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Hydrogen Peroxide Influence on Microbial Survivorship Jacob Cebulak Central Catholic Pittsburgh Grade 9 Problem • Humans use excess hydrogen peroxide to clean wounds. The concentration used is often damaging to normal tissues of the body. • At what concentrations does hydrogen peroxide affect the survivorship of Escherichia coli and Staphylococcus epidermidis? Introduction • Variable • Walgreens Hydrogen Peroxide 3% First Aid Antiseptic • Types of Bacteria Used • Escherichia coli • Staphylococcus epidermidis Walgreens Hydrogen Peroxide (H2O2) • First aid to help prevent infection. • For use on minor cuts, scrapes, and burns. • Aids removal of secretions in mouth. E.coli • Rod shaped cells, usually 2 nanometers in length • Prokaryotic • Gram-negative • Found in the intestines of many mammals • Commonly used model • Reproduces rapidly, usually within thirty minutes • Many strains, most non-pathogenic Staphylococcus epidermidis • Bacteria that is mostly harmless and lives normally on skin and mucous membranes of humans • Gram-positive • Many forms are considered non-pathogenic • Pathogenic forms can be lethal Gram+ vs. Gram- Bacteria Gram+ Gram- • Most pathogenic • Simple cell wall. • Antibiotics work against the formation of the cell wall. • Staphylococcus epidermidis • The cell wall is a thin layer of lipopolysaccharide, which adds extra protection. • This layer protects the cell from certain antibiotics. • Escherichia coli Recent Studies • Scientists in North Carolina explored the role of hydrogen peroxide in cell health. • Scientists in Oklahoma have studied the role of hydrogen peroxide in environmental adaptation of oral microbial communities. Rationale • The rationale of this experiment was to test the survivorship of bacteria when exposed to various concentrations of hydrogen peroxide. Hypotheses • Null Hypothesis: Hydrogen peroxide will not reduce the survivorship of E. coli and Staph. e • Alternative Hypothesis: Hydrogen Peroxide will significantly reduce the survivorship of E. coli and Staph. e. Materials • • • • • • • • • • • • • LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) LB-Agar Plates Escherichia coli Staphylococcus epidermidis Pipets Walgreens Hydrogen Peroxide 3% Antiseptic Test Tubes Vortex Spreaders Ethanol Matches Bunsen Burner 15 mL Sterile conical tubes with Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Procedure 1. 2. 3. 4. 5. 6. E. coli and Staph were grown overnight in sterile LB media. Samples of the overnight culture were added to fresh media in a sterile sidearm flask. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108-109 cells/ml. The cultures were diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml. The hydrogen peroxide was diluted with sterile dilution fluid to concentrations of 0%, .01%, .001%, .0001%, and .05% to total 9.9 ml. 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. Procedure 7. 8. 9. 10. The tubes were allowed to incubate at room temperature for 10 minutes. After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on LB agar plates. The plates incubated at 37°C overnight. The resulting colonies were counted. Each colony is assumed to have arisen from one cell. Survivorship Graph [# of Colonies Surviving] Peroxide Effects on Bacteria 900 800 700 600 500 400 300 200 100 0 P=3.2E-23 Control 0.0001 P=2.73E-20 0.001 E. coli S. epidermidis [H2O2%] 0.01 0.05 Dunnett’s Tests Escherichia coli Staphylococcus epidermidis Alpha – 0.05 Alpha – 0.05 T-Critical – 3.26 T-Critical – 3.26 0.0001% - 22.92 Significant 0.0001%- 15.33 Significant 0.001%- 26.02 Significant 0.001%- 20.89 Significant 0.01%- 36.36 Significant 0.01%- 27.58 Significant 0.05%- 38.99 Significant 0.05%- 29.03 Significant Survivorship Chart [% Survivorship] % Survivorship 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% 0.0001 0.001 0.01 E. coli Staph [H2O2 %] 0.05 Conclusion • The null hypothesis was rejected for all concentrations of H2O2. The concentrations affected the survivorship significantly. Limitations • Only tested 2 types of bacteria. • Only had 4 different concentrations. • Only 1 type of exposure used. • Plating process slightly unsynchronized. • Only survivorship was tested, not growth effects. Extensions • Test different brands of hydrogen peroxide. • Test growth effects. • Test more concentrations of hydrogen peroxide. • Possible synergistic effects. • More replicates. • More species of bacteria. References • http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con20032105 • http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis /sepidermidis.html • https://en.wikipedia.org/wiki/Gram-positive_bacteria • https://en.wikipedia.org/wiki/Gram-negative_bacteria • http://dictionary.cambridge.org/us/dictionary/english/hydrogen-peroxide • http://www.wakehealth.edu/NewsReleases/2008/Research_Explores_Role_of_Hydrogen_Peroxide_in_Cell_Health. htm • http://www.hindawi.com/journals/omcl/2012/717843/ Concentration Chart 0% HP 0.0001% HP 0.001% HP 0.01% HP 0.05% HP Bacteria 0.1mL 0.1mL 0.1mL 0.1mL 0.1mL SDF 9.9mL 9.89 mL 9.89mL 9.89mL 9.85mL 0mL 0.01mL 0.01mL 0.01mL 0.05mL 10mL 10mL 10mL 10mL 10mL (Sterile Dilution Fluid) HP (Hydrogen Peroxide) Total Volume Escherichia coli Data Control 0.0001% 0.001% 0.01% 0.05% Plate 1 832 276 224 61 0 Plate 2 896 308 327 42 0 Plate 3 824 376 242 46 0 Plate 4 792 428 307 47 0 Plate 5 784 372 276 68 0 Plate 6 856 296 283 72 0 Staphylococcus epidermidis Data Control 0.0001% 0.001% 0.01% 0.05% Plate 1 580 252 176 47 11 Plate 2 596 356 152 35 1 Plate 3 676 300 208 39 0 Plate 4 760 312 220 30 0 Plate 5 592 280 196 44 0 Plate 6 664 332 140 13 0 Escherichia coli ANOVA Anova: Single Factor SUMMARY Groups Control 0.0001% 0.001% 0.01% 0.05% Count Sum 6 6 6 6 6 4984 2056 1659 336 0 Average 830.6667 342.6667 276.5 56 0 Variance 1725.867 3415.467 1493.9 160.4 0 ANOVA Source of Variation SS df MS Between Groups 2601054 4 650263.5 Within Groups 33978.17 25 1359.127 Total 2635032 29 F 478.4422 P-value 3.2E-23 F crit 2.75871 Staphylococcus epidermidis ANOVA Anova: Single Factor SUMMARY Groups Control 0.0001% 0.001% 0.01% 0.05% Count Sum 6 6 6 6 6 3868 1832 1092 208 12 Average 644.6667 305.3333 182 34.66667 2 Variance 4796.267 1367.467 1003.2 149.8667 19.6 ANOVA Source of Variation Between Groups Within Groups Total SS df MS 1619982 4 404995.5 36682 25 1467.28 1656664 29 F 276.0178 P-value 2.73E-20 F crit 2.75871