Chick Development in a Windowed Egg
The objective of this lab is to observe the development of a chick embryo from the first day of incubation onwards. Rather than follow Hippocrates' advice which was to obtain 20 fertilized eggs and two hens, and then crack an egg at daily intervals, we will make "windows" in the eggshell which will permit repeated observations on the same embryo. The procedure is not difficult, but it does require practice. We will try the procedure first on non-fertilized, grocery store eggs, and then repeat the procedure on fertilized eggs from the hatchery.
Procedure
1. Make a viewing stand for the egg by cutting a piece of a styrofoam or cardboard egg carton so that it holds the egg firmly as it lies on its side (broadside, or on its long axis). Try the stand for size and for suitability for using with the dissecting microscope.
2. Assemble the following materials: egg holder, dissecting needle for punching hole in air sac, pair of fine dissecting forceps, slide coated with agar and neutral red dye, glass dish with chick Ringer solution, pipette and pipette bulb to wet agar slide, scalpel handle and blade for cutting agar chips, pencil, lab notebook, copy of Hamburger and Hamilton Staging Series, dissecting microscope, fiber optics light.
Common use Supplies: glass coverslips, jar of 70% ethyl alcohol, Kimwipes, melted paraffin, alcohol lamp, matches.
3. Practice opening an egg first on infertile, store-bought eggs. Wipe the outside of the shell with 70% ethyl alcohol. Wipe all your instruments with alcohol, too. Allow the alcohol to evaporate completely before you proceed. Using the dissecting needle, punch a small hole in the blunt end of the egg. Remove some of the egg shell using the egg sander (this will be demonstrated in class). Using a pair of forceps, carefully break the tough egg shell membranes and allow the yolk to orient spontaneously with the blastoderm on the upper surface.
4. Open a fertilized egg. Locate the blastoderm or the embryo using the dissecting microscope. Distinguish the area pellucida and the area opaca. Can you tell which is the head end? Stain the embryo with a chip of neutral red-impregnated agar. Make a quick sketch, and note its stage. Can you find a primitive streak? Examine your embryo under your dissecting microscope and identify the omphalomesenteric arteries and veins. Has the heart formed? If so, count the rate at which the heart beats.
5. Cover the hole in the egg with melted paraffin and a glass coverslip. Put a ring of melted paraffin around the circular opening. Dip a coverslip in the alcohol, wipe it off, and flame it briefly in the alcohol burner. Place the coverslip over the hole, sealing it in place with the remelted wax. A tight seal is important to protect the embryo from dehydration and infection.
6. Place the labeled, windowed, fertile eggs in the egg incubator. Follow directions from the instructor as to how the eggs should be positioned and spaced in the incubator. Note: you will not use your egg stand in the incubation, but keep your stand for subsequent viewings. Remember the location of your eggs in the incubator so that you won't have to keep the door ajar for a long time while looking for them. This will let heat and moisture out of the incubator and will reduce viability of the eggs. This incubator is set at 99 degrees F and has pans of water in the bottom to keep the atmosphere humidified. Please do not leave the door of the incubator open when you remove your embryo to look at it. And please slide the drawers in and out gently. Don't knock or jar the embryos.
7. Repeat the procedure with two or more fertile eggs, marking each with A, B, etc., and your initials.
8. Return tomorrow and the next day to view your eggs. For viewing, remove your eggs from the incubator, one at a time, and view them through the window with your dissecting microscope. Don't remove the glass window. If moisture condenses on the coverglass, rock the embryo gently to remove the drops. Embryos can tolerate being out of the incubator for approximately 10 minutes at a time. If you wish to observe your embryo for longer periods of time, return them to the incubator for 5-10 minutes before removing them again. Sketch the embryo each time and label the parts that can be viewed through the window. Important: Do not open the incubator door unnecessarily because this cools the air within. Move as quickly as is reasonable when taking the egg out of the incubator or when placing it inside. When making observations, keep in mind that the egg is cooling down to room temperature. Therefore, shorten the viewing period as much as possible and return the egg promptly to the incubator.
9. Incubate your egg for up to five days, returning to observe it daily. If you are lucky, your embryo will position itself under the window and your view will not be obscured by the yolk sac or chorioallantoic membrane. The normal time to hatching is 21 days. Healthy embryos (older than 3 days of incubation) have bright red blood vessels. Dead embryos look yellow or whitish. Keep a record (daily log) of your observations. These need not be extensive, but they should be complete enough to allow you to describe the major changes occurring during development. These embryos can serve as unoperated control for your experiments on blastoderm totipotency.
Use the following questions to guide your observations:
Day 2: Can you see somites and the closed neural tube?
Day 3: Can you find the limb buds? How much of the embryo is covered by amnion? The large bulging vesicles in the brain are the future optic tecta (parts of the mesencephalon). Can you see the somites?
Day 4: How have the limb buds changed their shape? When do the eyes become pigmented? Has the heart changed its shape? Notice the size and position of the allantois.
Day 5: How many of the extraembryonic membranes can you identify? Can you see any embryonic movements? Try to distinguish between embryonic movements and contractions of the amniotic sac which will rock the embryo about in the amniotic fluid. What parts of the embryo begin to move first?
Day 6 and older: When do the limbs begin to move? When do wings start looking different from legs? When do feather germ first begin to form? What about real feathers? When do the birds eyelids form?
Reference: Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88:49-92.
This page was posted 16 March 2003