Opening Eggs for Manipulations Windowing

1. Disinfect eggs by spraying with 70% v/v ethanol, allow to dry, and then pierce the blunt end using an egg-pricker or the point of a #11 scalpel blade to open the air space to the atmosphere (see Note 1). You may prepare eight eggs at a time.

2. Insert a 19-gage needle on a 5-mL syringe through the hole, and direct the tip toward the bottom inside surface of the egg, avoiding the thick albumen that surrounds the yolk. Withdraw 1-2 mL of the thin albumen.

3. With the point of a pair of curved scissors, carefully drill a hole at the middle of the upper side without pushing the scissor tip through the shell membrane. Carefully tear the shell membrane, and leave egg aside for a few moments while the embryo, which is immediately beneath the shell, subsides into the space created by the withdrawal of albumen. A drop of Howard's Ringer over the hole may accelerate the process.

4. Cover the top side of the egg with a piece of 25-mm wide Sellotape, stretched out before sticking down so that it will conform to the double curvature. Press it down firmly, and smooth out creases.

5. Use the curved scissors to make a spiral cut, starting with a small hole and leaving a circular aperture in shell about 12-15 mm in diameter. Direct your cut, as it progresses, so that the aperture overlies the blastoderm (see Note 2).

6. Examine under the stereoscope and stage the embryo by somite count (use neutral red to visualize if required; see Chapter 15).

7. Select those eggs in which the embryo lies more or less centrally in the window as hosts; use the remainder as donors if undertaking isochronic grafting.

8. Hosts: place a drop of Howard's Ringer on the embryo, and reseal egg with loosely applied Sellotape. Keep eggs on the bench—they are happy to be out of the incubator for several hours at this stage.

9. Donors: When doing isochronic grafts, you can economize by using as donors those embryos that are found, on windowing, to be less accessible. Cut around perimeter of the area opaca with Vannas scissors, taking care not to rupture the yolk. Lift embryo with prewetted spatula, and transfer to dish of Howard's Ringer. Float off the vitelline membrane, shake the blastoderm with forceps to free it off adherent yolk, and transfer to fresh Ringer. Subdissect as required (see Subheading 3.3.1.).

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