Effects of Eco-friendly Cleaners on Bacterial Survivorship Max Kaufmann Central Catholic High School 9th grade

Problem Do environmental friendly cleaning products have a negative impact on bacterial survivorship and do they work as well as cleaning products using traditional chemistry?

Mrs. Meyer's Clean Day Multi-Surface Everyday Cleaner Ingredients - 98% derived from plants -All ingredients are biodegradable -Sodium Methyl 2-Sulfolaurate is the main cleaning agent -All ingredients are safe for pets and children

GS Neutral Disinfectant Cleaner (NDC) -Industrial use cleaning product -Ingredients are “Hazards to Humans” -Ingredients : Alkyl dimethyl benzyl ammonium chloride Octyl decyl dimethyl ammonium chloride Dioctyl dimethyl ammonium chloride Didecyl dimethyl ammonium chloride

Escherichia coli (E. coli) -The Strain that was used is E.coli C -Lives in the intestinal tract of many mammals - Most forms are harmless, but some can become pathogens - Serves as a common prokaryotic cell model - Widely used in many experiments

Purpose The purpose of this experiment is to test if an eco-friendly cleaner, Mrs. Meyer's Clean Day Multi-Surface Everyday Cleaner, affects bacterial survivorship, and how it compares to a normal cleaning product.

Hypothesis Alternative hypothesis: Each of the cleaners will reduce E.coli survivorship. The NDC will reduce survivorship more than the Mrs.Meyers product. Null hypothesis Neither of the cleaners will reduce survivorship and will not vary significantly in effect.

Materials -E. Coli C -Incubator -Vortex -GS Neutral Disinfectant Cleaner -Mrs. Meyers Clean Day Multi-Surface Everyday Cleaner -Tubes -Various sized pipettes -56 LB agar plates (1% Tryptone,0 .5% Yeast Extract, 1% NaCl) -Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH2PO4 , 100mM K2HPO4 , 10mM MgSO4 , 1mM NaCl) -Sterile pipette tips - Cell spreaders -Ethanol alcohol -Open flame

Cleaning product exposures Agar infusion: Adding variable to plate before placing the microbe Liquid pulse: Exposure created by adding variable and microbe in SDF then spreading onto plates

Procedures Agar Infusion The ingredients were mixed to create the exposure of the variable For a low exposure NDC and 10% Meyers, 20 ul of variable and 180 ul of SDF was pipetted onto 6 plates, then spread evenly with a sterile cell spreader For a high exposure NDC and Meyers, 200 ul of variable was pipetted onto 6 plates, then spread evenly with a sterile cell spreader After two hours, 100 ul of E.coli was added from control tube then was spread onto all of the plates Plates were incubated for 2 days at 37 degrees celsius Number of bacterial colonies were counted visually

Concentration Table of Agar Infused Plates Meyers 100% Meyers 10% NDC 100% NDC 10% Variable 200 ul 20 ul SDF 0 ul 180 ul Total

Procedures Liquid Pulse The ingredients were mixed to create the exposure of the variable using the concentration table and placed in a tube All solutions were vortexed 0.1 ml of all solutions was pipetted onto six plates for each group Plates were then spread evenly with a sterile cell spreader Plates were then incubated for two days at 37 degrees celsius Number of bacterial colonies were counted visually

Concentration table of Liquid Pulse 100% stock of cleaners created (variable) Meyers 10% Meyers 1% NDC 10% NDC 1% Control E. Coli 0.1 ml SDF 8.9 ml 9.8 ml 9.9 ml Variable 1 ml 0.1ml 0 ml Total 10 ml 10ml

ANOVA of Liquid Pulse SUMMARY Groups Count Sum Average Variance Control 6 901 150 38.16666667 Meyers 10% 6 141 24 15.5 Meyers 1% 6 776 129 22.26666667 NDC 10% 6 0 0 0 NDC 1% 6 0 0 0 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 128805.5333 4 32201.38333 2120.372037 0 2.75871047 Within Groups 379.6666667 25 15.18666667 Total 129185.2 29

Dunnett's test of Liquid Pulse T-crit = 3.26 Groups T-Value Interpretation Meyers 10% 56.5 Significant Meyers 1% 9.4 NDC 10% 67.3 NDC 1%

ANOVA of Infused Plates SUMMARY Groups Count Sum Average Variance Control 6 901 150 38.16666667 Meyers 100% 6 258 43 14 Meyers 10% 6 600 100 25.6 NDC 100% 6 0 0 0 NDC 10% 6 771 128.5 18.7 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 92104.33333 4 23026.08333 1193.473566 0 2.75871047 Within Groups 482.3333333 25 19.29333333 Total 92586.66667 29

Dunnett's Test of Agar Infused Plates against control T crit = 3.26 Groups T-value Interpretation Meyers 100% 42.3 Significant Meyers 10% 19.8 NDC 100% 59.3 NDC 10% 8.5

Dunnett's test of results between the two variables T Crit = 3.26 Infused Plates T-value Interpretation 100% var. concentration 19.3 Significant 10% var. concentration 12.8 Liquid Pulse Plates T-value Interpretation 10% var. concentration 51 Significant 1% var. concentration 9.5

Conclusion The null and alternative hypotheses are rejected, Both cleaners significantly reduced bacterial survivorship, but the NDC had only killed off more bacterial colonies for ¾ of the concentrations compared to the Mrs. Meyers cleaner. -Meyer’s LD50 for the agar infused is approximately 100 ul and LD50 for the liquid is 6% -The Neutral Disinfectant Cleaner LD50 could not be determined due to at least one group per liquid/infused had eliminated all of the bacteria.

Limitations -During infusion, solution might not have completely absorbed into agar before microbe was plated -Incubation time period -Only the effect on survivorship was tested -Bacteria colonies grew to close together to on a few of the plates to get an accurate cell count -Only high and low concentrations used

Further experiments -More trials -Different bacteria -More concentrations and doses -Other effects on bacteria -Different ways of testing

References -https://www.mrsmeyers.com/lemon-verbena-multi-surface-everyday-cleaner/ -https://www.lung.org/our-initiatives/healthy-air/indoor/indoor-air-pollutants/cleaning-supplies-household-chem.html -https://homeguides.sfgate.com/household-cleaner-affect-environment-79335.html -https://www.care.com/c/stories/5919/7-benefits-of-green-cleaning/