22 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



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

 dehydration, the proportions being half-in-half, and the fixation time 12-24 hours. 

 The mixture prevents shrinkage and hardening that often attends the use of other 

 reagents. Other ratios used are Bouin-2 parts, Dioxan-1 part. If it is necessary 

 to decolorize, transfer directly to ammoniated 70% alcohol, later to pure dioxan 

 for dehydration. 



d. Michaelis' fluid : Fixation for 8 hours, after which jelly must be rernoved before 

 transferring to alcohols or dioxan for dehydration. 



e. Gatenby's fluid : Used in ratio of about 2 cc. per egg for 12-24 hours during 

 which time the jelly capsules will fall off the eggs. 



f. Gilson' s fluid : Short fixation (15-45 minutes) for cytological studies, recom- 

 mended particularly for oogenesis. 



g. Chroni-acetic fixative : Excellent for cytological studies of amphibian egg and 

 early embryos. 



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



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

 sectioned may be accomplished in 4% formalin, preferably made up in the same 

 medium used for the living organisms. Prokofieva, 1935 - Cytologia 6:148 rec- 

 ommends 50% formalin 8 pts. ; 5% chromic acid 2 pts. as fixative for chromo- 

 some structure of Anuran eggs. For Urodele larvae he recommends 10% forma- 

 lin 7 pts. , and 1% chromic acid 3 pts. 



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

 Goldsmith, 1929. Trans. Am. Micr. Soc. 48:216.) 



(1) Fix in Goldsmith's fluid: 



Chromic acid 1% 15 parts 



K Bichromate 2% 4 parts 



Glacial acetic 1 part 



(Fix small pieces 2 hours, large pieces 24 hours) 



(2) If eggs are left in fixative 24-48 hours, the jelly will be removed. 



(3) Wash 24 hours in running water. 



(4) Carry to 70% alcohol through gradual changes. 



(5) Change to 1 part aniline oil, and 2 parts 70% alcohol for 2 to 6 hours. 



(6) Change to 2 parts aniline oil, and 1 part 95% alcohol for 2 to 6 hours. 



(7) Change to pure aniline oil until clear - 1 or more hours. 



(8) Change to 50% aniline oil plus 50% toluene for 1 to 6 hours. 



(9) Change to 100% toluene, for 1 to 3 hours. 



(10) Place in 100% toluene for 1 to 3 hours. 



(11) Place in saturated 53° paraffin in toluene for 1 to 4 hours. 



(12) Place in 53° paraffin for 3 to 4 hours, at 55°C. 



(13) Embed in 53° paraffin. 



k. The following procedure has been used with considerable success by Dr. C. L.. 

 Parmenter in studying the cytology of the amphibian egg. 



(1) Fix for 24 hours in Smith's fluid. 



(2) Preserve eggs in jelly in 5% formalin. 



(3) To remove jelly: Use 20% solution chlorox in distilled water for 3 to 4 min- 

 utes. Watch and stop before the cortex is injured. 



(4) Rinse thoroughly in distilled water, several changes. 



(5) Dehydrate to 70% alcohol with b minute changes in ascending. 



(6) Dehydrate further: (Leave in this overnight) 



80% alcohol 96 cc. 



Phenol 4 cc. 



(See King & Slifer, 1933; Science 78.) 



