Part 91

OUTLINE FOR LABORATORY STUDY OF THE CHICK

The objects of laboratory study of the embryos are as follows:

1. To study the living embryo.

2. To study the entire embryo:

With the dissecting microscope, as an opaque object.

With the compound microscope after killing, hardening, clarifying, and mounting.

3. To study embryos by dissection, in later stages.

4. To study the serial sections with the compound microscope.

THE LIVING EMBRYO

The egg is opened under warm physiological salt solution: 0.8 per cent. sodium chlorid in distilled water heated to a temperature of 38°C.

Gradually pick away the shell at the large end. Note that there are two membranes and an air cell. Strip off the membranes. When sufficient shell and membranes have been removed from the large end, invert the open end of the egg in the salt solution and allow the contents to flow out. Care must be taken not to break the yolk. The embryo, or blastoderm, lies upon the surface of the yolk, which is usually turned with this body uppermost. Separate the blastoderm by cutting around the outside of the area vasculosa. In doing this a small pair of slightly curved scissors is needed. After the embryo, or blastoderm, has been separated, gently float it into a watch crystal with the flat bottom submerged in the salt solution. The watch crystal with its contents may now be gently lifted out. Next remove the vitelline membrane. The vitelline membrane is the delicate transparent membrane covering the blastoderm, The embryo is now ready for study.

THE PREPARATION FOR STUDY OF ENTIRE EMBRYOS AND SECTIONS

The following processes may be used in killing embryos up to four days, or ninety-six hours, of age. After removing the embryo as described above, spread the blastoderm out in the watch crystal and pipette off the salt solution. Allow it to stand till the edge of the tissue begin slightly to adhere. Then slowly add the killing fluid by aid of a pipette, dropping it on the center of the embryo. The pipette must be held low or the mechanical interference will dislocate the parts.

Older embryos are submerged with their membranes intact into the killing fluid. The quantity of fluid should be several times the bulk of the specimen. Kleinenberg’s picrosulphuric acid may be used as a killing fluid. This fluid is a saturated solution of picric acid plus 2 per cent. sulphuric acid, to which is added twice its volume of water.

Chick embryos from one to two days old should be left in this fluid from one and one-half to six hours. Embryos from two to four days old two and one-half to six hours. Remove the specimen from the killing fluid, and place it in 70 per cent. alcohol. Change the alcohol every twenty-four hours until the color ceases to come out of the embryo. Preserve in 80 per cent. alcohol.

If the specimen is to be mounted whole, transfer it from 80 per cent., then to 50 per cent., then to 35 per cent., and, finally to water. Small embryos should remain in each fluid thirty minutes and large ones sixty minutes.

The following method may be used for staining embryos: Dilute Delafield’s hematoxylin with four times its volume of water. To every 6 cubic centimeters of this diluted hematoxylin, add one drop of Kleinenberg’s undiluted picrosulphuric acid, and leave specimen in the fluid thus prepared until it is stained through. This will require from one to three hours. Now pass up through the series of alcohols to 70 per cent. Next extract the excessive stain with 1 per cent. hydrochloric acid in 70 per cent. alcohol. Wash repeatedly with 70 per cent. alcohol to free from the acid; then transfer the specimen to 80 per cent. alcohol and leave in this for several hours for complete removal of the acid; then transfer to 95 per cent. alcohol for thirty minutes. Allow the specimen to remain in absolute alcohol for one hour. Introduce a layer of oil of cloves or xylol beneath the alcohol. This may be done by gradually allowing the fluid to run down the side of the bottle. After the embryos have sunk into the oil and begun to appear transparent, remove the fluid and add fresh oil. After the specimen is sufficiently transparent, mount in balsam, supporting the cover slip so that it will not rest on the embryo.

In staining for section, place the embryo in borax carmine from the 50 per cent. alcohol and leave twelve hours. Then wash in 50 per cent. alcohol; after which transfer to 70 per cent. alcohol for six hours. Clarify in oil of cedar or oil of cloves, and place in melted paraffin for two hours. Imbed in paraffin, and section.

Embryos may be hardened, imbedded, and sectioned after the usual methods, using either paraffin or celloidin, and the sections stained with hematoxylin and eosin after sectioning.

POINTS TO BE OBSERVED IN THE STUDY

1. THE EMBRYO TWENTY-NINE TO THIRTY-FOUR HOURS OLD WITH FROM TEN TO FOURTEEN SOMITES.

A study of the egg.

In opening the egg, observe that the shell membranes are double.

Observe that the shell is porous.

Note the air cell at the large end of the egg and note that the space or cell lies between the outer and the inner shell membrane.

After the egg contents have dropped into the salt solution, note extending from the ends of the yolk the twisted denser cords of albumen. These are the chalazæ, which act as stays to the yolk. Note that the yolk is surrounded by a delicate membrane. This is the vitelline membrane. The yolk is the true ovum and serves as food for the developing embryo.

A study of the living embryo.

Note the amount of yolk that is covered by the blastoderm. Note the slipper-shaped, transparent center of the blastoderm. This is the area pellucida. In the center of this there is a narrow white streak. The area opaca is the area lying external to the area pellucida. In this there is the area vasculosa.