Hydrogen Peroxide Influence on Microbial Survivorship. Jacob Cebulak Central Catholic Pittsburgh Grade 9

Transcription

1 Hydrogen Peroxide Influence on Microbial Survivorship Jacob Cebulak Central Catholic Pittsburgh Grade 9

2 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?

3 Introduction Variable Walgreens Hydrogen Peroxide 3% First Aid Antiseptic Types of Bacteria Used Escherichia coli Staphylococcus epidermidis

4 Walgreens Hydrogen Peroxide (H2O2) First aid to help prevent infection. For use on minor cuts, scrapes, and burns. Aids removal of secretions in mouth.

5 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

6 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

7 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

8 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.

9 Rationale The rationale of this experiment was to test the survivorship of bacteria when exposed to various concentrations of hydrogen peroxide.

10 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.

11 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)

12 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 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 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.

13 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.

14 [# of Colonies Surviving] Survivorship Graph Peroxide Effects on Bacteria P=3.2E-23 P=2.73E-20 Control E. coli S. epidermidis [H2O2%]

15 Dunnett s Tests Escherichia coli Staphylococcus epidermidis Alpha 0.05 Alpha 0.05 T-Critical 3.26 T-Critical % Significant % Significant 0.001% Significant 0.001% Significant 0.01% Significant 0.01% Significant 0.05% Significant 0.05% Significant

16 [% Survivorship] Survivorship Chart 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% % Survivorship E. coli Staph [H2O2 %]

17 Conclusion The null hypothesis was rejected for all concentrations of H2O2. The concentrations affected the survivorship significantly.

18 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.

19 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.

20 References /sepidermidis.html Releases/2008/Research_Explores_Role_of_Hydrogen_Peroxide_in_Cell_Health. htm

21 Concentration Chart 0% HP % 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

22 Escherichia coli Data Control % 0.001% 0.01% 0.05% Plate Plate Plate Plate Plate Plate

23 Staphylococcus epidermidis Data Control % 0.001% 0.01% 0.05% Plate Plate Plate Plate Plate Plate

24 Escherichia coli ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control % % % % ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

25 Staphylococcus epidermidis ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control % % % % ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total