26 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



egg, may be necessary. Apparently a small amount of water invades the paraffin and 

 egg and reduces brittleness. 



Another modification is to paint each section with a very thin coating of celloidin and 

 mastix. Rubber -paraffin mixtures have been used. The early stages of amphibian de- 

 velopment are difficult to section satisfactorily. 



MOUNTING SECTIONS 



The conventional method is to coat the slide with a thin layer of egg-albumen prior to 

 mounting the sections. Some stains will show up the unevenness of the albumen and it is 

 difficult to control the amount applied. A more satisfactory method is to float the sections 

 on the slide and over albumen water made up of 10 cc. of (boiled) distilled water which 

 has been cooled and to which has been added 1 drop of egg albumen. Enough of this albu- 

 men-water should be used to allow complete expansion of the sections over the warming 

 oven, held at 40°-45 C. Adherence should be complete in 1 2 hours. Sections may be 

 floated on a 40 C. water bath and mounted on slides submerged and brought up under 

 them. 



Thick sections and large yolk masses may require additional treatment before they will 

 adhere permanently to the slides. In the hydration process leading to the staining, the 

 mounted sections should be taken through xylol and absolute alcohol and then immersed 

 briefly in a very thin solution of cellodin before going into the lower alcohols. The alco- 

 hols and stains will penetrate the celloidin satisfactorily. 



During the hydration process (descent through the alcohols) the yolk-laden sections of 

 amphibian eggs often come loose from the slide, no matter what precautions in albumen- 

 fixation are taken. To avoid this, just before going into the 95% alcohol from the 100% 

 (absolute) alcohol, dip the slides into the following mixture: 



Celloidin 8% 50 cc. 



Absolute alcohol 450 cc. 



Ether 450 cc. 



This will provide the sections with a very thin coating of celloidin which will hold them 

 in place but will in no way interfere with staining, and subsequent dehydration. 



HYDRATION OF MOUNTED SECTIONS 



This may be accomplished with 1 -2 minute shifts in the various alcohols after the em- 

 bedding substance (paraffin) has been completely dissolved off. Dioxan may be used in 

 hydration as well as dehydration. 



STAINING 



The choice of stain depends entirely upon the end results desired. 



Nuclear stains : (Where destaining is necessary, used acidified 70% alcohol.) 



a. Delafield's haematoxylin - should be deep wine colored, aged for months, and 

 used in concentrated form for 3-10 minutes. Follow with wash in tap water to 

 blue the stain. Cytoplasmic stains not necessary since Delafield's gives the 

 cytoplasnn a slight pink color. /• 



b. Harris' haematoxylin - excellent for chromosome studies in tail tips. Use like 

 Delafield's although better to dilute (4x) and stain longer. Destain with 35% acid 

 alcohol and blue in tap water. 



