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					Procedure
				Preparation: Prep sheet 1) Warm Dulbecco's modified Eagle Medium (DMEM, Sigma) and Howard's Ringers solution to 37 degrees C. Diliute the 1 mg/ml stock caffeine solution in DMEM. 2) Pour solutions into five 2 ml plastic dishes. Solutions: Howard Ringers Solution, DEM, caffeine solutions: 0.1 mg/ml. 0.2 mg/ml, 0.3 mg/ml. 3) An additional 2 dishes should be filled with warmed Howard Ringers solution for additional group controls. 4) Make a chart for recording the heart beats that includes the following information: Egg # Beats per minute Howard Ringers solution 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) Remove the heart region, leaving the heart intact and place the heart in a dish of Howard Ringers solution. 8) Try to record at least 3 or 4 hearts. 9) The beats of each heart should be recorded for a minute in one of the solutions containing caffeine and the controls. Protocol sheet (Lab Handout) 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 caffiene 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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