Notes

1. Any glassblower will produce glass rings from glass rods but the best rings are cut from glass tubes with the desired diameter, anything from 22-28 mm inner diameter, 3-4 mm thick. They are oblong when viewed from the side, and are either left rough or better still baked to give a smooth finish on the cut surface.

Here is a word of warning: If any kind of treatment with compounds is envisaged, rings with a rough cut edge should not be used. Despite thorough cleaning they will retain traces of compound and contaminate the next experiment. Not all egg yolks are the same size. Particularly eggs from very young hens are small and may not fit onto rings 26 mm in diameter. A few smaller rings with an inner diameter of 22 or 23 mm are recommended. Smaller rings are also easier to handle for the novice operator, and a few larger rings (28 mm inner diameter) come in handy if the eggs happen to be on the generous side.

2. Watchmaker's forceps may be straight or curved to the individual's preference. Curved forceps are handy for the release of the membrane when folded under the ring and in tricky situations.

3. The incubation time depends on the developmental stage required for the intended experiment (see ref. 6). Incubating the eggs on their sides is preferable to on their points, but it is not necessary. The blastoderm will always rise to the top of the yolk because of its lower specific gravity compared to the yolk. If the eggs are incubated on their tips, the blastoderm will be directly under the air sack and is more likely to be damaged when the egg is opened.

4. Antibiotic/antimycotic solution (100X) from (Gibco) contains

10,000 U of penicillin (base).

10,000 |g of streptomycin (base).

25 |g of amphotericin B/mL. Use 1% (1 mL in 100 mL BSS).

5. When separating the albumin from the yolk, do not pull on the chalazae too hard or the vitelline membrane will break. The chalazae are two thick gycoprotein threads by which the yolk is tied to the vitelline membrane and which allow it to rotate when the egg is turned so that the blastoderm may rise to the top.

6. Do not try to pour the yolk slowly and carefully from the shell into the BSS. That will certainly tear the yolk.

7. Keep the pie dish on a black surface to show up contours of the yolk, adhering albumin, and so forth.

8. Hold on to the vitelline membrane while lifting the watch glass out of the liquid.

9. A dry ring will instantly stick to the vitelline membrane when put down, and make it difficult to rearrange.

10. As mentioned before, just the cells on the periphery of the blastoderm adhere to the vitelline membrane. It is important not to destroy these delicate contacts.

Carefully pipet small amounts of BSS at the time against the inner edge of the ring to wash away yolk particles, and suck liquid up by slowly moving the pipet backward along the ring. Embryos younger than stage 4 have very delicate contacts to the vitelline membrane, and extra care is needed to transplant these stages successfully.

Best results are achieved after incubation times of 20-36 h. Embryos will develop normally and can be compared to embryos of the same stage in ovo, though not in the same time. New Culture embryos will be between one and two stages younger having their development interrupted by the explanting procedure and the following manipulation. Longer incubation times than 36 h may be tolerated, but the development will slow down and the incidence of abnormalities increases dramatically. Lack of nutrients and space to expand for the embryo may all play a part.

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