EXPERIMENTAL EQUIPMENT AND PROCEDURES 15 



HISTOLOGICAL HINTS FOR EGGS AND EMBRYOS 

 INTRODUCTION 



Satisfactory slides of embryonic material are difficult to ottain. This Is true of 

 toth chorionated and yolk-laden eggs as well as the early stages of development. The es- 

 sential steps are the removal of membranes, proper fixation, incomplete dehydration, and 

 short embedding in wax-paraffin mixtures. 



ANESTHETICS 



In general, anesthetics are not necessary when eggs or embryos are to be fixad im- 

 mediately. Occasionally it is desirable to fix an embryo in a certain position, or to 

 reduce body movements during fixation, when anesthesia is in order. 



a. 1^-222 : This imported poison is the most satiafactoi-y anesthetic available, 



used in 1/5OOO concentration either in Standard ( Holtfreter' s) Solution, 

 Spring Water, or Locke's solution (for chick embryos). The embryos are 

 immobilized in about 1 minute and, after retiim to normal medium, recover 

 in about 10 minutes without ill effects. Must be used fresh. 



b. Chloretone : Generally 0.5^ concentration in whatever medium the embryo is 



accustomed, will give slow but quite satisfactory anesthesia. 



c. Magnesium sulphate : (Epsom salts) Simply drop a few crystals into small volume 



of water containing the embryo and await immobilization. 



d. Cyanide : KCN l/lOOO In salt solutions acts as an anesthetic. 



e. Ether and chloroform : These volatile anesthetics are for air breathing forma; 



hence will find little use with embryonic material. 



f . Chilling : Embryos are rapidly retarded in all of their activities by adding 



to their media some cracked ice. Such embryos may be operated upon and will 

 recover, upon return to normal temperatures, without ill effects. It may 

 take 10-15 minutes to adequately stupefy the organiama. 



REMOVAL OF JELLY CAPSULES 



It is easier and allows better fixation if the Jelly membranes are removed from eggs 

 and embryos prior to the killing process. 



The Urodele egg is provided with a distinct Jelly capsule which may be punctured with 

 needles or sharp watchmaker's forceps, and pulled off of the egg. If a tear is made by 

 means of a pair of forceps, the embryo will usually "shell out". If the capsule is placed 

 on a piece of paper towelling, filter or blotting paper, to which it will adhere, this 

 operation may be facilitated. 



The Anuran egg generally has looser but more adherent Jelly capsules. This Jelly may 

 be removed by cutting single eggs away from the mass and placing them- on coarse paper and 

 rolling them along the flat side of a scalpel until the bulk of the Jelly rolls off onto 

 the paper. A better method Is to pierce the Jelly with one prong of the #5 watchmaker's 

 forceps, slide a prong of a second pair of forceps along the first, and, with an outward 

 motion cut through the Jelly. It may then be peeled off. 



It has been reported that ultra-violet light will dissolve off the Jelly capsules of 

 eggs but it la very likely that the same irradiation will damage the egg or embryo. Chem- 

 ical removal of the Jelly, after fixation, may be achieved by placing the embryo and cap- 

 sule into 10^ sodium hypochlorite or chlorox diluted with 5-6 volumes of water. The Jelly 

 can be shaken off within a few minutes. Javelle water (potassium hypochlorite) diluted 

 3-1+ times may also be used. Following fixation in Gilson's fluid, the Jelly hardens suf- 

 ficiently so that it may be picked off the embryo which la subsequently hardened in 

 alcohol. 



