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					Material & methods
				Materials needed 1. About 3 three-day old eggs per participant 2. Dulbecco's Eagle Medium (Sigma) at 37 degrees Celsius, 20 ml per participant recommended, including that needed to make caffeine solutions 3. An incubator, to heat the solutions and to keep the eggs prior to experiment 4. stock 1 mg/ml caffeine solution based in DEM (to make concentrations ranging between 0.1mg/ml and 1.0 mg/ml with DMEM), about 10.5 ml recommended per person 5. 70% ethanol 6. Styrofoam egg cartons (to hold the eggs during extraction of the embryos) 7. Tools for dissection: blunt forceps, two pairs of fine forceps, small scissors, a plastic spoon etc 8. Howard Ringers solution 9. 2ml Plastic Petri dishes, one per solution for each participant 10. Clock, preferably a stopwatch or timer 11. If possible, dissection microscope with video camera to record embryonic heart beats Procedure 1) Warm Howard Ringers balanced salt solution and Dulbecco's modified Eagle Medium (DMEM, Sigma) to 37 degrees C. Dilute the 1 mg/ml stock caffeine solution in DMEM to final concentrations of 0.1 mg/ml. 0.2 mg/ml, 0.3 mg/ml, 0.5 mg/ml and 1 mg/ml in DMEM (or a subset of these concentrations). 2) Pour each solution ( Howard Ringers Solution, DMEM, caffeine solutions ) into a 60 mm sterile plastic petri dish. 3) An additional 3 dishes should be filled with Howard Ringers solution for embryo dissection, and heart isolation and storage. 4) Make a chart for recording the heart rates that includes the following information: Egg # Heart Rate (beats per minute) Howard Ringers solution Media Base (DMEM) 0.1 mg/ml Caffeine 0.2 mg/ml Caffeine 0.3 mg/ml Caffeine 0.5 mg/ml Caffeine 1.0 mg/ml Caffeine . . 5) Take the eggs out of the incubator and place in Styrofoam tray on the bench. Extraction of the embryos: please see Basic Protocol Removal of the heart: 6) Using the fine forceps, remove the trunk region of the embryo above and below the heart. 7) Trim trunk away with fine forceps, leaving the heart intact. Transfer the hearts in a dish of Howard Ringers solution with a cut-off transfer piper. 8) Record the heart rate in Howard Ringers. Transfer the heart into DMEM and record the heart rate. 9) Transfer the heart into the solution containing the lowest concentration of caffeine tested. Record the number of heart beats for a minute. Repeat for for each solution of caffeine tested. 10) Repeat for at least 2 hearts. Download Lab Handout Alternate Lab Handout using intact chick embryos. Three-day-old chick embryos used in this study were divided into two groups, labeled A and B. The hearts in group A were placed in DMEM , then caffeine solutions 0.1 mg/ml, 0.5 mg/ml and 1.0 mg/ml successively. Hearts in group B were added to HR, DEM and caffeine solutions 0.1 mg/ml, 0.2 mg/ml and 0.3 mg/ml.
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