1 2 3 4 5 6 7 8 9 10

Heart Rate
7 eggs were collected and incubated for 6 days. Using forceps the embryos were removed from the eggs and placed in warm Howard's ringers solution. With fine forceps, the trunk was severed above and below the heart region. The dorsal region of the embryo was removed and the heart left intact. The heart was transferred to fresh warm Howard's ringers solution. 1.0 mg of nicotine was added directly to 4 of the hearts. Heart rate was measured by counting the beats every 15 seconds and then allowing 15 seconds to pass before the next reading. The procedure for the 3 controls was the same, except they were treated with 1.0 mg of saline solution instead of nicotine.

Staining for Limb Cartilage
2, 13 day old nicotine treated embryos and 3, 13 day old control embryos were isolated for staining. Legs and wings were fixed in 5% TCA for 1 hour. The limbs were then washed 3 times with PBS and immersed in Alcian green stain for approximately 18 hours. They were washed 3 times with 70% ethanol, and once with 85% ethanol, 95% ethanol, and 100% ethanol. Limbs were then washed 3 times with methyl salicylate and left in the solution for 4 days. Staining was observed and images were captured using an Olympus DP10 camera.

©Cebra-Thomas, 2000

Last Modified: 5 May 2000

[Lab Protocols | Students | Cebra-Thomas | Course | Links ]