VITAL STAINING AND MORPHOGENET I C MOVEMENTS 219 



STAINING PROCEDURE* 



It is not alwaya possible to stain an exact area with a particular dye. The usual 

 procedure is to place various small vitally stained pellets within the wall of a depres- 

 sion in the Permoplast (or paraffin) and in the appropriate medium and then to fit the 

 egg or embiyo into the depression, moulding the material to hold the embryo firmly in 

 place. It is not particularly important to use any special configuration as long as a 

 record is made, immediately after staining, of the exact distribution of the stained areas 

 on the egg or embryo. 



Bemove the Jelly membranes from the egg, or embryo, and place it in the depression. 

 Gravity will orient the egg so that the vegetal pole takes most of the stain in any de- 

 pression. This position can be varied by holding the egg in position with a hair loop 

 while building up a closely confining cover of the Permoplast (or paraffin) with a ball 

 tip. If the colored pellets are properly alternated within the depression, and properly 

 spaced, the transferred marks on the embryo will not become confluent. Cover the egg or 

 embryo with Standard Medium and leave undisturbed for h^ to 6o minutes at the laboratory 

 temperature. Gently uncover the embryo and shake it out of the depression. 



STAGES AND AREAS TO BE STAINED 



1. Grey Crescent : Within 20 to 30 minutes after insemination a grey crescent will 

 appear between the animal and the vegetal hemispheres of the frog's egg, the more 

 pronounced the longer the eggs are aged in the uterus. Using but a single (Neu- 

 tral Bed) pellet, attempt to orient the grey crescent region adjacent to the dye 

 and allow it to stain for half an hour. 



A second method of staining -the grey crescent is to utilize the Jelly of the 

 egg by spreading it gently onto a small square of filter paper Just enough to hold 

 the egg firmly in place. Take a stiff piece of dry and colored agar pellet 

 held with forceps and, using it as a pencil, mark the region of the grey crescent 

 by applying and holding the dye against the egg for as long a period as possible. 

 (Do not overstain. ) 



Study the movement of the stained grey crescent, and determine Its relation 

 to the first cleavage furrow and to the subsequent position of the initial involu- 

 tion of gastrulation. 



2. Blastula Stage : At about the 614--128 cell stage (stage #8 Sana) apply h stain 

 marks of two colors around the germ ring, alternating the colors. These spots 

 should appear circumferentlally placed. On other blastulae of similar stage, ap- 

 ply a line of alternating colors from the germ ring of one side, through the dorsal 

 hemisphere, to the germ ring of the other side. 



These stained areas should not only move but should change shape, depending 

 upon their location in relation to the morphogenetlc movements of gastrulation. 

 (See Goerttler, I925 and Vogt, 1925.) 



3. Gastrula Stage : At the first indication of gastrulation (stage #10) mark the dor- 

 sal, the lateral, and the (presumptive) ventral lip regions of the future blasto- 

 pore. If you are successful in this work, attempt to repeat Goerttler' 3 work of 

 staining a line of spots both dorsal and ventral to the Initial involution of the 

 blastopore. 



h. Yolk Plug Stage : (stage #11 Sana or Amblyatoma) 



a. Presumptive notochord : Carefully apply a stain to the medium upper lip of 

 the early blastopore. When this embryo has reached the tall-bud stage 

 (Bana, stage #1? or #l8) dissect it with needles to locate the position of 

 the iftvaginated colored cells. 



* Either Anuran or Urodele eggs (or embryos) may be used, but the latter are preferred, 

 because of the reduced natural pigmentation. 



