METHODS FOR OBSERVING THE DEVELOPMENT 419 



opening by sawing through the shell by a slight 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 br.eak straight 

 so that cuts along all edges are advised. Saw gently, and only through the shell, brush- 

 ing away the shell particles as they are dislodged. When the four sides are sawed 

 through, make the corner breaks with a needle or scalpel, and gently grasp the square 

 piece of shell and remove it, intact. 



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

 Moisten the shell membrane with one drop of sterile Locke's solution. Re-sterilize 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 axis. 

 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. 



Remove the mounting ring from the 95% alcohol, let it air-dry briefly, dip it into 45 C. 

 melted paraffin, and place it on the egg so that it encircles the shell opening. With a 

 small water-color paint brush paint melted paraffin onto the outer margins of the mount- 

 ing ring so that it is thoroughly sealed to the shell and no air can pass beneath. Do not 

 allow any paraffin to get into the egg. 



Secure a circular coverslip, previously cleaned, and holding it with forceps pass it 

 through a gas flame, exposing both surfaces. The coverslip should just fit the mounting 

 ring. While still warm, bring the coverslip into position on the mounting ring and gently 

 press it into place. The paraffin adherent to the mounting ring should melt and fasten 

 the coverslip tightly to it. Paint a ring of paraffin on the outer edge of the coverslip, 

 further sealing it to the egg shell. The entire operation consists simply of providing a 

 sealed window in the place of a limited amount of egg shell. Return the egg to the incu- 

 bator, in the same position, for the first 24 hours. Thereafter the egg may be rotated 

 somewhat, but the embryo and its membranes should be kept away from the window. 



Two modifications of the above procedure have been practiced, but neither is necessary. 

 One is to puncture the air space so that it will be deflated and the embryo will be further 

 depressed away from the upper shell membrane. If this is done, simply cover the punc- 

 ture with paraffin or scotch tape. The second modification consists of adding egg-albumen 

 from a second egg, to fill up the space between the embryo and the glass window. While 

 albumen is bacteriolytic, this practice is ill advised, because it tends to add to the in- 

 fection hazard through handling, and it generally clouds up the window. 



Of course, a glass window is not the only useful type. Pliofilm, cellophane, and even 

 Scotch tape have been used. If there is apt to be considerable delay between the prepara- 

 tion of the host, and the graft tissues, it is advisable to protect the embryo with a tem- 

 porary Scotch tape covering of the shell opening. Square and round coverslips may be 

 used without a mounting ring, but paraffin sealing is the more difficult. 



The embryo may be examined from time to time but it must be remembered that with 

 each removal of the shell cap there is opportunity for bacterial infection and increased 

 evaporation. Embryos may be carried through to hatching. 



