Ill 



PROTOCOLS ON FIXATION AND STAINING 



In order to facilitate the use of the directions on fixation, 

 imbedding, and staining given In this book, I present them in 

 summary and topical form as "protocols" for (i) staining with 

 iron haematoxylln, (2) with safranin, and (3) for staining of 

 mitochondria. For each an egg in metaphase of first cleavage 

 is suggested as the object to be employed; any other small 

 object, as Insect testis, a bit of mammalian tissue, may however 

 be substituted — in which case the making of an outline drawing, 

 as recommended for the egg, Is omitted. 



Protocol I. 



Staining with iron haematoxylin. 



Example of object: Eggs of a nereid or of an echlnid In 

 metaphase of first cleavage. 



1. With the aid of a camera lucida make as quickly and as 

 carefully as possible an outline drawing of an egg showing Its 

 shape, extent of its spindle area and the disposition and size of 

 the cytoplasmic components. (This Is done to compare the 

 fixed egg with the living.) 



2. To a drop of egg suspension in a vial, add 2 cc. of the 

 modified Meves. (Avoid use of cork stoppers!) 



3. At 5 minute intervals during the next 40 minutes, gently 

 turn the bottles upside down two or three times to insure even 

 penetration of the fixative. 



4. After 40 minutes, decant fixative carefully. Fill bottles 

 with distilled water. (If distilled water Is acid, use tap water.) 

 As soon as the eggs settle, decant water and add water anew. 

 Repeat this process, gently turning the bottles upside down after 

 each renewal of water. In this wise wash eggs for an hour. 



5. Run up eggs in alcohol: 35 per cent, 50 per cent, 70 per 

 cent, and 80 per cent — one hour each. 



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