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 Types of Bacteria Used
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
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 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 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.

13. 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

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

15. Dunnett’s Tests Escherichia coli Staphylococcus epidermidis
Alpha – 0.05
T-Critical – 3.26
0.0001% Significant
0.0001% Significant
0.001% Significant
0.001% Significant
0.01% Significant
0.01% Significant
0.05% Significant
0.05% Significant

16. Survivorship Chart
[% Survivorship]
[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. (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

22. 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

23. 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

24. Escherichia coli ANOVA
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 6
4984
0.0001%
2056
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 4
3.2E-23
Within Groups 25
Total 29

25. Staphylococcus epidermidis ANOVA
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 6
3868
0.0001%
1832
0.001%
1092
182
1003.2
0.01%
208
0.05% 12 2
19.6
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 4
2.73E-20
Within Groups
36682 25
Total 29