The effect of 2-Butoxyethanol on microbial life

1. The effect of 2-Butoxyethanol on microbial life Alex Kochman ‘21 Central catholic high school

2. 2-butoxyethanol • Primary alcohol • Clear, colorless, sweet smelling • Flammable • Used mainly in paints, solvents, inks, cleaning products and surface cleaners • Usually used in amounts less than or equal to 700 ppm • Recommended 25-50ppm/8 hour day • General population usually exposed through household cleaners

3. Toxicity of this chemical • Has negative effects on the following: • Liver • Reproductive health • Lungs • Skin • DNA • Hemoglobin levels • Chemical balance in the blood • Various cancers?

4. Microbial flora • Various eukaryotes and prokaryotes; mostly bacteria • Flora effects on human health is a hot area for research • Positive micro flora effects- nutrition, digestion, immune regulation, and anti-foreign pathogens • Most chemicals ingested by humans aren’t evaluated for their effects on microbial flora

5. Gram positive vs. gram negative Gram Negative Gram Positive Most pathogenic bacteria in humans are Gram (+) organisms Simple cell wall Some antibiotics work against the formation of the cell wall • Cell walls contain an extra layer of lipopolysaccharides for extra protection • Outer membrane protects against local environments

6. Escherichia coli (e.coli) • Microbial flora commonly found in the human intestines and stomach • Gram-negative • Non-pathogenic (most strains) • Prevents colonization of the intestine with pathogenic bacteria

7. Staphylococcus epidermidis (staph) • Part of normal human flora, typically skin flora, and less commonly in mucosal flora • Gram-positive • Non-pathogenic • Helps protect skin from pathogenic bacteria

8. Purpose • To determine if 2-butoxyethanol has a significant impact on the survivorship of E.coli and Staph

9. Hypotheses • Null: 2-butoxyethanol will not have a significant impact on the survivorship of E.coli and Staph • Alternative: 2-butoxyethanol will significantly reduce survivorship among E.coli and Staph

10. Materials • Sterile dilution fluid(10 mM KH2PO4, 10 mM K2HPO4,1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) • Sterile test tubes • Luria broth agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) • Pipettes • Incubator • Micro pipettes • Sterile pipette tips • Sterile spreader bars • Ethanol • Bunsen burner • Vortex • Labeling tape • 2-butoxyethanol • E.coli (DH5 Alpha) • Staphylococcus epidermidis (Ward’s Science)

11. Procedures (liquid pulse) • 1. The E.coli and Staph were grown overnight in sterile LB media at 37° Celsius • 2. The cultures were diluted until certain concentrations of cells were reached (approximately 10⁵ cells/ml for both E.coli and Staph) • 3. Used the following ratios and a 10% 2-butoxyethanol stock solution

12. Concentration chart

13. Procedures (liquid pulse Continued) • 4. Cell suspensions were vortexed • 5. Tubes were incubated at room temperature for 10 minutes • 6. 0.1mL was transferred and spread onto each plate (8 replicates for each concentration) • 7. Plates were incubated for 24 hours, and then colonies were counted • 8. Statistics were performed to check for significant variation

14. 2-butoxyethanol effect on e.coli (liquid pulse) E.Coli Surviorship P-value= 2.32E-07 -10.8% 252.5 225.3 -28.1% 181.6 -49.2% Average Surviving Colonies 128.3 0% 0.01% 0.1% 1% Concentration

15. Dunnett’steste.coli (T-crit= 3.03)

16. 2-butoxyethanol effect on staph (liquid pulse) Staph Surviorship 840.3 P-value=1.69E-09 Average Surviving Colonies -47.5% 441.3 -70.0 -68.5% 251.8 264.3 0% 1% 0.1% 0.01% Concentration

17. DunneTt’steststaph (t-crit= 2.21)

18. Procedures (agar infusion) • 1. 200 microliters of the 10% 2-butoxyethanol stock was spread directly onto plates to create a high concentration • 2. 20 microliters of stock and 180 microliters of pure water were spread onto other plates to create a low concentration • 3. Plates were incubated for one hour • 4. 0.1mL from the liquid pulse control tube was spread onto plates • 5. Plates were incubated for 24 hours

19. Combined survivorship graph (agar infusion) E. Coli P-value= 0.000195 Staph P-value= 6.24E-05 831.8 840.3 Average Surviving Colonies 367.5 252.5 223.3 173.5 Low Control High Concentration E.Coli Staph

20. Agar infusion Dunnett’s test (e.coli=3.03/Staph=2.21

21. Conclusions • 2-butoxyethanol had a significant impact on the survivorship of microbial flora • Data suggests that the null hypothesis was rejected for E.coli in all cases except 0.01% and the low concentration agar infusion test • Data also suggests that the null hypothesis was also rejected for Staph in all cases but the low concentration agar infusion test

22. Limitations • Only used a maximum of 8 replicates • Didn’t analyze where or how the agent damaged the cell • Only one exposure time • Only liquid pulse and agar infusion tests were performed • Reproduction was not examined • Unknown concentration of 2-Butoxyethanol in body

23. Sources of possible error • Small measurement errors • Synchronization errors during plating processes • Small differences in exposure time

24. Extensions • Add more replicates or concentrations • Perform other types of tests (E.g. disk assay) • Perform a detailed analysis of cell damage • Add other types of flora

25. References • https://www.atsdr.cdc.gov/phs/phs.asp?id=345&tid=61 • https://www.ewg.org/guides/substances/152484#.WhHjFLpOmhA • https://pubchem.ncbi.nlm.nih.gov/compound/8133

26. Raw data

27. Anova (e.coli)

28. Anova (staph)