Chlorine and UV Effects on Bacteria Survivorship Oscar Shaver Grade 9 Central Catholic High School
Electromagnetic Spectrum Diagram showing the range of all types of frequencies of electromagnetic radiation The types of radiation are radio waves, microwaves, infrared, visible, ultraviolet, x-rays, and gamma rays
UV Light Rays Ultraviolet (UV) rays are light rays that have shorter wavelengths than visible light Range from 150nm 300nm They are naturally given off by the sun, but most are absorbed by the ozone layer The wavelength used in this experiment is 254nm light
Effects of UV Light Heavy exposure to UV light without protection can lead to skin cancer and photokeratitis Thought to interfere with many important biological molecules including DNA Used for sterilization
Chlorine common household cleaning agent contains active ingredients Ø Sodium Hypochlorite Ø Sodium Chlorate Ø Sodium Hydroxide Ø Sodium Polyacrylate Ø Sodium Carbonate
Sodium Hypochlorite NaClO Sodium cation and hypochlorite anion Soluble in water
Staphylococcus epidermidis Gram positive bacteria Part of normal human flora Found on skin Not pathogenic Coccal bacteria When incubated, forms white colonies
Purpose To determine the individual and synergistic effects of chlorine and UV light on staph survivorship
Null: Hypotheses 1. Chlorine and UV light individually will have not reduce staph survivorship 2. Chlorine and UV light will not have synergistic effects at reducing staph survivorship Alternate: 1. Increased chlorine concentration and UV light exposure individually will reduce staph survivorship 2. Chlorine and UV light will synergistically reduce staph survivorship
Materials LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Incubator Ethanol Spreader bars Staphylococcus epidermidis Clorox bleach UV lamp (254 nm light) Test tubes Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Sterile pipette tips Micropipettes Sterile capped test tubes with sterile distilled water Sidearm flask
Procedures 1. Bacteria (Staph) was grown overnight in sterile LB Media 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask 3. The culture was 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 culture was diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/ml. 5. Various concentrations (0%, 3x10^-4%, and 3x10^-5%) of chlorine bleach were created in test tubes containing SDF, resulting in 9.9ml per tube
Concentration Chart Final % in Tubes 3x10^-5% 3x10^-4% 0% Sterile Water 9.8 ml 9.8 ml 9.9 ml Bacteria 0.1 ml 0.1ml 0.1 ml Chlorine Bleach 0.1ml (3x10^-5% stock) 0.1 ml (3x10^-4% stock) 0 ml Total Volume 10 ml 10 ml 10 ml
Procedures(continued) 6. 0.1 ml of cell culture was then added to the test tubes, yielding a final volume of 10 ml. 7. The solutions were mixed by vortexing and allowed to sit at room temperature for 10 minutes. 8. After vortexing to evenly suspend cells, 0.1 ml was removed from the tubes and spread on LB plates. 9. Plates were then placed under a UV lamp for certain times (0,4, and 8 seconds) 10. The plates were incubated at 37 C for 24 hours 11. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
Concentrations and Radiations 0 4 sec (L) 8 sec (H) 0 0 0 L 0 H 0 3x10^-4 (L) 3x10^-5 (H) L 0 L L L H H 0 H L H H
Chlorine Effects on Staph P-Value: 0.00031
Dunnett's Test: Chlorine Group t-value t-critical Result 3x10^-5% 4.88 3.03 Significant 3x10^-4% 5.04 3.03 Significant
UV Effects on Staph P-value: 2.18e-08
Dunnett's Test: UV Group t-value t-critical Result 4 seconds 12.45 3.03 Significant 8 seconds 13 3.03 Significant
Synergistic Effects of Chlorine and UV on Staph P-values: Sample-2.15e-24 Columns- 1.55e-07 Interaction- 1.15e-06
Conclusions The null hypotheses can be rejected for every group Chlorine and UV had significant negative effects individually and synergistically on the survivorship of staph Both variables proved to be lethal even at very low concentrations
Limitations Spread plating was not performed in perfect synchronization, therefore some bacteria had a slightly higher exposure time Only one species used Only survivorship assessed Plating was done in room with partial sunlight Extensions Other forms of chlorine will be tested and their results will be compared The active ingredients in the different forms of chlorine will be compared A graph will be made to illustrate which forms of chlorine disinfect most effectively More species of bacteria will be used Spread plating will be performed more synchronously UV exposure time will be at smaller increments
Works Cited Chlorine Residual Testing." SpringerReference (2011): n. pag. Cdc.gov. Web.27. Dec.2015 Electrolytic Cells and Electrolysis." Chem1 Electrochemistry: Electrolysis. N.p., n.d. Web. 27 Dec. 2015. Lucas, Jim. "What Is Ultraviolet Light?" LiveScience. TechMedia Network, 31. Mar.2015. Web. 27 Dec. 2015 "Staphylococcus Epidermidis." - Wikipedia, the Free Encyclopedia. N.p., n.d. Web. 27 Dec. 2015. "The Element Chlorine." It's Elemental -. N.p., n.d. Web. 27 Dec. 2015. "What Is Germicidal Ultraviolet? Ultraviolet.com." Ultraviolet.com. N.p., n.d. Web. 27 Dec. 2015.
ANOVA: Chlorine
ANOVA: UV
Two Factor ANOVA: UV and Chlorine