47. EXPERIMENTAL PROCEDURES 

 WITH FISH EGGS AND EMBRYOS 



Fish material has been generally considered too difficult for experimental (operative) 

 procedures by graduate students of embryology. However, Nicholas, Oppenheimer, 

 Luther, Eakin and others have demonstrated that the fish egg and embryo can be studied 

 much in the manner of the classical experiments with amphibian eggs and embryos. 

 Through the very generous help of Dr. Oppenheimer, the following procedures are out- 

 lined: (1) Vital staining of presumptive areas, (2) Excision and reconstitution, (3) Ex- 

 plantation and culturing in vitro, and (4) Transplantation. 



In all of this work there are three very important considerations. 



1. Preparation of the egg or embryo: The fish egg is provided with a tough chorion, 

 or outer shell. This must be removed for most experiments, or a window must 

 be provided through which the vital dye or a graft may be inserted. The method 

 is described by Nicholas (1927) as follows: 



a. Mortality is greatest during the cleavage stages. Shell removal after the 

 embryo has formed a distinct cap on the upper surface of the yolk will be the 

 more successful, and should be attempted first. 



b. It may help to hold the egg in a Permoplast or other soft depression. Use 

 very sharp-pointed scissors (iridectomy) and insert one blade between the 

 egg and its shell so that its point is at a tangent to the egg. Enter the shell 

 to the right of the embryo above the omphalomesenteric vein, but forward. 



If one point of the scissors is longer than the other, this may be used for the 

 puncture and invasion of the shell. If the invasion is properly made, the em- 

 bryo should not be injured. A small amount of fluid will escape from around 

 the embryo. 



c. Avoid any pressure against the egg or yolk sac, either of which will rupture 

 with the slightest pressure. The chorion is relatively so tough that it will 

 hold the egg to the scissors, so that the latter may now be rotated into such a 

 position that a cut may be made. Assist in the rotation of the egg with a hair 

 loop or spear-point needle. When properly oriented on the scissors, cut 

 through the chorion and continue the cut around the egg (or embryo) dividing 

 the shell, as nearly as possible, into equal halves. 



d. Discard those eggs or embryos which have been ruptured or are in any way 

 damaged. (If prepared, some parts of such damaged embryos may be used 

 for in vitro experiments described below. ) 



e. The removed eggs and embryos of Fundulus will develop in distilled, fresh, 

 or sea water providing the yolk membrane is intact. It is probably best to 

 use filtered water of the normal environment for eggs of the various species 

 used. 



2. Operating medium: It has just been stated that the Fundulus embryo can survive 



a wide range of osmotic conditions. (See Kao et al 1954). This may not be equally 

 true of freshwater forms. In general, for marine forms, Holtfreter's (Standard) 

 Solution is used but in concentrations twice or three times the normal and in nor- 

 mal or double concentration for freshwater forms. In general, therefore, the 

 slightly hypertonic media are advised. Trinkaus ('56) uses double -strength bi- 

 carbonate-free Holtfreter' solution with phosphate buffer to which egg yolk from 

 Fundulus eggs is added. 



3. Asepsis : Prior to decapsulating the egg (or embryo) wash it in 8 to 1 changes of 

 large volumes of sterile water, in a sterile finger bowl. After decapsulating, 



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