Chlorine Bleach on Microbial Resistance Daniel Crawford Grade

Chlorine Bleach on Microbial Resistance Daniel Crawford Grade 11 Central Catholic High School

Antibacterial Agents • Well-documented evidence that bacteria are able to develop resistance to antibiotics Are Bacteria able to develop resistance to household Antibacterial Agents? • Different antibacterial agents commonly used Does initial exposure to one antibacterial agent affect resistance to another?

Gram (-) vs. Gram (+) Bacteria • Cell was contains an extra layer of lipopolysaccharides for extra protection. • Outer membrane protects bacteria from several antibiotics. • Most pathogenic bacteria in humans are Gram (+) organisms. • Simple cell wall • Some antibiotics work against the formation of the cell wall.

Escherichia coli (E. coli) • • • Large and diverse group of gram (-) bacteria Free living, symbionts, or pathogens Live in the intestinal tract of many mammals Most strains are non-pathogenic Serve as a common prokaryotic cell model

Staphylococcus epidermidis (Staph. ) • Common surface symbiont in many mammals (Human). • Gram (+) bacteria • Most strains considered non-pathogenic. • Pathogenic strains can be life-threatening.

Chlorine Bleach • Sodium Hypochlorite, Na. OCl • Forms hypochlorous acid (HOCl) with water – Na. OCl (s) + H 2 O (l) → HOCl (aq) + Na. OH- (aq) • Hypochlorous acid divides into hydrochloric acid (HCl) and oxygen (O). • Strong Oxidizer – effective at killing bacteria, fungi, and viruses. – Denaturization, Low p. H

Hydrogen Peroxide, (H 2 O 2) • Standard [3%] of H 2 O 2 • Clear liquid • H 2 O 2 readily becomes H + HO 2 or 2 OH. – Free radicals, very reactive – Powerful oxidizing agent • Hydrazine fuel, First Aid, Bleaching Agent

Purpose To determine if Escherichia coli or Staphylococcus epidermidis initially exposed to Chlorine Bleach Are able to develop resistance to Hydrogen Peroxide

Hypotheses • Null Hypothesis: Initial exposure WILL NOT increase E. coli and Staph. survivorship, when exposed to Hydrogen Peroxide • Alternative Hypothesis: Initial exposure WILL increase E. coli and Staph survivorship, when exposed to Hydrogen Peroxide

Materials • Clorox Brand Bleach (8. 75% Na. OCl) • Standard Hydrogen Peroxide ([3%]) • Micro and Macro pipettes + Sterile tips • Spreader bars • LB agar plates (1% Tryptone, 0. 5% Yeast extract, 1% Na. Cl) • Escherichia coli DH 5 -alpha (E. Coli) (Obtained from Doonan Lab, CMU) • Staphylococcus epidermidis (Staph) (Obtained from Ward’s Scientific) Burner Sidearm Flask Vortex Incubator Gloves/goggles Klett Spectrophotometer SDF (Sterile Dilution Fluid)(100 m. M KH 2 PO, 100 m. M K 2 HPO 4, 10 m. M Mg. SO 4, 1 m Na. Cl) • Sterile Test Tubes • Ethanol • •

Procedure (Liquid Exposure) Steps 1 -4 referred to a Dilution Process 1. Bacteria (E. coli and Staph. ) were grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/m. L. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/m. L.

Procedure (Pt. 1) 5. Tubes were mixed, with the amounts shown on the table below, to result in *[10 %] was a concentrations of Chlorine Bleach of*: used -3. 5 [0%] [10 -5%] [10 -4%] [10 -3%] [10 -2%] SDF 9. 9 m. L 9. 8 m. L 8. 9 m. L Microbe 0. 1 m. L Chlorine Bleach 0 m. L 0. 1 m. L @ [10 -3%] [10 -1%] Total Volume 10 m. L p. H 7 7 7

Procedure (Pt. 1) Steps 6 -8 referred to a Plating Process 6. The solutions were mixed by vortexing and allowed to sit at room temperature for 3 minutes. 7. After vortexing to evenly suspend cells, 0. 1 m. L aliquots were removed from the tubes spread on LB agar plates. 8. The plates were incubated at 37 C overnight and the resulting colonies were counted.

Procedure (Pt. 1) 9. The Dilution Process was completed and the following concentrations of Hydrogen Peroxide, at the following concentrations, and Plating Process was performed [0%] [10 -2%] [10 -1%] SDF 9. 9 m. L 9. 8 m. L 8. 9 m. L 0 m. L Microbe 0. 1 m. L Hydrogen Peroxide 0 m. L 0. 1 m. L @ [1%] 9. 9 @ Total Volume 10 m. L p. H 7 7 6 5 [1%]

Number of Colonies Results (Pt. 1) Bleach Effects on Bacterial Survivorship 700 p-value < 0. 05 E. coli 611. 5 600 537 p-value < 0. 05 Staph. 489. 25 500 433. 5 364 From test with comparable control 378. 5 300 t-value 45. 85 56. 38 200 100 0 6. 333 [0%] 0 [10 -5 -5%] [10 -4%] -4 [10 -3. 5%] -3. 5 Bleach Concentration 0 7 [10 -3%] -3 0 0 [10 -2%] -2

Results (Pt. 1) Number of Colonies 600 500 400 Hydrogen Peroxide Effects on Bacterial Survivorship 489. 25 463. 66666 67 414 364 t-value 35. 14 15. 34 300 200 144. 75 90. 5 100 0 p-value < 0. 05 0 0 [0%] [10 -2 -2%] [10 -1 -1%] 1 [1%] Hydrogen Peroxide Concentration E. coli Staph.

Procedure (Pt. 2) 10. Cloning: 1 colony transferred to sterile LB media and incubated for 2 days – Colonies From: E. coli exposed to [10 -3. 5%] Bleach Staph. exposed to [10 -3%] Bleach

Procedure (Pt. 2) 11. The Dilution Process was performed with: – E. coli Strain exposed to Bleach (now, “Exposed”) – Staph. Strain exposed to Bleach (now, “Exposed”) – E. coli Strain NOT exposed to Bleach (“Not Exposed”) – Staph. Strain NOT exposed to Bleach (“Not Exposed”) • (both from original strain)

Procedure (Pt. 2) 12. Each strain (Exposed and Not-Exposed) was exposed to appropriate concentration, based on species survivorship: E. coli : Staph. : • [0%] (Control) • [10 -1%] Hydrogen Peroxide 13. The Plating Process was completed, statistical tests were performed, and conclusions drawn

Hydrogen Peroxide Effects on E. coli survivorship 500 Number of Colonies 450 E. coli, Not Exp. E. coli, Exp. 438. 25 400 350 300 250 200 301. 33333 Percent Survivorship 1. 83% 4. 31% ANOVA p-value 4. 30*10 -7 1. 40*10 -6 150 100 50 0 8 13 [0%] [10 -1%] Hydrogen Peroxide Concentration

Hydrogen Peroxide Effect on Staph. Survivorship Number of Colonies 350 Staph. , Not Exp. Staph. , Exp. 316. 667 300 250 200 150 183. 25 Percent Survivorship ANOVA p-value 1. 74% 1. 48*10 -8 0% 7. 28*10 -7 100 50 0 5. 5 0 [0%] [10 -1%] Hydrogen Peroxide Concentration

Conclusion • Initial Exposure to Bleach appeared to NOT INCREASE resistance of Staph. to Hydrogen Peroxide effects Null Hypothesis Accepted • Initial Exposure to Bleach appeared to INCREASE resistance of E. coli to Hydrogen Peroxide effects Null Hypothesis Rejected

Limitations • Plating slightly unsynchronized • Only Liquid Pulse Procedure utilized • Only one clone: more “generations” may be needed to develop resistance Extensions • More variables, concentrations, exposure times, etc. • Different Exposure methods– agar infusion • Study growth, over time • More cloning; more “generations” • Develop Resistance to Bleach

Bleach Effects on E. coli Survivorship 500 Number of Colonies 450 400 350 300 250 Percent 444. 33333 E. coli, Not Exp. 33 438. 25 Survivorship E. coli, Exp. 101. 39% 91. 68% 301. 33333 ANOVA p-value 33 276. 25 0. 82 0. 14 200 150 100 50 0 [0%] [10 -4%] Bleach Concentration

Bleach Effects on Staph. Survivorship 350 316. 67 Staph. , Not Exp. Number of Colonies 300 Staph. , Exp. 250 200 150 100 183. 25 Percent Survivorship 0. 09% 0. 95% ANOVA p-value 1. 24*10 -6 8. 14*10 -7 50 0 0. 25 1. 25 [0%] [10 -3%] Bleach Concentration

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p. H • Measure of Acidity or Basicity of aqueous solution • Hydrogen ions (H+) and hydroxide ions (OH-). – More H+, More Acidic – More OH-, More Basic • measured on a scale of 0 -14 • p. H < 7: Acidic > 7: Basic = 7: Neutral

Anova: Single Factor Bleach on Staph SUMMARY Groups Count Sum Average Variance 0% 2 728 364 50 10 -5% 4 1734 433. 5 777 10 -4% 4 1514 378. 5 91 10 -3% 4 28 7 10 10 -2% 4 0 0 0 ANOVA Source of Variation Between Groups Within Groups Total SS df MS 699258. 4 4 174814. 6 2684 13 206. 4615 701942. 4 17 T-value 35. 13692 F P-value 846. 7176 F crit 1. 44 E-15 3. 179117

Dunnett’s test • Dunnett’s test – comparison of two groups to determine significant variation. – t-crit. ≈2. 7 – t-value • > t-crit → SIGNIFICANT • < t-crit → NOT SIGNIFICANT

Percent Survivorship E. coli Not Exp. Bleach 101. 39% 91. 68% Hydrogen Peroxide 1. 83% 4. 31% Staph. Not Exp. Bleach 0. 09% 0. 95% Hydrogen Peroxide 1. 74% 0%

[10 -3. 5%] SDF 6. 74 Microbe 0. 1 Chlorine Bleach 3. 16 m. L @ [10 -3%] Total Volume 10 p. H 7