Joe Stern Pittsburgh Central Catholic Grade 9 Sodium Hypochlorite Effects on Escherichia coli and Staphylococcus epidermidis Joe Stern Pittsburgh Central Catholic Grade 9

Research Questions Does an environmentally friendly cleaning agent perform better, similar, or worse than a chemically based cleaning agent? How do different concentrations of cleaning agents compare to each other in reducing microbial growth?

Cleaning Agents LA’s Totally Awesome Bleach Javellisant: Chemical Based pH = 12 Contains active ingredients: Sodium hypochlorite - 5.25% Sodium chlorate Sodium hydroxide Sodium polyacrylate Sodium carbonate

Cleaning Agents Cont. Simple Green All-purpose Cleaner: Environmentally friendly Biodegradable U.S. EPA Safer choice product pH = 9 Contains active ingredients Sodium hypochlorite - 5.25% Sodium citrate Sodium carbonate

Sodium Hypochlorite NaOCl Sodium cation and hypochlorite anion Forms a hypochlorous acid when with water Found in cleaning agents Strong oxidizer Proficient in killing bacteria Base

Gram (-) vs. Gram (+) Bacteria Gram Positive Bacteria (Staph) Most pathogenic bacteria in humans are Gram (+) organisms Thick layer of peptidoglycan in the cell wall Antibiotics can impede the ability of the peptidoglycan to link Gram Negative Bacteria (E. Coli) Thin layer of peptidoglycan The outer membrane is an extra thin layer of lipopolysaccharide, further adding protection to the cell Outer membrane provides protection from some antibiotics

Model Bacteria: Escherichia coli Gram negative bacteria Rod-shaped Intestinal symbiont Lives in our intestinal tract Commonly used as a model for microbial life in the human body Some strains are pathogens

Model Bacteria: Staphylococcus epidermidis Gram positive bacteria Skin symbiont Found on human skin Commonly used as a model for microbial life in the human body Strains are non-pathogenic

Purpose Determine effects of two common cleaners on E. coli and Staph (each containing sodium hypochlorite) Investigate the reliability of an environmentally friendly cleaning agent when compared to a chemical based cleaning agent

Hypotheses Null Hypotheses: Various concentrations of sodium hypochlorite will have no toxic effect on the survivorship of the microbes There will be no significant difference in microbial survivorship between an environmentally friendly cleaner and a chemical based cleaner Alternative Hypotheses: Various concentrations of sodium hypochlorite will have a toxic effect on the survivorship of the microbes There will be a significant difference in microbial survivorship between an environmentally friendly cleaner and a chemical based cleaner

Materials Sidearm flask Spreading turntable LB agar plates Spreader bars Ethanol Burner Sterile capped test tubes with sterile distilled water LA’s Totally Awesome Bleach Javellisant Simple Green All-purpose Cleaner LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile Dilution Fluid Micropipettes Sterile micropipette tips E. coli C strain (Obtained from CMU) Staphylococcus epidermidis (Obtained from Ward’s Science) Vortex Incubator

Procedure (Liquid Exposure) Both bacteria were grown until a density of 50 klett spectrophotometer was reached. This was approximately 10^8 cells/mL The cultures diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL Cleaners were diluted to a concentration of 1% in SDF. The solutions shown in the table of concentrations were created in sterile test tubes Solutions were vortexed

Table of Concentrations: Liquid Exposure 0% (Control) 0.001% 0.01% 0.1% Microbe 0.1 ml Sodium Hypochlorite Solution 0 ml 0.01 ml 1 ml SDF 9.9 ml 9.89 ml 9.8 ml 8.9 ml Total volume 10 ml

Procedure cont. (Liquid Exposure) 0.1 ml of each tube was spread onto agar plates Plates were grown overnight at 37 C and the resulting colonies produced were counted

Procedure (Infused Agar) Aliquots of cleaners were spread onto agar plates Plates were then incubated for 1 hour to absorb 100 μL samples from the control tube were then added to the infused plates The table below shows the concentrations of the infused agar plates Plates were grown overnight at 37 C and the resulting colonies produced were counted

Table of Concentrations: Infused Agar (1%) 200 uL 20 uL Percentage of Sodium Hypochlorite 1% 0.1% SDF 0 uL 180 uL Total volume 0.2 ml

Hypochlorite Effects on E. coli Survivorship (Liquid Exposure) P-value: 8.66E-06 < 0.05 Individual p-values: Chemical: 5.3E-05 < 0.05 Environmental: 0.018 < 0.05

Hypochlorite Effects on Staph Survivorship (Liquid Exposure) P-value: 3.29E-14 < 0.05 Individual p-values: Chemical: 5.91E-10 < 0.05 Environmental: 1.18E-07 < 0.05

Dunnett’s Test (E. coli Liquid Exposure) Chemical Based Bleach T- Value T- critical Significance? 0.1% 2.56 3.49 Not sig. 1% 4.62 Significant 10% 5.50

Dunnett’s Test (E. coli Liquid Exposure) Cont. Environmentally Friendly Bleach T-value T-critical Significance? 0.1% 0.26 3.49 Not sig. 1% 2.20 10% 3.67 Significant

Dunnett’s Test (Staph Liquid Exposure) Chemical Based Bleach T-value T-critical Significance? 0.1% 0.75 3.49 Not sig. 1% 2.13 10% 7.22 Significant

Dunnett’s Test (Staph Liquid Exposure) Cont. Environmentally Friendly Bleach T-value T-critical Significance? 0.1% 0.07 3.49 Not sig. 1% 0.10 10% 4.42 Significant

Hypochlorite Effects on E. coli Survivorship (Infused Agar) P-value: 1.59E-5 < 0.05

Hypochlorite Effects on Staph Survivorship (Infused Agar) P-value: 1.0E-5 < 0.05

Dunnett’s Test (E. Coli Infused Agar) Chemical Based Environmentally Friendly T-value T-critical Significance? 0.1% 0.46 3.48 Not sig. 1% 3.53 Significant T-value T-critical Significance? 0.1% 6.45 3.48 Significant 1% 7.53

Dunnett’s Test (Staph Infused Agar) Chemical Based Environmentally Friendly T-value T-critical Significance? 0.1% 40.7 3.48 Significant 1% 42.59 T-value T-critical Significance? 0.1% 42.98 3.48 Significant 1% 43.77

Conclusions Reject null hypothesis for various concentrations of sodium hypochlorite having a toxic effect on microbial survivorship Microbial survivorship decreased as concentration of sodium hypochlorite increased Reject null hypothesis for the common cleaning agents not having a significant difference in reduction of microbial survivorship The environmentally friendly bleach allowed for more microbial survivorship than the chemical based bleach in liquid exposure tests In infused agar tests, the chemical based bleach allowed for more microbial survivorship than the environmentally friendly bleach

Explanations The different pH levels in the two cleaners could have caused different microbial growth. This could be a result from the environmentally friendly bleach using more organic material instead of synthetic Possible ingredient in one of the variables caused it to reduce or allow microbial growth When active ingredients are applied in higher concentrations, they permit for less microbial survivorship

Limitations Spread plating could have possibly not been synchronized due to human error The timespan of collecting data may have not been completely synchronized due to time constraints Only two species used Only survivorship was tested Only 4 concentrations were tested

Extensions Other cleaning agents and bacteria will be tested Active ingredients of cleaning agents will be compared A more diverse pool of microbes will be tested Synchronized spread plating Perform experiment over longer periods of time to test cleaning agent duration A variety of concentrations will be tested

Works Cited www.lenntech.com/processes/disinfection/chemical/disinfectants-sodium-hypochlorite.htm. www.foodsafety.gov/poisoning/causes/bacteriaviruses/ecoli/index.html. www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria www.ukessays.com/essays/biology/estimation-inhibitory-effect-of-clorox-on-escherichia-coli-biology-essay.php. https://nj.gov/health/eoh/rtkweb/documents/fs/1707.pdf www.eurekalert.org/pub_releases/2008-11/cp-hhb110708.php. https://owlcation.com/stem/How-Bleach-Works-Disinfection-and-Stain-Removal www.jstage.jst.go.jp/article/bbb/73/1/73_70722/_article. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643108/

Chemical Bleach ANOVA: E. Coli (Liquid Exposure)

Chemical Bleach ANOVA: Staph (Liquid Exposure)

Natural Bleach Anova: Staph (Liquid Exposure)

Cleaning Agents Staph Microbial Survivorship ANOVA (Liquid Exposure)

Cleaning Agents E. coli Microbial Survivorship ANOVA (Liquid Exposure)