596 EXPERIMEHTAL FISH EMBRYOLOGY 



VITAL STAINING OF FISH EMBRYOS 



Procedure : 



1. Prepare vital-dye stained cellophane. Grubler'a Nile Blue Sulphate and Neutral 

 Red are to be used separately, and together. The cellophane (thinnest available) 

 should be soaked In 1^ aqueous solutions for a day or more, then dried on clean 

 white paper. The cellophane Is preferable to the agar because it remains in one 

 piece and can be removed. Store the red, blue, and the combination-stained cel- 

 lophane in clean marked envelopes until used. These vital dyes are not considered 

 to be toxic. 



2. Pass the eggs (in their sheila) through sterile media. Marine forms are to be 

 treated in double strength Holtfreter's and freshwater forms in normal Holt- 

 freter's (Standard) Solution. When tap or fresh water are used the stain pene- 

 trates too rapidly and may even damage some of the cells. 



3. Following the (Nicholas, 192?) technique described above, cut a minute window 

 through the chorion of the egg to be studied. 



h. With watchmaker's forceps insert a small piece of stained cellophane through the 

 window and maneuver it into the desired position with a fine hair loop. The 

 chorion will generally hold the cellophane in position, where it should be left 

 for from 15 to 1+5 minutes. The penetration and diffusion of the dye can be ob- 

 served directly. The dye penetration is best in hypertonic media. 



5. After. adequate staining has occurred, carefully remove the stained cellophane 

 with watchmaker's forceps, wash the egg with chorion in one change of sterile 

 medium and place it in a covered #2 Stender, containing the normal medium, and 

 at appropriate temperatures for that embryo. 



6. Observe during the next 56 to 1+8 hours, keeping the embryo at the lower limit of 

 viable temperatures if it is desired to prolong the early stages. The record 

 consists of a series of drawings of the changing position of the stained areas, 

 beginning Immediately after, applying the dye. 



SKETCHES OF VITALLY STAINED FISH EMBRYOS SHOWING MOBPHOGENETIC MOVEMENTS 



