1. Hydrogen Peroxide Anti-Microbial Effects
Michael Fantini
Grade 9
Central Catholic High School

2. Hydrogen Peroxide Commonly used as a household wound disinfectant
Highly damaging to tissue
Used at high concentration 3%
Has peroxide group
Powerful oxidizer
Corrosive at higher concentrations

3. Model Bacteria
◆ Escherichia coli – gram negative – rod-shaped – intestinal mammalian symbiont – widely studied – easy to maintain, high growth rate, simple nutritional requirements

4. Model Bacteria Cont.
◆ Staphylococcus epidermidis – gram positive – round shaped – form clusters – exterior human flora – easy to grow in a lab setting – can produce biofilms –form clusters

5. Purpose
Investigate the effects of hydrogen peroxide at various concentrations on E. coli and Staph
Rationale
Can hydrogen peroxide be used as a disinfectant at lower concentrations?

6. Hypotheses Null: Alternative:
Hydrogen Peroxide will not have a significant effect on the survivorship of the bacteria
Alternative:
Hydrogen peroxide will have a significant negative effect on the survivorship of the bacteria

7. Materials LB agar plates Incubator
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Sidearm flask
Spreader bars
Sterile Dilution Fluid
ethanol
Sterile pipette tips
Sterile capped test tubes with sterile distilled water
Micropipettes
Staphylococcus epidermidis
Hydrogen peroxide
Escherichia coli
SDF(100mM KH2PO, 100mM K2HPO4, 10mM MgSO4, 1m NaCl)
Vortex

8. Procedures Liquid Exposure
Both bacteria were grown until a density of 50 klett spectrophotometer density was reached. This was approximately 10^8 cells/mL
Both cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL
The stock was added to tubes containing 8.9 mL of sterile water to create concentrations of [3*10^-4], [3*10^-3], [0.03], [0.15], and [0.3]
Sterile water was added to the tubes to bring all of the quantities of the tubes up to 9.9 mL
0.1 mL of bacteria were added to their respective tubes
The tubes were allowed to stand at room temperature for 10 minutes
0.1 mL of each tube were plated onto LB agar, 6 plates per tube

9. Procedures Cont.
8. The plates were grown overnight at 37 C and resulting colonies counted 9. Stat tests were completed
Total Percent in Tubes 0%
3*10^-4%
3*10^-3%
0.03%
0.15%
0.3%
Sterile Water
9.9 ml
9.899 ml
9.89 ml
9.8 ml
9.4 ml
8.9 ml
Hydrogen Peroxide
0 ml
0.001 ml
0.01 ml
0.1 ml
0.5 ml
1 ml
Bacteria
Total Volume
10 ml

10. Hydrogen Peroxide Effects on Microbial Survivorship
LD50
~ [0.01]
~ [0.02]
P-Values
3.39E-24
9.95E-17
Explain Anova

11. Dunnett’s Test Results: Staph
Concentration
T-Value
T-Critical
Results
 3*10^-4% 15
3.39
Significant
3*10^-3% 27
0.03% 28
0.15% 29
0.3%
Explain Dunnetts

12. Dunnett’s Test Results: E. Coli
Concentration
T-Value
T-Critical
Results
 3*10^-4% 7
3.39
Significant
3*10^-3% 14
0.03% 15
0.15%
0.3%
Explain Dunnetts

13. Procedures Cont. Direct Infusion
200uL of hydrogen peroxide was added to 2 LB agar plates per bacteria
20uL of hydrogen peroxide and 180uL of water were added to 2 LB agar plates per bacteria
The LB agar plates were put in an incubator for one hour
0.1 mL of each bacteria were added to their respective infused plates
The plates were incubated at 37 C and the resulting colonies were counted

14. Direct Infusion 0 ul 20 ul 200 ul 420 378 0 ul 20 ul 200 ul 273 296
Staph Survivorship Data
E. Coli Survivorship Data
0 ul
20 ul
200 ul
420
378
0 ul
20 ul
200 ul
273
296
P-Values

15. Conclusion The null hypothesis was rejected for all concentrations
H2O2 had a significant negative effect on the survivorship of bacteria
H2O2 was observed to be very lethal even at low concentrations

16. Possible Limits and Extensions
Slight variance in spread plating synchronization
Only survivorship assessed
Only two species used
Possible non-uniform spreading of hydrogen peroxide
One exposure time
More trials and exposure times
Different microbes
Different concentrations of hydrogen peroxide
Synergistic effects of hydrogen peroxide and other variables

17. Works Cited
"E. Coli Bacteria Infection Symptoms, Causes, Treatments." WebMD. WebMD, n.d. Web. 06 Jan "E. coli (Escherichia Coli)." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 21 Dec Web. 06 Jan "Escherichia Coli." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan "Hydrogen Peroxide | Chemical Compound." Encyclopedia Britannica Online. Encyclopedia Britannica, n.d. Web. 06 Jan "Hydrogen Peroxide." Hydrogen Peroxide. N.p., n.d. Web. 06 Jan "Hydrogen Peroxide." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan "Staphylococcus Epidermidis." - MicrobeWiki. N.p., n.d. Web. 06 Jan "Staphylococcus Epidermidis." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan

18. Anova: Single Factor
Staph Liquid Pulse
SUMMARY
Groups
Count
Sum
Average
Variance 6
1397
0.0001
689
0.1
109 1 41 5 10
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
3.39E-24
Within Groups 30
Total 35

19. Anova: Single Factor
E. Coli Liquid Pulse
SUMMARY
Groups
Count
Sum
Average
Variance 6
1202
0.0001
663
110.5
359.5
0.1 87
14.5
115.5 1 29 5 10
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
9.95E-17
Within Groups 30
Total
221173 35

20. Anova: Single Factor
Staph Direct Infusion
SUMMARY
Groups
Count
Sum
Average
Variance 2
798
399
882 20
200
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
212268
106134
361
Within Groups 3
294
Total
213150 5

21. Anova: Single Factor
E. Coli Direct Infusion
SUMMARY
Groups
Count
Sum
Average
Variance 2
569
284.5
264.5 20
200
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
Within Groups 3
Total 5