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+ 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 1. E. coli and Staph were grown overnight in sterile LB media. 2. Samples of the overnight culture were added to fresh media in a sterile sidearm flask. 3. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8-10 9 cells/ml. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. The hydrogen peroxide was diluted with sterile dilution fluid to concentrations of 0%,.01%,.001%,.0001%, and.05% to total 9.9 ml. 6. 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 10 3 cells/ml.
Procedure 7. The tubes were allowed to incubate at room temperature for 10 minutes. 8. After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on LB agar plates. 9. The plates incubated at 37 C overnight. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
[# of Colonies Surviving] Survivorship Graph 900 800 700 600 500 400 300 200 100 0 Peroxide Effects on Bacteria P=3.2E-23 P=2.73E-20 Control 0.0001 0.001 0.01 0.05 E. coli S. epidermidis [H2O2%]
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] Survivorship Chart 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% % Survivorship 0.0001 0.001 0.01 0.05 E. coli Staph [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/
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 (Sterile Dilution Fluid) HP (Hydrogen Peroxide) Total Volume 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
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 Count Sum Average Variance Control 6 4984 830.6667 1725.867 0.0001% 6 2056 342.6667 3415.467 0.001% 6 1659 276.5 1493.9 0.01% 6 336 56 160.4 0.05% 6 0 0 0 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% 6 1832 305.3333 1367.467 0.001% 6 1092 182 1003.2 0.01% 6 208 34.66667 149.8667 0.05% 6 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