EXPERIMENTAL FISH EMBRYOLOGY 395 



EXPERIMENTAL PROCEDURES WITH FISH EGGS AND EMBRYOS 



Fish material has been generally conaldered too difficult for experimental (oper-Mve) 

 procedures by graduate students of embryology. However, Nicholas, Oppenhelmer, 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. Oppenhelmer, the following procedures are outlined: (1) Vital stain- 

 ing of presumptive areas, (2) Excision and reconstitutlon, (5) Explantatlon and culturlng 

 in vitro, and (h) 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 nmat be 

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

 described by Nicholas (192?) 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 will be necessary to hold the egg In a Permoplast 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 embryo 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 egual 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 la 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 

 almost any osmotic condition. This may not be equally true of freshwater forms. 

 In general, for marine forms, Holtfreter's (Standard) Solution la used but in con- 

 centrations twice or three times the nonnal and In normal or double concentration 

 for freshwater forms. In general, therefore, the slightly hypertonic media are 

 advised. 



5. Asepsis : Prior to decapsulatlng the egg (or embryo) wash it in 8 to 10 changes of 

 large volvunes of sterile water, in a sterile finger bowl. After decapsulatlng, 

 transfer the embryo (gently) with wide-mouthed and sterile pipette through several 

 changes of sterile medium. All glassware and solutions should be autoclaved, in- 

 struments and pipette should be flamed before use. Steel instruments are used 

 exclusively. (Oppenhelmer has reduced mortality from the usual 50^ to almost 0^ 

 by using aseptic precautions - private commurdcatlon. ) 



