HjEMATOXYLIN AND EOSIN 501 



tion may be used, but it is preferable to use a solution made by adding 

 to 95 per cent, alcohol enough of a stock solution, consisting of 5 gm. of 

 eosin dissolved in 300 c.c. of 50 per cent, alcohol, to give a deep yellowish- 

 red color to the alcohol. 



Sections are transferred from water to the haematoxylin solution for 

 about 5 minutes. They are then washed in several changes of tap water 

 until the sections are deep blue in color. If this change does not occur 

 rapidly, a few drops of ammonium hydrate may be added to the water 

 They are then examined with the microscope. If the sections are over- 

 stained (a condition recognizable in the staining of the cytoplasm as well 

 as the nuclei) the sections are washed in a 0.5 per cent, solution of hydro- 

 chloric acid in 70 per cent, alcohol until the section is reddish-brown, 

 usually about i minute. Wash in water made alkaline with a few drops 

 of ammonia and re-examine. If the nuclei are not well stained, the sec- 

 tions are returned to the haematoxylin solution for 5 to 10 minutes longer, 

 after which they are examined as before. 



When the stain is sharply limited to the nuclei and is of satisfactory 

 depth, the sections are washed for 15 to 30 minutes in tap water and then 

 are passed through 50 per cent., 70 per cent., 80 per cent, and 95 per cent, 

 alcohol, remaining about i minute in each. Stain in the alcoholic solution 

 of eosin for i to 5 minutes. Wash in 95 per cent, until red clouds no longer 

 leave the section. 



Paraffin sections are then passed through absolute; absolute and xylol 

 (equal parts) ; and xylol, in which they remain for about 5 minutes, and 

 are then ready for mounting. Celloidin sections are transferred from 

 95 per cent, to oil of origanum for about 5 minutes, before mounting. 



Eosin and Methylene Blue. This method is highly recommended, 

 especially for tissues fixed in Zenker's fluid and sectioned in paraffin. 



Stain the sections for 20 minutes or longer in a 5 or 10 per cent, aqueous 

 solution of eosin. The tissue must be overstained, as the eosin is partially 

 extracted in the subsequent treatment. Wash out the excess of the stain 

 in water. 



Stain for 10 to 15 minutes in Unna's alkaline methylene blue (i gm. 

 of methylene blue and i gm. of potassium carbonate dissolved in 100 c.c. 

 of water) diluted i : 3 or i : 5 with water. Wash in water. Differentiate 

 and dehydrate in 95 per cent, alcohol, keeping the section in constant 

 motion so that the decolorization is uniform. When the pink color re- 

 turns to the section and when, as seen under the microscope, the blue is 

 limited to the nuclei, the section is quickly washed in absolute, passed 

 through absolute and xylol, and put in xylol for about 5 minutes. 



Heidenhain's Iron Haematoxylin. The best results are obtained with 

 very thin paraffin sections. From water the sections are transferred to 

 a 2-2.5 P er cent - aqueous solution of ferric ammonium sulphate for 4 to 



