METHOD OF CHORIO-ALLANTOIC GRAFTING 439 



THE PROCEDURE: 



1. Preparation of the hosts: Begin incubation of fertile host eggs, properly marked, 

 about 8 days before the operation. Turn the eggs twice daily. 



2. Preparation of the donors: Begin incubation of donors on the 5th or 6th day of 

 host incubation, so that the donors will be either 48 or 72 hours along at the time 

 the hosts are 8 days incubated. Mark the eggs, and rotate twice daily. 



3. Preparation of the host for the graft: (Work fast under the most sterile condi- 

 tions) 



a. Candle the host eggs, select and mark the regions most suitable for a graft. 

 The forked junction of large blood vessels at some distance from the embryo 

 should be indicated by a pencil drawing on the shell. 



b. Prepare a window in the shell, under aseptic conditions (see directions for 

 this above). Remove the shell in one piece. Moisten the shell membrane 

 with several drops of sterile Locke's or chick Ringer's solution. This is 

 necessary in order to avoid injury to the underlying and sometimes adherent 

 chorio-allantoic membrane, and consequent rupturing of the blood vessels. 

 Cover the aperture with a sterile cover glass, or replace the shell, and re- 

 turn the (host) egg to the incubator while preparing the graft. 



4. Preparation of the t ransplant: (Eye - 48 hours, limb - 72 or more hours) 



a. Candle the donor to determine the approximate age. Crack the underside of 

 the shell on the side of a finger bowl containing about 100 cc. of sterile chick 

 Ringer's solution. When the blastoderm has moved around the dorsal posi- 

 tion, quickly excise it, separate it from the vitelline membrane, and transfer 

 it in a wide-mouthed pipette to a sterile watchglass under a dissection micro- 

 scope. Confirm the age and stage of development. 



b. Ouickly, and with sterile needles and watchmaker's forceps, excise the anlage 

 to be studied and transfer it in a drop of the sterile medium to the chorio- 

 allantois of the prepared host. Replace the shell, and seal it with a ring of 

 melted paraffin. Replace the host egg in the incubator without shifting its 

 gravitational axis, and leave it unmoved for 6 to 8 hours. After this interval, 

 treat the egg as any regularly incubated egg. 



The eye: The eye graft may be taken from any stage after about the 33 

 hour stage. The retina, pigmented layer, and lens may be 

 easily distinguished in the graft, upon recovery. It is, of course, 

 possible to study the independent differentiation of parts of the 

 early optic vesicle, cup, etc. Final study may be made after 

 clearing with oil of wintergreen, when the pigment and retina 

 show up well, and then after sectioning and staining. 



The limb bud : Under the dissecting microscope, locate the wing and leg 

 buds (72 hours or older). Make transverse cuts with a sharp 

 needle through the embryo, one through the neck level and the 

 other between the wing and the leg buds. Then dissect out each 

 appendage bud in the following manner: Cut parallel to the body 

 at the base of the bud, but include some of the body somites. 

 Then cut (at right angles to the first cut) at the anterior and the 

 posterior limits of the bud. Finally make the cut parallel to the 

 first, beyond the outer limit of the bud, thus excising the bud in 

 a rectangular piece of tissue. Remove all yolk and loose tissue 

 adherent to the bud. A single donor may provide several ap- 

 pendage buds, and occasionally a single host may survive the 

 implantation of several buds. 



c. Make a complete record, including sketches, of the condition of the chorio- 

 allantois and the donor (explant) at the time of the operation. 



