h26 ■ EXPERIMENTAL CHICK EMBRYOLOGY 



the ahell over the air space (at the blunt end of the egg) and to provide a removable 

 (shell) cover, so that the embryo can be watched at stated intervals for developmental 

 changes. 



These procedures have two uses: First, it is not possible to maintain an excised 

 embryo on culture media and to have it develop perfectly normally for more than several 

 days (and at early stages only). By these methods the embryo may be observed under per- 

 fectly normal conditions and morphogenesis can be studied, at least until the embryo as a 

 whole becomes opaque from the development of its organs. Such embryos can be carried 

 through to hatching. Second , when grafts are added to the chorio-allantois, they can be 

 observed through the window, at least for a number of days. 



THE WINDOW METHOD : 



Secure an egg of less than 55 hours of incubation (7 or 8 day embryos may be similar- 

 ly treated when making chorio-allantoic grafts) and place it in a bed of cotton in a 

 finger bowl. Orient the smaller or pointed end of the egg to the right, and tilt it 

 slightly below the horizontal so that the blastoderm will float slightly toward the blunt 

 (air-space) end. Leave the egg in this position for a few minutes. Holding the egg in 

 this exact position, remove it (without Jarring) to the candler and locate the blastoderm. 

 Mark its position with pencil on the ahell above. Eetum the egg, in exactly the same 

 position, to the cotton bed. 



With forceps apply a cotton swab, soaked in 95^ alcohol plus 1^ iodine (or chlorazene 

 solution), to the upper surface of the egg, including the area of the blastoderm. Wipe 

 dry with sterile cotton. Arrange the microscope lamp so that its light shines at an angle 

 onto the egg surface but have the light far enough away so that its heat is barely felt on 

 the back of the hand. Check the temperature of the light at the egg surface by a ther- 

 mometer. It should be leas than 105°F. 



Secure a previously sterilized moujating ring of non-toxic material such as glass, 

 celluloid, pliofilm, pyralin, or thin rubber washers with a maximum diameter of not more 

 than Ji/h of an inch. Place the ring on the shell over the region of the blastoderm and, 

 with a sharp and sterile needle, scratch the shell along the inner margin of the ring. 

 Replace the ring in 95^ alcohol in a covered Stender, and brush away any shell particles 

 with sterile cotton. A portion of the shell within the demarked area is now to be removed. 

 Eemember that all instruments used muat be sterile, the hands must be clean, and the 

 operator should avoid breathing into or allowing any dust to blow Into the egg while 

 opened. 



There are various tricks to removing the egg shell, gained largely through experience. 

 Aids to the removal, however, are a dental drill with circular disc; ordinary hack-saw 

 blade; ampoule saws; or merely a sharp scalpel. The ahell is to be sawed through, without 

 damage to the underlying shell membrane. Bectangular, triangular, square or circular open- 

 ings have been used. Probably the aimpleat procedure la to make a square opening by saw- 

 ing through the shell by a alight rotary motion of the saw, following the outer curvature 

 of the shell, on each of the four sides but working along parallel sides of the square for 

 the first two cuts. Avoid cutting through the shell membrane. Some workers leave the 

 fourth side as a sort of hinge, but it does not generally break straight so that cuts 

 along all edges are advised. Saw gently, and only through the shell, brushing away the 

 shell particles as they are dislodged. When the four sides are sawed through, make the 

 comer breaks with a needle or scalpel, and gently grasp the square piece of shell and 

 remove it, intact. 



Before invading the shell membrane aee that there are no shell fragments lying on it. 

 Moisten the shell membrane with one drop of sterile Locke's solution. Be-aterilize in- 

 struments And the margins of the shell opening, if it seems desirable. (The host egg is 

 left at this stage in chorio-allantoic grafting to await the preparation of the graft.) 

 With a sharp (sterile) needle puncture the center of the shell membrane, directing the 

 point of the needle under the membrane and away from you, and at right angles to the egg 

 axle. If the needle is sharp, it can be brought upward and thus be used as a knife to 

 cut a slit-like opening in the shell membrane. With sharp (sterile) scissors, cut away 

 the shell membrane to the margins of the shell opening. 



