higher concentrations of vitamin c
Survivorship was lower when not exposed to vitamin c

18. Possible Limitations Spread plating was not perfectly synchronized
Only E. coli was tested
Only UVC radiation was tested
Only one antioxidant was tested
Only two concentrations of vitamin c were tested

19. Extensions
Efforts will be made to achieve more synchronous spread plating
More species will be tested
More wavelengths of UV radiation will be tested
Other antioxidants will be tested
More concentrations of vitamin c will be tested

20. Resources
"How to Count Bacteria.” disknet. n.p., n.d. Web. 20 January 2017.

“Culturing E. Coli.” newton.dep. US Department of Energy Office of Science, n.d. Web. 15 December 2016.
̈E. coli (Eschericia coli). ̈ cdc.gov. US Center for Disease Control and Prevention, 29 December Web. 19 December 2016.
Krotec, Mark. Personal interview. 28 October, 2016.
Zeman, Gary. "Ultraviolet Radiation." Health Physics Society. Web. 14 December
Bailey, Regina. "Cellular Respiration." Biology. Sat. 15 December q

21. ANOVA (concentration of vitamin c)

22. ANOVA (exposure time)

23. Two factor ANOVA