Anti-Microbial Effects of Household Cleaners - PowerPoint Presentation

Anti-Microbial Effects of Household Cleaners Clayton Gentilcore Grade 9 Pittsburgh Central Catholic High School

ProblemMillions of dollars spent on infection control and food safety in both commercial and residential environments An effective method of elimination needs to be determined Environmentally-friendly cleaner ( i.e. , Green Works) Anti-microbial cleaner ( i.e. , Lysol)

ObjectiveDetermine effectiveness of household cleaners on survivorship of Escherichia coli (“E. coli”)

BacteriaOne cell prokaryotic organism 4 Basic shapes Cocci (round) Bacilli (rods) Vibrios (bent rod) Spirilla (spirals) Reproduces rapidly Primarily asexualAble to adapt and mutateMany are capable of infecting humans

E. coliGamma proteobacteria Discovered by Theodor Escherich in 1885 Gram-negative Facultative anaerobic N0n-sporulating Rod shaped Most studied prokaryotic organism (“lab rat ”) Found in soil, water and intestines of endothermsHelps break down food in intestinesProduces Vitamin K in intestines Over 700 types of E. coli

Harmful E. coliCausesGastroenteritis Urinary tract infections Neonatal meningitis Strain 0157:H7 (EHEC) causes Life threatening hemolytic-uremic syndrome (HUS) Effects can vary based on age and immune system strength

Outbreaks of E. coli CDC estimates 73,000 cases on average per year of 0157:H7 Resulting in approximately 60 deaths Illness costs are $405 million

Preventative MeasuresMillions of dollars spent per year to protect public Ionizing radiation or chemical treatments to eliminate bacteria Educating public on food preparation and handling

CDC RecommendationsCleaning only removes germs Disinfecting and sanitizing destroys 99.9% of germs Approved disinfectants are given a number by the EPA

Anti-Microbial Cleaner Active ingredients that compromise cell activities: N-Alkyl C8-18 dimethyl benzyl ammonium chloride Benzalkonium Chloride C12-16 Alkyldimethyl aminesEPA approved disinfectant No. 777-66 APPROVED BY University of Missouri – St. Louis, Department of Biology FDA National Institute of Health

Green Cleaner All purpose cleaner Main ingredient is alkyl polyglucoside (coconut derivative) Potassium citrate added for increased cleaning performance Non-ionic surfactant Environmental compatibility 97% natural ingredients No EPA number

Null HypothesisLysol and Green Works cleaners will not significantly reduce E. coli survivorship. Alternative Hypothesis 1. Lysol and Green Works cleaners will significantly reduce E. coli survivorship. 2. Lysol will reduce survivorship more effectively than Green Works.

Materials Lysol Brand II All Purpose Complete Cleaner Green Works All Purpose Cleaner Escherichia coli B Micropipettes, micro rack, micro tubes Turn table, spreader bars, matches , ethanol, Bunsen burner LB media and agar plates (1% tryptone, 5% yeast extract, 1% NaCl, 2 mL 1M NaOH, 1.5% agar)20 mL Sterile capped test tubes with Sterile Dilution Fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) 0.22 micron syringe filters + 10 mL syringe Incubator Vortex Sidearm flask, and Klett spectrophotometer

Procedure Escherichia coli B was grown overnight in sterile LB media. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. The culture was incubated at 37 ° C until a density of 50 Klett spectrophotometer units was reached. (Cell density approximately 10 8 cells/mL).The culture was diluted in a sterile dilution fluid to a concentration of approximately 105 cells/mL.The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL. 0.1 mL. of cell culture was then added to the test tubes, yielding a final volume of 10 mL. and a cell density of approximately 10 3 cells/mL. The solution was mixed by vortexing and allowed to sit at room temperature for 15 minutes . After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on 64 LB agar plates. The plates were incubated at 37°C for 24 hours. The resulting colonies were counted. Each colony is assumed to have risen from one cell .

Chart of Concentrations 0% Cleaner 0.10% Cleaner 1% Cleaner 10% Cleaner E. coli 0.1mL 0.1mL 0.1mL 0.1mL SDF (Sterile Dilution Fluid) 9.9mL 9.89mL 9.8mL 8.9mL Cleaner ( Lysol / Green Works ) 0mL 0.01mL 0.1mL 1mL Total 10mL 10mL 10mL 10mL

Cleaner Effects on E. coli Survivorship Concentration of Variable Average Number of Colonies 1 3.37876E-11 3.29175E-13 1.00115E-06 P-values: P-values: 1.73832E-36 3.4428E-27 Lysol LD50: 0.43% Green Works LD50: 0.88%

Anova: Single Factor Lysol Groups Count Sum Average Variance 0% 8 2421 302.625 103.125 0.1% 8 1440 180 74.57142857 1% 8 30 3.75 9.071428571 10% 8 0 0 0 Source of Variation SS df MS Between Groups 518846.3438 3 172948.7813Within Groups 1307.375 28 46.69196429 Total 520153.7188 31 F P-value F crit 3704.037384 1.73832E-36 2.946685266

Anova: Single Factor Green Works Groups Count Sum Average Variance 0% 8 2421 302.625 103.125 0.1% 8 1823 227.875 196.9821429 1% 8 799 99.875 209.8392857 10% 8 400 50 30 Source of Variation SS df MS Between Groups 322051.0938 3 107350.3646Within Groups 3779.625 28 134.9866071 Total 325830.7188 31 F P-value F crit 795.2667813 3.4428E-27 2.946685266

Significant Variation (Dunnett’s Test) T-critical value (significant difference): 2.88 Alpha: .05 Green Works Concentration T-value Interpretation 0.10% Concentration 12.87 Significant 1% Concentration 34.90 Significant 10% Concentration 43.49 Significant Lysol Concentration T-value Interpretation 0.10% Concentration 35.89 Significant 1% Concentration 87.48 Significant 10% Concentration 88.58 Significant

Conclusions The results suggest that the null hypothesis can be rejected. The results suggest that the alternative hypothesis can be accepted. The Lysol with its anti-microbial agents performed more effectively in eliminating the E. coli as opposed to Green Works, as shown through the LD50s.At 1%, Lysol eliminated 99% of the E. coli colonies, based on the results of the control. Green Works never achieved an elimination of 99% of the E. coli colonies.

Limitations and Extensions Limitations Only 2 cleaning products were used. Only 4 concentrations were used. E. coli was the only bacteria that was used. The plating of E. coli was not fully synchronized. Extensions Test with different brands of cleaning products.Test with different types of bacteria, molds, and algae.Test with different temperatures, spices, and oils to determine a better “green” method to eliminate E. coli. Directly infuse the cleaner into the agar for a long exposure test. Perform a “wipe test.”

ReferencesDyer, Betsey Dexter. A Field Guide To Bacteria. Ithaca, New York: Cornell University, 2003.  Facklam, Howard and Margery. Bacteria. New York: Twenty-First Century Books, 1994.Manning, Shannon D. Escherichia Coli Infections.Philadelphia: Chelsea House, 2005.Singleton, Paul. Introduction To Bacteria. New York: John Wiley & Sons, 1992. Volk, Wesley A. and Margaret E Wheeler. Basic Microbiology. New York: Harper & Row, 1988. Zimmer, Carl. Microcosm: E. Coli and the New Science of Life. New York: Pantheon Books, 2008.

0% 0.10% 1% 10% 0% 0.10% 1% 10% #1.1 289 172 3 0 #1 Total 1195 712 16 0 #1.2 293 166 7 0 #2 Total 1226 728 14 0 #1.3 312 183 6 0 #1 Average 298.75 178 4 0 #1.4 301 191 0 0 #2 Average 306.5 182 3.5 0 #2.1 296 175 1 0 Total 2421 1440 30 0 #2.2 309 179 4 0 Total Average 302.625 180 3.75 0 #2.3 319 185 8 0#2.430218910 Lysol Data

0% 0.10% 1% 10% 0% 0.10% 1% 10% #1.1 289 226 79 57 #1 Total 1195 935 356 203 #1.2 293 258 86 51 #2 Total 1226 888 443 197 #1.3 312 219 84 49 #1 Average 298.75 233.75 89 50.75 #1.4 301 232 107 46 #2 Average 306.5 222 110.75 49.25 #2.1 296 213 111 48 Total 2421 1823 799 400 #2.2 309 231 106 43 Total Average 302.625 227.875 99.875 50 #2.3 319 228 117 59#2.430221610947 Green Works Data