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

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

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

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

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?

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

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

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

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

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

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

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

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

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 0.000266 0.000121

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

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

Works Cited "E. Coli Bacteria Infection Symptoms, Causes, Treatments." WebMD. WebMD, n.d. Web. 06 Jan. 2016. "E. coli (Escherichia Coli)." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 21 Dec. 2015. Web. 06 Jan. 2016. "Escherichia Coli." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016. "Hydrogen Peroxide | Chemical Compound." Encyclopedia Britannica Online. Encyclopedia Britannica, n.d. Web. 06 Jan. 2016. "Hydrogen Peroxide." Hydrogen Peroxide. N.p., n.d. Web. 06 Jan. 2016. "Hydrogen Peroxide." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016. "Staphylococcus Epidermidis." - MicrobeWiki. N.p., n.d. Web. 06 Jan. 2016. "Staphylococcus Epidermidis." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016.

Anova: Single Factor Staph Liquid Pulse SUMMARY Groups Count Sum Average Variance 6 1397 232.8333 442.1667 0.0001 689 114.8333 374.9667 0.1 109 18.16667 225.3667 1 41 6.833333 109.3667 5 10 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 267768.2 53553.64 278.9575 3.39E-24 2.533555 Within Groups 5759.333 30 191.9778 Total 273527.6 35  

Anova: Single Factor E. Coli Liquid Pulse SUMMARY Groups Count Sum Average Variance 6 1202 200.3333 2420.267 0.0001 663 110.5 359.5 0.1 87 14.5 115.5 1 29 4.833333 48.56667 5 10 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 206453.8 41290.76 84.15713 9.95E-17 2.533555 Within Groups 14719.17 30 490.6389 Total 221173 35  

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 0.00026618 9.552094496 Within Groups 3 294 Total 213150 5  

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 107920.3333 53960.16667 612.0245747 0.00012089 9.552094496 Within Groups 3 88.16666667 Total 108184.8333 5