VITAL STAINING AND MORPHOGENETIC MOVEMENTS 



205 



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 any 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 can be made easily in a paraffin 

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



STAINING PROCEDURE* 



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

 dure is to place various small vitally stained pellets within the wall of a depression in 

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

 embryo 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. 



Remove 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 depres- 

 sion. 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 em- 

 bryo with Standard Medium and leave undisturbed for 45 to 60 minutes at the laboratory 

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



Animal pole 



Sperm entrance 

 point 



Vtntral 



side \ 10°^ 



Z0'\ 



Ventral blastopore up 



Limit of involution 



Dorsal blastopore lip 

 Gray crescent 

 Vegetal pole 

 Surface changes during gastrulation. (Modified from Pasteels.) 



STAGES AND AREAS TO BE STAINED 



Grey Crescent : Within 20 to 30 minutes after insemination a grey crescent will ap- 

 pear 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 (Neutral 

 Red) pellet, attempt to orient the grey crescent region adjacent to the dye and allow 

 it to stain for half an hour. 



* Either Anuran or Urodele eggs (or embryos) may be used, but the latter are preferred, because of the reduced natural pigmenta- 

 tion. 



