1. EFFECT OF GREEN CLEANER ON MICROBIAL POPULATION Mark Carnivale Pittsburgh Central Catholic High School 12 th Grade

2. Clorox Green Cleaner  Green Works® Cleaners:  Environmentally safe ingredients  Ingredients are natural, biodegradable, nontoxic, and do not emit VOCs (volatile organic compounds)  Sold at relatively low price  Claimed to perform as well as conventional non-green cleaners  Uses:  Intended for surfaces such as counters, appliances, stainless steel, and sealed granite  Not stated as a antibacterial cleaning agent  Used to remove dirt, grease, grime, and other messes

3. Green Cleaner Ingredients IngredientWhat it isWhat it does Filtered waterA good solventIncorporates all ingredients into working solution Coconut-based cleaning agent (nonionic surfactant, alkyl polyglucoside) Surfactant, aka cleaning agent Made from coconuts Cleans various messes Corn-based ethanolA form of alcohol. Made from cornA drying agent GlycerineSolvent derived from plantsStabilizes product at low temperatures Essential oilsOil extracted from plantsFragrance Biodegradable preservativeDerived from a petrochemicalPrevents growing of bacteria ColorantStandard blue/green dyeMore appeasing appearance

4. Formula 409® Cleaner  Formula 409® All-Purpose Cleaner  Traditional house cleaner  Uses some hazardous ingredients  Proclaimed as very effective  Sold at relatively low price  Uses  Intended to remove dirt, grease, grime and other messes  Antibacterial cleaning agent  Used on surfaces such as granite, glass, and stainless steel

5. Formula 409® Ingredients IngredientWhat it isWhat it is used for SolventA solventDissolves various ingredients SurfactantsAmphiphilic compounds Reduces water surface tension Anti-bacterial agentsA variety of compoundsKills various bacteria BuilderMultiple compoundsEnhance cleaning properties AlkanolamineCompounds that carry hydroxyl and amino groups Solvents of cleaning solution DyecoloringMake product look appeasable Essential OilsOils extracted from plantsA fragrance WaterA good solventincorporates ingredients into a working solution

6. Escherichia Coli  Escherichia coli (E. coli) – very common, found in intestinal tract of most mammals  There are many strains of E. coli, most are non-pathogenic  Pathogenic strains can cause illness and death in humans  Frequently studied in biology  ubiquitous, simple structure, easily manipulated in the laboratory

7. Purpose  To compare the antibacterial capabilities of Clorox All Purpose Green Cleaner and Formula 409 All Purpose Cleaner

8. Hypotheses  Null: The survivorship between the E. Coli treated with 409 and the E. Coli treated with green cleaner will not vary significantly  Null: The cleaners will have no significant effect on E. coli survivorship  Alternative: The green cleaner’s effect on survivorship will vary significantly from 409’s effect on the survivorship and both agents will reduce bacterial survivorship

9. Materials  Clorox Green Works All Purpose Cleaner  Formula 409 All Purpose Cleaner  Sterile Dilution Fluid (SDF)  Klett Spectrometer  Micropipettes with sterile tips  Spreader bar  Sidearm flask  Sterile test tubes  Bunsen burner  Matches  Sterile Petri Dishes treated with LB media  E. coli at 10 3 cells/mL.  Vortex  Ethanol  Incubator (set at 37˚C)

10. Experiment Description  Experiment #1 Infusion of LB media with cleaner After absorbance was completed, E. coli was pipetted on to plates  Experiment #2 Liquid Pulse Exposure Application of solution onto LB media plates

11. Procedure  Experiment #1- Infusion of Petri Dishes  E. coli was grown overnight in sidearm flask to a density of about10 7 cells/mL  0.2mL of 409 cleaner was pipetted and spread onto 6 LB media Petri Dishes  0.2mL of green cleaner was pipetted and spread onto 6 LB media Petri Dishes  The 12 LB media Petri Dishes were placed in incubator for 2 hours

12. Procedure (cont.)  Experiment #1 (cont)  A tube of E. coli at 10 3 cell/mL was generated by serial dilution  The tube was vortexed  0.1mL of E. coli was pipetted onto each of the 12 Petri dishes  Petri dishes were placed back into incubator for 24 hours  Cells colonies were counted and recorded  All cell colonies were assumed to have arisen from one cell

13. Procedure (cont)  Experiment #2 (liquid pulse exposure)  E. coli was grown overnight in a sidearm flask to a density of 10 7 cells/mL  Concentrations of the two cleaners were made with SDF and the E. coli accordingly (2 tubes per concentration):

14. Procedure (cont)  Experiment #2 (cont) Concentration 10% Concentration 1% Concentration 0.1% Control Group SDF8.9 mL9.8 mL9.89 mL9.8 mL 409 cleaner1mL0.1mL0.01mL0 mL E. coli0.1mL TOTAL10 mL Concentration 10%Concentration 1%Concentration 0.1% SDF8.9 mL 9.8 m L 9.89 mL Green Cleaner1mL0.1mL0.01mL E. coli0.1mL TOTAL10 mL

15. Procedure (cont)  Experiment #2 (cont)  Each concentration was vortexed  0.1mL from each tube was pipetted onto each plate  10 plates were plated for each concentration (60 total) With two tubes of a same concentration, 5 plates were plated from each tube  12 plates were plated from the control group With two tubes of the control group, 6 plates were plated from each tube  Plates were placed in an incubator at 37˚C for 24 hours  Colonies were counted and assumed to have arisen from one cell

16. Data Impreg. w/ green Impreg. w/ 409 920 450 530 950 610 940 MEAN 73.333333330 Experiment #1 Experiment #2

17. E. coli (Infusion exposure)

18. E. coli (liquid pulse exposure)

19. Dunnett’s Test If t > t-crit, then data significant, reject null VariableT-valueT-critSignificance Formula 40916.9012.26Significant Green Cleaner24.1582.26Significant VariableT-valueT-critSignificance Green 10%24.0172.20Significant Green 1%5.7832.20Significant Green 0.1%2.9142.20Significant 409 10%29.8602.20Significant 409 1%29.0632.20Significant 409 0.1%8.5042.20Significant Experiment #2 (Liquid Pulse Exposure) Experiment #1(Infusion)

20. Conclusions  Experiment #1  The null was rejected because the statistical analyses showed that there was significant variation between the the E. coli treated with 409 and the E. coli treated with green cleaner  Experiment #2  The null was rejected since the statistical analyses showed that both cleaners had a significant effect on the survivorship of the E. coli  The Alternative was accepted since the green cleaner’s effect on survivorship varied significantly from the 409 cleaner’s effect on the survivorship

21. Limitations and Extensions  Limitations  409 is intended for antibacterial purposes while green cleaner is not stated for that purpose  Not all plates could be spread synchronously  Extensions  Other students could be used to plate in synchrony to ensure more accurate results  Different bacteria species could be used  Cells grown from culture could be more closely monitored to ensure more precise densities are achieved  Perform experiment in a field test for practicality

22. References  http://www.greenworkspresskit.com/ http://www.greenworkspresskit.com/  http://en.wikipedia.org/wiki/Greencleaning http://en.wikipedia.org/wiki/Greencleaning  http://www.green-technology.org/what.htm http://www.green-technology.org/what.htm  http://www.biosci.ohio- state.edu/safety/MSDS/FORMULA%20409%20CLEANER.htm http://www.biosci.ohio- state.edu/safety/MSDS/FORMULA%20409%20CLEANER.htm  http://www.americanelements.com/AEgreentechnology.html http://www.americanelements.com/AEgreentechnology.html  http://www.about-ecoli.com/ http://www.about-ecoli.com/