What follows is a general protocol to be executed before performing experimental surgery or descriptive anatomy on a developing chicken embryo of any age. It will enable you to do something useful with eggs other than scramble them.
This protocol is not directly useful for performing vivisection. I do not know or want to know how to dissect a living animal. But I do know how to access to a living embryo, after which it is my policy either to minimize the invasiveness of my manipulations or kill the embryo quickly. Because the embryos I work with regularly are often only a few cell layers thick, it is possible to expose them to all sorts of experimental stimuli noninvasively. Additionally, before the development of thier peripheral nervous systems, embryos do not feel any pain.
- Collect a freshly laid, fertile egg. Store the egg at 15 º C for no longer than one week. The low temperature will prevent the zygote within the egg from beginning to divide, however, after a week its chances of ever developing normally dwindle.
- Place the egg in a tray in which it lies horizontally, and mark the uppermost edge of the egg with a marker in order to remember which way is up. As it develops, the embryo will float above the heavy yolk. If you avoid rotating the egg, the position of the embryo will be easy to ascertain later.
- Incubate the egg at 36-38 º C until it has developed to the desired age. The humidity inside the incubator should be at 70%; you can achieve this by leaving a pan full of deionized water in the incubator.
- At this stage, you need to decide whether you will allow the egg to equilibrate to room temperature before opening it. If the egg is warmer than the air when it's opened then it is likely to lose a lot of moisture to evaporation, and subsequently dry out and die. However, embryos are sensitive to temperature perturbations, and the longer a given embryo is left at room temperature, the more likely it is not to resume normal development when it is returned to incubation. The deciding factor in this case is usually the age of the embryo: if it's old enough to have extraembryonic vasculature then it will not be so sensitive to evaporation, and your best option to leave the embryo at 36-38 degrees right up until you're ready to open it. If the embryo is young enough to still lack that vasculature, remove the egg from the incubator now.
- Obtain a box of tissues, a squirt bottle full of 70% ethanol, a beaker full of 95% ethanol, and an alcohol burner. Light the burner.
- Obtain a hot plate and a metal beaker. Fill the beaker with solid parrafin wax and place it on the hot plate so that the wax melts. Obtain or prepare a cotton swab wrapped around a wooden dowel and leave it dipped in the melted wax.
- Wipe all surfaces at your workstation with 70% ethanol. Pay particular attention to any shelves overlying the work area, if these aren't clean then dust resting on them can float into egg after you open it, contaminating the embryo with germs.
- Prepare and flame sterilize the following instruments: A pair of sharp, curved scissors, two pairs of sharp forceps, a glass ball-tip rod, and a needle made of sharpened tungsten wire attached to a glass handle. Obtain a syringe with a beveled 18 gauge needle, and clean it thoroughly with 70% ethanol. Do not flame sterilize it, or you will melt the glue holding the needle onto its base.
- Prepare an aliquot of neutral red solution. This should be 0.2% dye powder in saline, filtered and subsequiently autoclaved.
- Prepare one empty sterile tube, and one sterile tube full of phosphate buffered saline.
- If you have not done so yet, remove the egg from incubation
- Wipe the surface of the egg with 70% ethanol. After the ethanol evaporates, affix a small square of transparent tape to the top surface of the egg. This will prevent the shell from crumbling when you puncture it later.
- Using the syringe, gently puncture the narrow end of the eg and withdraw approximately 1 ml of thin albumin. This will cause the embryo to sink low enough that you won't pucture it when you cut the egg open. Save this fluid in the sterile tube. Paste the small hole left by the needle closed with the melted parrafin wax.
- Using the scissors, cut a circular hole approximately 1 cm in diameter in the top edge of the egg. Whenever possible, cut so that the scissor blades are parallel and not perpendicular to the shell. This will minimize your likelihood of puncturing the yolk membrane underneath. If you do puncture the yolk membrane, your embryo is almost certain to die.
- Examine the egg under a microscope. If it is indeed fertile, you will now see a series of white concentric circles on the yolk's yellow surface. This is the blastodisk, and within it is the embryo, which will be transparent if it younger than about three days old. If it is older than this, the embryo will be plainly visible and in the place of the blastodisk you will see a network of extraembryonic vasculature.
- Dip the glass ball-tip prod in the neutral red solution and touch it to the center of the innermost white circle. Neutral red is a phototoxin, so use as little of it as possible, and once you have added it to the embryo set your microscope so that it emits as little light as possible.
- Dilute the thin album one to one with phosphate buffered saline. Squirt the mixture back into the egg. This will return essential fluid to the embryo, and will also cause it to rise to a more accessible height.
- Use the tungsten needle to tear the viteline membrane, a thin, papery membrane that lies directly over the embryo. If the embryo is older than about two days old, it will also lie underneath the thick chorionic membrane. If you need to access a portion of the embryo that is underneath the chorion, use two forceps to tease apart the two sections of the chorion, which meet at a very evident seam. Once again, be careful that as your tear the surface membranes you don't tear the yolk membrane underneath them.
You now have an embryo, ready for surgery or dissection. If you are performing experimental surgery and wish the embryo to develop further, then after the surgery is completed, cover the aperture at the top of the shell tightly with transparent tape and return the egg to incubation.
This protocol is modified from techniques I learned from Dr. Drew M. Noden