l6 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



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 corroeive-acetic or 

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

 mended; and for later embryos and tissues in general, Bouin or Bouin-dioxan mixtures are 

 suggested. Fixation may be speeded up by the addition of I'jt Turgitol (Carbide & 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 



together, 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 12-2U hours, followed by thorough washing (12-21+ 

 hours) in running water. If the material is discolored, follow bleaching 

 directions below (bichromate bleach). Tissues or embryos fixed in Smith's 

 may be permanently preserved in k$ 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); 2k hours for whole embryos; or much longer if it is inconvenient to 

 change because Bouin' s is a preservative as well as a fixative. The yellow 

 of the picric acid is best removed by adding about 2']^ NHj^OH to the 705t alcohol 

 when dehydrating, changing the solution every hour until the color is entire- 

 ly gone. Lithium carbonate acts more slowly and may leave crystals, while the 

 ammonia will eventually all evaporate. If chromophlls are to be studied, such 

 tissues must subsequently be properly neutralized by long exposure to pure 

 70^ alcohol. 



c. Bouin- Dioxan : This is a rapid and entirely satisfactory method of fixation and 



dehydration, the proportions being half-ln-half , and the fixation time 12-2lv 

 hours. The mixture prevents shrinkage and hardening that often attends the 

 use of other reagents. Other ratios used are Bouin-2 parts, Dloxan-1 part. 

 If it is necessary to decolorize, transfer directly to ammoniated 70^ alcohol, 

 later to pure dioxan for dehydration. 



d. Mlchaelis' fluid : Fixation for 8 hours, after which Jelly must be removed be- 



fore transferring to alcohols or dioxan for dehydration. 



e. Gatenby's fluid : Used in ratio of about 2 cc. per egg for 12-21+ hours during 



which time the Jelly capsules will fall off the eggs. 



f. Gllson's fluid : Short fixation (15-^5 minutes) for cytological studies, recom- 



mended particularly for oogenesis. 



g. Chrom-acetic fixative : Excellent for cytological studies of an^hibian egg and 



early embryos. 



h. Acetic-alcohol : Fix tissues for 8 hours, transfer directly to absolute alcohol. 



1. Formalin fixatives : Gross fixation of embryos or tadpoles which are not to be 

 sectioned may be accomplished in k<f, formalin, preferably made up in the same 

 medium used for the living organisms. Prokofleva, 1955 - Cytologla 6:ll+8 

 recommends ^Cff> formalin 8 pte.; 5^ chromic acid 2 pts. as fixative for chromo- 

 some sti-ucture of Anuran eggs. For Urodele larvae he recommends 10^ formalin 

 7 pts., and 1^ chromic acid 5 pts. 



J. The following procedure has proven to be very good for amphibian eggs. (See 

 Goldsmith, I929. Trans. Am. Mlcr. Soc. U8:2l6.) 

 1. Fix in Goldsmith's fluid: 



Chromic acid 1^ 15 parts 



K Bichromate 2^ h parte 



Glacial acetic 1 part 



(Fix small pieces 2 hours, large pieces 2k hours) 



