MORPHOCENETIC MOVEMENTS AS 

 DETERMINED BY VITAL STAINING 



PURPOSE: To stain the early embiyo with vital dyes hy means of which movements of various 

 cell areas can be followed to their final location in organogenesis. 



MATERIALS : 



Biological : Blastula stages of Anura and Urodela. 



Technical : Powdered or crystalline agar, cellophane, sind vital dyes (Nile blue sul- 

 phate and neutral red, preferably Gruebler's). 



METHOD : 



Precautions : 



a. These vital dyes are water-soluble. It is therefore wise to soak the pieces of 

 stained agar or cellophane in distilled water briefly before using them for stain- 

 ing embryos, to remove excess dye. 



b. The smallest pieces of stained medium should be used. These can be prepared in 

 the dry state by cutting into small beads beneath a dissection microscope. 



c. While being stained the embryo should be as dry as is compatible with the main- 

 tenance of normal conditions. 



d. All membranes except the vitelline membrane must be removed. The vitelline 

 membrane can be punctured if it is otherwise difficult to hold the embryo in 

 position. 



e. The Permoplast or soft paraffin base must be rigid enough to hold the egg in 

 place for k'^ to 6o minutes without applying abnormal pressure. 



Control : This is an exploratory and qualitative type of experiment so that controls 

 are not possible. Localized injury of cell areas could be used to impede such cell 

 movements as seem evident from the vital staining observations. 



Procedure : 



PREPARATION OF STAINING MEDIUM 



Bring 100 cc. of distilled water to a boil in each of 2 Erlenmeyer flasks. To each, 

 add 2 grams of pure powdered or shredded agar, and dissolve congjletely by further boiling. 

 Avoid burning by constantly stirring with a glass rod. 



To one flask add 1 gram of Nile blue sulphate (Gruebler's) and to the other add 1 gram 

 of Neutral Red (Gruebler's). Heat gently until the solutions are homogeneous. 



Tilt some clean lantern slide covers (or other glass plates) slightly on paper towel- 

 ling, and pour the warm and stained agar mixture onto the plates so that there is a thin 

 and even layer. Allow the agar to dry thoroughly in a dust- free environment, and then 

 wrap the plates in white typewriter paper and label for future use. (See Vogt, 1925 •) 



Generally the thin layer of stained and dried agar can be chipped off of the glass 

 plate with a scalpel, but if this proves difficult, simply add a drop of distilled water 

 to the edge of the agar film and allow it to swell, after which it is possible to cut out 

 a small strip of stained agar. This can be further subdivided with sharp scissors. 



A recent modification of this original procedure is to use the thinnest sheets of 

 cellophane or pliofilm which take up the stain and can be cut Into small pellets. Such 

 pieces of stained cellophane can be kept in envelopes until needed. 



The staining dishes are generally Syracuse dishes provided with Permoplast or soft 

 paraffin bases. Permoplast is softer and easier to mould than paraffin, but it Is apt to 

 crumble when left in water for aqy length of time. It is well to prepare 10 to 12 dishes 

 well in advance of these experiments, each provided with depressions of various sizes in 

 anticipation of various sized embryos. Depressions cein be made easily in a paraffin base 

 by means of a warmed ball-tip, while the dish Is partially filled with water. 



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