25. MORPHOGENETIC MOVEMENTS AS 

 DETERMINED BY VITAL STAINING 



PURPOSE: To stain the early embryo with vital dyes by means of which movements of 

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

 frontispiece) 



MATERIALS: 



Biological: Blastula stages of Anura and Urodela. 



Technical : Powdered or crystalline agar, cellophane, and vital dyes (Nile blue 

 sulphate and neutral red, preferably Gruebler's). 



METHOD: 



Precautions : 



1. 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 

 staining embryos, to remove excess dye. 



2. The smallest pieces of stained medium should be used. These can be pre- 

 pared in the dry state by cutting into small beads beneath a dissection mi- 

 croscope. 



3. While being stained the embryo should be as dry as is compatible with the 

 maintenance of normal conditions. 



4. All membranes except the vitelline membrane must be removed. The vitel- 

 line membrane can be punctured if it is otherwise difficult to hold the em- 

 bryo in position. 



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

 in place for 45 to 60 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 completely 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 towelling, 

 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. 



* Culture media contain salts which may precipitate the dyes. 



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