Download CCHS Jacob Cebulak Hydrogen Peroxide Influence on Microbial

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
•
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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