53. METHODS FOR OBSERVING THE 

 DEVELOPMENT OF THE CHICK EMBRYO 



The hen's egg is fertilized at the upper end of the oviduct so that by the time it is layed 

 the embryo has reached the stage of gastrulation, at least. The blastoderm of such an 

 egg shows no visible structures at this time but after 18 hours of incubation the primitive 

 streak may be discerned. Candling will not generally indicate any development before 

 the 33 hour stage. 



It is now possible to replace a portion of the shell with a cover-glass window through 

 which development can be observed from day to day. A second method is to remove the 

 shell 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 perfectly 

 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 33 hours of incubation (7 or 8 day embryos may be similarly 

 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 shell above. Return the egg, in exactly the same position, to the cot- 

 ton 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 less than 103° F. 



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

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

 than 3/4 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. Re- 

 place 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. 

 Remember that all instruments used must 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 di-ill with circular disc; ordinary hack-saw 

 blade; ampoule saws; or merely a sharp scalpel. The shell is to be sawed through, with- 

 out damage to the underlying shell membrane. Rectangular, triangular, square or cir- 

 cular openings have been used. Probably the simplest procedure is to make a square 



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