Disinfectant Wipe Extract Effects on Bacteria Ethan Williams Grade 9 Central Catholic High School

Rationale Any disinfectant wipe should be able to significantly reduce survivorship of microbes The more expensive Clorox wipes should be more effective in reducing survivorship so that they can make up for the price difference

Purpose To determine if Clorox and Great Value wipes greatly reduce survivorship of gram positive and gram negative bacteria If so, which is more effective and at what concentration?

Clorox Wipes Higher-end, more expensive - $4.99 per canister – 78 wipes per character Active Ingredient - Dimethyl Benzyl Ammonium Chloride Common Household chemical used to clean surfaces Clorox claims that these wipes will kill 99.9% of both Staph and E. coli on hard surfaces

Great Value Wipes Lower-end wipes, cheaper - $1.99 per canister- 75 wipes per canister Active Ingredient – Octyl Decyl Dimethyl Ammonium Chloride Also used commonly in cleaning products Claim that it kills 99.9% of bacteria

Comparison of Inactive Ingredients Clorox Great Value Water Substrate Hexoxythenol Citric Acid-Lemon scented Fragrance d-Limonene Isopropanol Sodium Bicarbonate Water Substrate Citric Acid-Lemon scented Fragrance

Escherichia coli (E. coli) Gram (-) Commonly found in the human gut Most strains are not pathogenic Most widely used bacteria model (represents other gram (-) bacteria)

Staphylococcus epidermidis (Staph) Gram (+) Normal human flora - typically skin flora Can cause staph infections when it enters the body Can cause staph food poisoning

Hypotheses Null: Neither the Clorox nor the Great Value Wipes will have an effect on the survivorship of the bacteria Alternative: The Clorox and Great Value wipes will significantly reduce survivorship of the E. coli and Staph Alternative #2: The Clorox wipes will reduce survivorship more than Great Value

Materials 250 mL beaker 500 gram weight Spreader bars Ethanol Sterile Water Test Tubes + Rack SDF (10 mM KH2PO4, 10 mM K2HPO4,1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Incubator Forceps Hole puncher Clorox brand disinfectant wipes Great Value brand disinfectant wipes E. coli Staph YEPD Plates (0.5% yeast extract, 2% peptone, glucose Micropipettes Sterile Pipette Tips Test Tubes Sterile Tubes

Procedure: Stock Solution 45 mL of sterile water was placed in a sterile 50 mL conical tube A wipe was placed in the tube for 5 seconds The tube was inverted 10 times to ensure that the solution was evenly spread in the water The wipe was removed with sterile forceps

Procedure: Liquid Pulse (Cont.) 0.1 mL of each concentration was pipetted onto 5 plates per concentration per brand resulting in 20 plates per brand of E. coli and 20 plates of staph per brand The solutions were spread onto the plates with sterile spreader bars The E. coli plates were incubated for 1 day at 37°C The staph plates were incubated for 2 days at 37°C to allow for sufficient growth

Procedure: Liquid Pulse E. coli and Staph were grown at a density of 10ˆ⁸ cells per mil They were then diluted in SDF to a density of 10ˆ⁵ cells per mil The following ingredients were mixed to create the following exposures The solutions were incubated at room temperature for 10 minutes 0% 0.1% 1% 10% SDF 9.9 mL 9.89 mL 9.8 mL 8.9 mL Wipe Extract 0 mL 0.01 mL 0.1 mL 1 mL Bacteria Total Volume 10 mL

Procedure: Sterile Disk Assay 0.1 mL of bacteria was plated with a sterile spreader bar A hole punch was used to create sterile disks from the wipe 4 sterile disks were placed in a cross shape on each plate The plates were incubated for 1 day at 37°C for E. coli and 2 days at 37°C for staph After incubation, the zone of inhibition was measured (mm)

Clorox vs Great Value E. coli Survivorship

Dunnet’s Test Evaluation T Value T crit Significant/ Not Significant Clorox 0.1% 10.98 2.77 Sig Clorox 1% 17.86 Clorox 10% 24.23 Great Value 0.1% 9.97 Great Value 1% 15.21 Great Value 10% 22.23

Clorox v. Great Value Staph Survivorship

Dunnet’s Test Evaluation T Value T-crit Significant/ Not Significant Clorox 0.1% 1.95 2.81 Not Sig Clorox 1% 26.24 Sig Clorox 10% 39.67 Great Value 0.1% 11.92 Great Value 1% 19.87 Great Value 10% 25.05

E. coli Zone Inhibition

Staph Zone Inhibition

Conclusions The null can be rejected, as the effects on the bacterial survivorship were very significant, and the p-values were far below the alpha of .05 The alternative hypothesis was supported, as the Clorox wipes had far less bacterial survivorship than the Great Value wipes

Limitations and Extensions Test more brands of wipes More replicates for each concentration Use a lower concentration than 10% as the high, possibly 5% Use different types of bacteria Limitations: Possible error in pipette technique Possible error in spreading on the plates Possible error in counting of colonies Only 5 replicates were used Study does not reveal other effects on bacterial health

Works Cited “Ammonium Chloride.” National Center for Biotechnology Information. PubChem Compound Database, U.S. National Library of Medicine, pubchem.ncbi.nlm.nih.gov/compound/ammonium_chloride#section=Top. “E. Coli (Escherichia Coli) | E. Coli | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, www.cdc.gov/ecoli/index.html. Foodsafety.gov. “E. Coli.” FoodSafety.gov, U.S. Department of Health and Human Services, 24 Aug. 2009, www.foodsafety.gov/poisoning/causes/bacteriaviruses/ecoli/index.html. “Gateway on Pesticide Hazards and Safe Pest Management.” Beyond Pesticides, www.beyondpesticides.org/resources/pesticide-gateway?pesticideid=89. “Healthcare-Associated Infections.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 17 Jan. 2011, www.cdc.gov/hai/organisms/staph.html. “Ingredients Inside.” The Clorox Company, 9 Dec. 2016, www.thecloroxcompany.com/en-us/what- were-made-of/ingredients-inside/clorox/clorox-disinfecting-wipes1-crisp-lemon-44600017273/. “Staphylococcal (Staph) Food Poisoning | Food Safety | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, www.cdc.gov/foodsafety/diseases/staphylococcal.html.

Raw Data- Clorox E. coli SUMMARY Groups Count Sum Average Variance 0% 5 1470 294 509 0.10% 697 139.4 662.8 1% 471 94.2 187.7 10% ANOVA Source of Variation SS df MS F P-value F crit Between Groups 225757.8 3 75252.6 221.4125782 3.0948E-13 3.238871517 Within Groups 5438 16 339.875 Total 231195.8 19

Raw Data- Great Value E. Coli SUMMARY Groups Count Sum Average Variance 0% 5 2943 588.6 340.3 0.10% 2084 416.8 339.7 1% 1433 286.6 387.3 10% 60 12 139.5 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 886758.8 3 295586.2667 979.7357198 2.45899E-18 3.238871517 Within Groups 4827.2 16 301.7 Total 891586 19

Raw Data – Clorox Staph SUMMARY Groups Count Sum Average Variance 0% 5 1447 289.4 449.3 0.10% 719 143.8 328.7 1% 511 102.2 10% 1 0.2 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 215794.2 3 71931.4 259.9382058 8.83275E-14 3.238871517 Within Groups 4427.6 16 276.725 Total 220221.8 19

Raw Data – Great Value Staph SUMMARY Groups Count Sum Average Variance 0% 5 2401 480.2 226.7 0.10% 1722 344.4 332.8 1% 941 188.2 482.2 10% 56 11.2 84.2 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 613020.4 3 204340.1333 725.9619268 2.66307E-17 3.238871517 Within Groups 4503.6 16 281.475 Total 617524 19