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 Types of Bacteria Used 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 Most pathogenic Simple cell wall. Antibiotics work against the formation of the cell wall. Staphylococcus epidermidis Gram- 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 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.

Escherichia coli B was grown overnight in sterile LB media. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. The culture was placed in a shaking water bath until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 or 109 cells/ml. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml. The petroleum was diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% 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. 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. Procedure

Survivorship Graph P=3.2E-23 P=2.73E-20 [# of Colonies Surviving] [H2O2%]

Dunnett’s Tests Escherichia coli Staphylococcus epidermidis Alpha – 0.05 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] [H2O2 %]

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/con- 20032105 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/News- Releases/2008/Research_Explores_Role_of_Hydrogen_Peroxide_in_Cell_Health. htm http://www.hindawi.com/journals/omcl/2012/717843/

(Sterile Dilution Fluid) Concentration Chart 0% HP 0.0001% HP 0.001% HP 0.01% HP 0.05% HP Bacteria 0.1mL SDF (Sterile Dilution Fluid) 9.9mL 9.89 mL 9.89mL 9.85mL (Hydrogen Peroxide) 0mL 0.01mL 0.05mL Total Volume 10mL

Escherichia coli Data Control 0.0001% 0.001% 0.01% 0.05% Plate 1 832 276 224 61 Plate 2 896 308 327 42 Plate 3 824 376 242 46 Plate 4 792 428 307 47 Plate 5 784 372 68 Plate 6 856 296 283 72

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 Plate 4 760 312 220 30 Plate 5 592 280 196 44 Plate 6 664 332 140 13

Escherichia coli ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 6 4984 830.6667 1725.867 0.0001% 2056 342.6667 3415.467 0.001% 1659 276.5 1493.9 0.01% 336 56 160.4 0.05% ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2601054 4 650263.5 478.4422 3.2E-23 2.75871 Within Groups 33978.17 25 1359.127 Total 2635032 29  

Staphylococcus epidermidis ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 6 3868 644.6667 4796.267 0.0001% 1832 305.3333 1367.467 0.001% 1092 182 1003.2 0.01% 208 34.66667 149.8667 0.05% 12 2 19.6 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1619982 4 404995.5 276.0178 2.73E-20 2.75871 Within Groups 36682 25 1467.28 Total 1656664 29