EXPERIMENTAL CHICK EMBRYOLOGY ^ 



t Quickly, and with sterile needles and watchmaker's forceps, excise the 

 anlage to he 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. Beplace the host egg in the incubator 

 without shifting its gravitational axis, and leave it unmoved for 6 to « 

 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 35 



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 dis- 

 sect 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 rectangu- 

 lar piece of tissue, Eemove all yolk and loose tissue ad- 

 herent 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. 



5. Becovery of the graft : , . v v ^ir, 



a. The graft must be removed before the host extra- embryonic membranes begin 



to diy up. This is generally by the l8th day of incubation, or about 9 

 to 10 days after the transplantation of the graft is made. 



b. Candle the host to attempt to locate the graft. This is generally diffi- 

 cult because of the opacity of the host. However, if the graft has 

 "taken" it will be found close to the original window. Make a cut 

 through the shell about 1 centimeter outside of the original window, and 

 remove the shell carefully, without rupturing the underlying chorio- 

 allantois. Examine the underside of the shell to see if the explant may 

 be adherent to it. The graft will generally appear as a fluid-filled 

 vesicle, opaque, and without discernible structure. Particularly in the 

 case of the appendage anlage, the detailed structure will not be seen 

 until the tissues are histologically cleared. 



c. Use the Lundvall technique (see above) for quick staining of cartilage, 

 etc. of differentiated limbs. The eyes should be sectioned after normal 

 fixation (Bouin or Klinenberg' a) and stained with Conklin' s Haematoxylin. 



6. Other e mbryonic areas to be tested by chorj o-allantolc grafting: It is sugges- 

 ted that the following additional structures be given the opportunity to develop 

 on the chorio-allantois. 



a. Anterior half of the primitive streak, Including Eensen s node. 



b. Neural crests and nerve cord, from embryos with 5 to 10 somites. 



c. Somite blocks. 



d. Heart anlage of h2 hour stage. 



e. The entire blastoderm of primitive streak stage. 



(Note: For further instructions the student ^^is advised to consult Hamburger's: 

 "A Manual of Experimental Embryology" p. 1^J>-) 



