Arranging Vitelline Membrane on the Ring

1. Use a stereoscopic, binocular dissecting microscope

2. Stretch vitelline membrane, with the blastoderm in the center, well out on the watch glass. Remember the vitelline membrane ought to be inside-out at this stage.

3. Wet a glass ring in BSS (see Note 9), and lay it on the vitelline membrane and fold the membrane over the ring, holding ring with straight forceps and pulling membrane with curved tweezers, working all the way around until all the membrane is securely draped over the ring. Hold the membrane only at the cut edge to avoid injury to it and the blastoderm (see Note 10).

4. Pipet off most of the liquid and the adhering yolk. With fine scissors, cut away the surplus membrane close to the inside of the ring.

5. Wash all yolk from under the ring with BSS, and pipet 1 mL of thin albumin onto the watch glass beneath the ring to supply the developing embryo with a source of water and protect it from bacterial infections. Albumin is rich in bactericidal proteins. Little or no liquid should cover the embryo.

6. Lay one or two strips of Kleenex tissue in Petri dish, and wet with BSS to create a moist chamber.

7. Now the embryo is ready for surgery or the designed treatment. If surgery is performed, leave the embryo to heal at room temperature for a couple of hours before further incubation. This helps to close the wound while the blastoderm is not expanding, and there is less tension in the fragile tissues. It may be appropriate to photograph embryos at this stage.

9. Incubate at 38 (±) 1°C in a tissue-culture incubator. Facilities for an air mix of 95% air and 5% CO2 are not essential.

10. Observe the development of the embryos the next day. They should have reached stage 10-12 (Fig. 3) and may be photographed again.

Fig. 3. New Culture after 24 h of incubation.

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