1. David McFall Grade 9 Central Catholic High School

2. Coffee Americans consume 400 million cups of coffee per year. Coffee has a high caffeine content due to its high concentration in its endosperm. Coffee can be decaffeinated by a treatment of methylene chloride.

3. The Effects of Coffee  Coffee’s side effects are drawn from the release of the hormone epinephrine, shown here. The most notable effects include:  high heart beat  high blood pressure  higher blood sugar content  tense muscles Epinephrine

4. How caffeine interacts with the body  The binding of the hormone adenosine to an adenosine receptor in the brain brings about sleep.  Caffeine blocks adenosine from being connected to the adenosine receptor.  The hormone epinephrine is then produced. This creates the notable effects of caffeine.

5. 20 mg of Caffeine per ounce0.6 to 1.4 mg of Caffeine per ounce Starbucks X-bold Sumatra Roast (CAFF) Starbucks Decaf House Blend (DCAFF)

6.  Usually, when an ingested material is used as a variable, its effects on the human that ingested it are recorded and observed.  However, this experiment deals with the ingested material’s effect on the flora of the human body.

7. Escherichia coli  Escherichia coli is a large and diverse group of gram (-) bacteria.  It is found in the intestinal tract of most animals, including humans.  Estimated to cause infection in more than 70,000 patients a year in the United States.  E. coli has been tested in conjunction with caffeine before in previous studies.

8.  A study from the Lithuanian State Science and Studies Foundation concluded that when in high concentration, caffeine lowers the population of E. coli.  Another study from the Indiana University School of Medicine supported the previous study.

9.  To determine the effect of coffee on human coliform populations.

10. Null Hypothesis: Neither caffeinated nor decaffeinated coffee will have an effect on the survivorship of E. coli. Alternative Hypothesis: Caffeinated and decaffeinated coffee will significantly affect survivorship.

11.  E. coli  Latex Gloves  Micropipettes  Micro tubes  Starbucks X-bold Sumatra Roast  Starbucks Decaf House Blend  Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  58 LB agar plates  LB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl)  2 syringe Sterile Filters  Bunsen Burners  Spread Bar  Incubator  Ethanol  Matches  Vortex  Klett Spectrophotometer

12. 1.E. coli was grown overnight in sterile LB agar. 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 (37°C) 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.Caffeinated and decaffeinated coffee was mixed with the appropriate amount Sterile Distillation Fluid to create coffee concentrations of 10%, 1%, and.1%. 6.100 µL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8.100 µL aliquots were removed from the tubes and spread on LB plates. 9.The plates were incubated at 37 degrees for 24 hours. 10.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 11.The appropriate statistical analyses were performed to adequately assess the hypothesis.

13. 1.Repeat steps 1-6 in the Preliminary Procedure. 2.Both brands of coffee were brewed as directed. 3.The coffee was then sterile filtered. 4.200 µL of sterile coffee were removed from the tubes and spread on LB plates. 5. 100 µL of E. coli (control tube) was removed from the tubes and spread on LB plates. 6.The plates were incubated at 37 degrees for 24 hours. 7.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 8.The appropriate statistical analyses were performed to adequately assess the hypothesis.

14. Control.01x.1xx E. coli0.1 ml Sterile Dilution Fluid (SDF)9.9 ml9.89 ml9.8 ml8.9 ml Coffee0 ml0.01 ml0.1 ml1 ml Total Volume10 ml Chart of Concentration

16. T Critical= 3.49 (Significant) Variable Concentration T Value Interpretation.01x CAFF2.2 Insignificant.1x CAFF3.46 Insignificant x CAFF4.78 Significant.01x DCAFF7.63 Significant.1x DCAFF4.54 Significant x DCAFF3.69 Significant

17.  Did liquid exposure to coffee significantly effect survivorship?  P-Value 7.29E-8 Significant  Which coffee exposures significantly effected survivorship?  10% Caffeinated Exposure T-Value 4.78 Significant  0.1% Decaffeinated Exposure T-Value 7.63 Significant  1% Decaffeinated Exposure T-Value 4.54 Significant  10% Decaffeinated Exposure T-Value 3.69 Significant  Did caffeinated coffee vary from decaffeinated in survivorship?  0.1% CAFF-P-Value 1.41E-3 Significant  1% CAFF-P-Value 0.28 Insignificant  10% CAFF-P-Value 0.62 Insignificant  Did continuous exposure to coffee (Infused Plates) alter survivorship?  CAFF-P-Value 0.12 Insignificant  DCAFF-P-Value 0.3 Insignificant

18.  It seems as if caffeinated coffee increased the population of E. coli, but only in the 10% concentration.  Decaffeinated coffee, on the other hand, seemed to increase the population of E. coli in all concentrations.

19.  Plating was not exactly synchronized(10 minute lag), which could have resulted in extra time for bacterial replication. A team of students could remedy this technical problem.

20.  Test more brands of coffee  Use a different model  Test coffee in conjunction with other caffeinated beverages, such as tea or energy drinks

21.  http://www.espressoforums.com/viewtopic.php?t=137  http://www.miyerbamate.com/content/Yerba%2BMate%2Bharmful%2Bi ngredients%2Bin%2Bcoffee%2Band%2Btea.htm  http://www.starbucks.com  http://www.coffeeresearch.org/science/decaffeination.htm  http://www.about-ecoli.com/  http://www.nlm.nih.gov/medlineplus/ecoliinfections.html