EXPERIMENTAL EQUIPMENT AND PROCEDURES 21 



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 oper- 

 ation 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 with 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 watch- 

 maker'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. 



Remove frog egg jelly with 10% Chlorox (see Shumway: 1942 Anat. Rec. 83:309) or by 

 brief Trypsinization. 



It has been reported that ultra-violet light will dissolve off the jelly capsules of eggs but 

 it is very likely that the same irradiation will damage the egg or embryo. Chemical re- 

 moval of the jelly, after fixation, may be achieved by placing the embryo and capsule 

 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 

 3-4 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 is subsequently hardened in 

 alcohol. 



KILLING AND FIXING PROCESSES 



The fixation method of choice depends upon the end results desired. It has recently been 

 discovered that decapsulated amphibian eggs can be briefly boiled to coagulate them, 

 prior to normal chemical fixation. For cytological preparations the corrosive-acetic or 

 chrom-acetic fixations are best; for early embryos with much yolk. Smith's fluid is rec- 

 ommended; and for later embryos and tissues in general, alcoholic Bouin or Bouin-dioxan 

 mixtures are suggested. Fixation may be speeded up by the addition of 1% Turgitol (Car- 

 bide & Carbon Company, N. Y. C. ) which reduces the surface tension of the fixative. 



a. Smith's fluid : This fixative is made up of two solutions which, when brought to- 

 gether, rapidly deteriorate. It is therefore necessary to mix them just before 

 use and to never use it when it has become discolored (dark). The fixative 

 should be used for 12-24 hours, followed by thorough washing (12-24 hours) in 

 running tap water. If the material is discolored, follow bleaching directions be- 

 low (bichromate bleach). Tissues or embryos fixed in Smith's may be perma- 

 nently preserved in 4% formalin directly after washing. This fixative is good for 

 yolk-laden eggs and will give a minimum of distortion. 



b. Bouin' s fluid : The most universally satisfactory fixative known, made up in 

 aqueous or alcoholic solutions. Fixation may be as short as 1 hour (tail tips); 

 24 hours for whole embryos; or much longer if it is inconvenient to change be- 

 cause Bouin' s is a preservative as well as a fixative. The yellow of the picric 

 acid is best removed by adding about 2% NH4OH to the 70% alcohol when dehy- 

 drating, changing the solution every hour until the color is entirely gone. Lith- 

 ium carbonate acts more slowly and may leave crystals, while the ammonia will 

 eventually all evaporate. If chromophils are to be studied, such tissues must 

 subseauently be properly neutralized by long exposure to pure 70% alcohol. 



