20 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



Some investigators expose the amphibian egg 'by cutting off one or two sections and 

 then soaking the entire block in water, overnight. Such an egg will expand beyond the cut 

 siu'face and several sections will be lost, but frequently very nice serial sections of the 

 remaining portion of the egg can be acquired. A second soaking, half-way through the 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 ceiloidln 

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

 development 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 

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

 oven, held at 4o°-'+5'-'C. Adherence should be complete In 12 hours. 



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 

 alcohols and stains will penetrate the ceiloidln 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: 



Ceiloidln 8$ 50 cc. 



Absolute alcohol _. k^O cc. 



Ether 1^50 cc. 



This will provide the sections with a very thin coating of ceiloidln 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 

 embedding 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 5-10 minutes. Follow with wash in tap 

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

 gives the cytoplasm a slight pink color. 



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

 like Delafield's although better to dilute C+x) and stain longer. Destain 

 with 55^ acid alcohol and blue in tap water. 



c. Harris' acid haemalum - dilute to 25^ with distilled water, stain as with 

 haematoxylin and rinse in tap water. 



