1. Siddarth Narayan Grade 9 North Allegheny Intermediate High School

2.  What effect does ethanol have upon E Coli and Staph cultures?

3.  More than 10% of your body mass is due to symbionts and pathogens  Mostly Prokaryotic cells  Some may cause diseases  Also protect the body from foreign invaders  Reinforce immune system, prevent allergies, and produce vitamins  Lie within skin, saliva, and gastrointestinal tracts  Break down abnormally large nutrients

4.  Large and diverse group of gram (-) bacteria  Free living, symbiontes, or pathogens  Live in the intestinal tract of many mammals.  Most strains are not pathogenic  Serve as a common prokaryotic cell model.

5.  Common surface symbiont in many mammals (Human).  Gram (+) bacteria  Most strains considered non-pathogenic.  Pathogenic strains can be life-threatening.

6.  Often made through the process of fermentation  Fermentation is the process by which yeast breaks down sugar into carbon dioxide and alcohol.  Common uses include: Household chemicals, alcoholic beverages, and medical chemicals and disinfectants  Very toxic substance with numerous effects on body

7.  To examine effects of ethanol upon endosymbiontes

8. Null Hypothesis: Ethanol will not significantly affect microbial survivorship Alternative Hypothesis: Ethanol will significantly reduce the survivorship of E coli and staph cells.

9.  LB agar plates (LB media + 1.5% agar)  LB media (0.5% yeast extract, 1% tyrptone, 1% NaCL)  Sterile pipette tips  Micropipettes  Vortex  Incubator  Sidearm flask  Spreading turntable  Spreader bar  Ethanol (Ethyl Alcohol)  Sterile capped test tubes with Sterile distilled water.  DH5 Alpha E coli Strain  Staphylococcus epidermidis (Ward’s)  0.22 micron syringe filters + 10mL syringe  Klett

10. 1. The microbes were grown overnight in sterile LB media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 1%, 10%, 25% and 50% to a total of 9.9 mL. 6. 0.1 mL of Staph/ E coli was then added to the test tubes, yielding a final volume of 10 mL and a cell density of 10 3 cells/mL per tube.

11. Concentration Chart 0%1%10%25%50% Sterile Dilution Fluid 9.9 mL9.8 mL8.9 mL7.4 mL4.9 mL Microbe0.1 mL Ethanol0 mL0.1 mL1 mL2.5 mL5 mL Total Volume 10 mL 7.The solutions were mixed by vortexing and allowed to sit at room temperature 8.After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on LB agar plates (3 plates per concentration)

12. 9. The plates were then incubated at 37 degrees for 24 hours. 10. The resulting colonies were counted.

13. P Value=1E-05

14. Anova: Single Factor (E Coli) SUMMARY GroupsCountSumAverageVariance 50%320.6670.333 25%310.333 10%3115238411788 1%3889296.33490 0%3773257.7246.3 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups4E+0549398830.271E-053.478 Within Groups31051103105 Total4E+0514

15. ConcentrationT- ValueInterpretation 1%.81Insignificant 10%3.3Insignificant 25%7.6Significant 50%7.6Significant

16. P Value=1.46E-11

17. Anova: Single Factor (Staph) SUMMARY GroupsCountSumAverageVariance 50%3000 25%3399133327 10%31422474468 1%31141380.333344.33333 0%31092364268 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups462882.34115720.6522.51911.46E-113.47805 Within Groups2214.66710221.4667 Total465096.914

18. ConcentrationT-ValueInterpretation 1%1.7Insignificant 10%11.7Significant 25%24.5Significant 50%38.7Significant

19. E coli~ 22% Staph~ 24%

20.  The null hypothesis can be rejected for the following concentration of ethanol and species: E coli (25%, 50%), Staph (10%, 25%, 50%)  Both microbes display similar survivorship

21.  Low sample sizes  Only one type of exposure (Liquid Pulse Exposure)  Limited concentrations of variable  Only one exposure time

22.  More replicates being used in same setup  Other exposures (diffusion assay)  Varied exposure time  Synergistic Effects  Can ethanol benefit survivorship in certain quantities?  Does ethanol only have an effect upon pathogenic endosymbiontes?

23. Plate 1Plate 2Plate 3 0% Ethanol263270240 1% Ethanol364255270 10% Ethanol420262470 25% Ethanol010 50% Ethanol011

24. Plate 1Plate 2Plate 3 0%360350382 1%382373386 10%480492450 25%114135150 50%000

27.  www.ncbi.nlm.nih.gov/pubmed/19609257 www.ncbi.nlm.nih.gov/pubmed/19609257  www.mayoclinic.com/health/staph- infections/DS00973 www.mayoclinic.com/health/staph- infections/DS00973  www.mayoclinic.com/health/e-coli/DS01007 www.mayoclinic.com/health/e-coli/DS01007  www.life.umd.edu/classroom/.../GramPosvsG ramNeg.htm www.life.umd.edu/classroom/.../GramPosvsG ramNeg.htm  www.ncbi.nlm.nih.gov/pubmed/15112929 www.ncbi.nlm.nih.gov/pubmed/15112929  http://jb.asm.org/content/157/1/233.abstra ct http://jb.asm.org/content/157/1/233.abstra ct