426 



MORPHCXJENETIC MOVEMENTS 



10. 



Make a sketch of the blastoderm, any identifiable structures of the embryo, 

 and the positions of the colored areas. If a temporary window is placed over 

 the shell opening (coverslip) the sketch may be made during a brief candling. 



With melted, and soft paraffin seal a circular coverglass onto the egg sur- 

 face, over the window. This need not be made with the care of transplanta- 

 tions for the duration of the experiment is short. 



After 24, hours, crack the underside of the shell on the side of a finger bowl 

 about 2/3 f^ll 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 de- 

 termine 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 24 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 24 hours later 

 excise 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 24 hours later. 



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

 quickly after 24 hours, and fix in an attempt to preserve the vital dye in 

 position (see section on Morphogenetic Movements for specific directions, 

 based on Detwiler's paper, for preserving vital dyes). 



THE METHOD OF CARBON -MARKING IN VITRO: (Spratt, 1947, 1948) 



By this method the entire blastoderm of an 18 to 20 hour incubated chick embryo is ex- 

 cised, placed on albumen medium and cultivated in vitro for 24 or more hours (at 103 F. ) 

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

 blood charcoal. 



1, 



2. 



To prepare the albumen medium separate the yolk 

 from the albumen of one unincubated egg and add the 

 albumen to 50 cc. of chick Ringer's solution (0.9% 

 NaCl, 0. 042% KCl, and 0. 024% CaCl2 made up in 

 glass distilled water) contained in a 500 cc. Erlen- 

 meyer flask. Stopper and shake the flask vigor- 

 ously for 1 minute. 



Excise the blastoderm from an 18 to 20 hour in- 

 cubated egg and place it in chick Ringer's at 103 F. 

 in a watchglass, and remove all excess yolk. 



3. With wide-mouthed pipette, remove the blastoderm 

 in minimum of medium, and transfer it to the sur- 

 face of the (above) albumen-Ringer's in a watch- 

 glass at 103 F. (on a warming stage). With micro- 

 pipette, remove all excess medium from the surface 

 of the blastoderm so that it floats freely on the sur- 

 face film of the culture medium. 



Caibon-marking in vitro: 

 Chick blastoderm Primitive 

 Streak Stage. 



