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.  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

5.  To examine effects of ethanol upon endosymbiontes

6. Null Hypothesis: Survivorship will not be significantly affected by the presence of ethanol Alternative Hypothesis: Ethanol will have a tangible effect upon survivorshop

7.  YEPD agar plates (YEPD media + 1.5% agar)  YEPD media (1% yeast extract, 2% peptone, 2% glucose)  Sterile pipette tips  Micropipettes  Vortex  Incubator  Sidearm flask  Spreading turntable  Spreader bar  Ethanol (Ethyl Alcohol)  Sterile capped test tubes with Sterile distilled water.  (Obtained from Jones Lab, CMU)  0.22 micron syringe filters + 10mL syringe  Klett

8. 1. The microbes were grown overnight in sterile YEPD 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 7 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.

9. 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 YEPD agar plates (3 plates per concentration)

10. 9. The plates were then incubated at 30 degrees for 48 hours. 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

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

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

13. 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

14. 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

15.  Upon examination of the data, the null hypothesis can be rejected  Data is indicative of an increase in survivorship for both colonies in the 0-10% ethanol concentrations, while survivorship drops considerably in the 25- 50% margin  Both microbes display similar survivorship in ethanol environment

16.  Incorrect concentrations of alcohol could have been produced.  Spreader bars couldn’t have been sterilized properly  Only one type of alcohol was used  Only three samples of each microbe were taken