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
Cell walls contain an extra layer of lipopolysaccharides for extra protection
Outer membrane protects against local environments
Most pathogenic bacteria in humans are Gram (+) organisms
Simple cell wall
Some antibiotics work against the formation of the cell wall

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
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)
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

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 9.9mL 0mL Concentration 9.89mL 9.8mL 8.9mL 0.01mL0%
0.01%
0.1% 1%
Sterile Dilution Fluid
9.9mL
9.89mL
9.8mL
8.9mL
Stock of 2-
Butoxyethanol
0mL
0.01mL
0.1mL
1mL
Microbe
Final volume
10mL

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% % % %
Concentration

15. Dunnett’s test e.coli (T-crit= 3.03)
Concentration
T-value
Significance
0.01%
1.72
Insignificant
0.1%
4.48
Significant 1%
7.86

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%
0.01%
0.1% 1%
Concentration

17. DunneTt’s test staph (t-crit= 2.21)
Concentration
T-value
Significance
0.01%
6.96
Significant
0.1%
10.27 1%
10.05

18. Procedures (agar infusion)
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=
Staph P-value= 6.24E-05
840.3
831.8
Average Surviving Colonies
367.5
252.5
223.3
173.5
Control
Low
High
Concentration
E.Coli Staph

20. Agar infusion Dunnett’s test (e.coli=3.03/Staph=2.21
Concentration
T-value
Significance
E.Coli Hi 5
Significant
E.Coli Lo
1.85
Insignificant
Staph Hi
8.25
Staph Lo
0.15

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=6 1
HjFLpOmhA

26. Raw data 0% (control) 0.01% 0.10% 1% Hi Lo 272 235 190 124 160 230 228
180
126
156
172
213
260
231
188
121
200
212
233
236
112
162
238
241
254
211
296
216
219
268
222
Staph
752
357
245
279
396
790
898
496
355
878
702
352
317
295
407
763
1009
560
214
223
312
896

27. Anova (e.coli) Anova: Single Factor SUMMARY Groups Count Sum Average
Variance
Column 1 8
2020
252.5
Column 2 6
1352
Column 3
1090
Column 4 4
513
128.25
368.25
Column 5
694
173.5
Column 6
893
223.25
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 5
E-07
Within Groups 26
Total 31

28. Anova (staph) Groups Count Sum Average Variance Column 1 4 3361 840.25
Column 2
1765
441.25
Column 3
1007
251.75
Column 4
1057
264.25
Column 5
1470
367.5
Column 6
3327
831.75
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 5
E-09
Within Groups 18
Total 23