EXPERIMENTAL CHICK EMBRYOLOGY 1^1 



10. After 2k hours, crack the underside of the shell on the side of a finger "bowl 

 ahout 2/3 full of Locke's (or saline) solution, cut out the entire blastoderm, 

 transfer it with a wide-mouthed pipette to a watchglass of saline solution, and 

 examine by reflected and transmitted light on a warming stage, to determine the 

 changes in the size and positions of the colored areas. 



Variations in the procedure : 18-20 hour incubation stage. 



1. Attempt to stain Hensen's Node specifically, and 2l4- hours later excise the 

 blastoderm, split the embryo lengthwise through the neural tube, invert the 

 blastoderm, and locate the stain. If the Node was stained, the notochord and 

 ventral neural tube should be stained. 



2. Stain the Primitive Streak posterior to Hensen's Node, and 2k hours later ex- 

 cise and examine the blastoderm for the position of the stain. 



3. Stain a spot directly anterior to Hensen's Node with Nile Blue Sulphate and a 

 second spot to either side of the midline, at the same level as the Nile Blue, 

 but use Neutral Red. In this way the movements of the Pre-Nodal areas may be 

 determined 2k hours later. 



k. Stain the Primitive Streak stage with several spots in the two colors, excise 

 quickly after 2k hours, and fix in an attempt to preserve the vital dye in posi- 

 tion (see section on Morphogenetic Movements for specific directions, based on 

 Detwiler's paper, for preserving vital dyes). 



THE METHOD OF CABBON- MARKING IN VITRO : (Spratt, I9U7, I9I48) 



Ejy this method the entire blastoderm of an 18 to 20 hour incubated chick embryo is 

 excised, placed on albumen medium and c\iltivated in vitro for 2k or more hours (at 105°F, ) 

 and the morphogenetic movements are determined by movement of adherent particles of blood 

 charcoal. 



1. To prepare the albumen medium separate the yolk 

 from the albumen of one unincubated egg and add 

 the albumen to 50 cc. of chick Binger's solu- , 

 tlon (0.9^ NaCl, 0.0^2^ KCl, and 0.02'+^ CaCl2 

 made up in glass distilled water) contained in 

 a 500 cc. Erlenmeyer flask. Stopper and shake 

 the flask vigorously for 1 minute. 



2. Excise the blastoderm from an I8 to 20 hour 

 incubated egg and place it in chick Binger's Carbon-marking In vitro: 

 at 103°?., in a watchglass, and remove all ex- chick Olastoderm Primi- 

 oeas yolk. tlve Streak Stage. 



5. With wide-mouthed pipette, remove the blastoderm in minimum of medium, and 

 transfer it to the surface of the (above) albumen-Blnger' 3 in a watchglass at 

 103°F. (on a warming stage). With mlcropipette, remove all excess medium from 

 the surface of the blastoderm so that it floats freely on the surface film of 

 the culture medium. 



k. Place carbon (blood charcoal) particles on the Hensen's Node, directly in front 

 of it, and at one or more levels of the Primitive Streak (see figures above). 

 Indicate by parallel charcoal marks on the medium exactly where these particles 

 are placed. These marks to be used as reference marks later. Watchmaker's 

 forceps may be used for this marking. The forceps may be dipped into the char- 

 coal, shaken of all excess particles, and then touched to the relatively dry 

 upper surface of the blastoderm. There is sufficient moisture to provide a 

 sticky surface so that the carbon particles will come off the forceps very 

 readily, and will remain adherent to the blastoderm. 



• 5 . After 2k to k8 hours examine the blastoderm, cultured in vitro, for any change 

 in the position of the carbon particles. It will, of course, be necessary (as 

 always) to make sketches at the beginning and at the termination of the experi- 

 ment. 



