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

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

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

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

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

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

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

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

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

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.

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, 0.7-0.9 mW cm2 at working surface)

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

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.

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

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

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

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

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

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

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 2013. Web. 19 December 2016. Krotec, Mark. Personal interview. 28 October, 2016. Zeman, Gary. "Ultraviolet Radiation." Health Physics Society. Web. 14 December 16 2016. Bailey, Regina. "Cellular Respiration." Biology. Sat. 15 December q6. 2016

ANOVA (concentration of vitamin c)

ANOVA (exposure time)

Two factor ANOVA