400 APPENDIX. 



procedure. Since the permanency of all hematoxylin preparations depends 

 upon the elimination of every trace of acid, prolonged and thorough washing 

 is essential, otherwise fading will occur. If after short washing the sections 

 are placed for a few minutes in water, to which a few drops of ammonia have 

 been added, and then washed thoroughly in, preferably running, water, per- 

 manency of the staining is assured. 



Bohmer's Hematoxylin. 



Hematoxylin crystals i gm. 



Absolute alcohol 10 cc. 



Ammonium alum 10 gm. 



Distilled water 200 cc. 



Dissolve the hematoxylin in the absolute alcohol and keep in stoppered 

 bottle for 24 hours. Dissolve the alum in the warm distilled water. When 

 cool, gradually add the dissolved hematoxylin while stirring. Place in an 

 open wide-mouth bottle or dish, protected from dust, for one week, when, 

 after filtering, the stain is ready for use. Keep in a tightly corked bottle 

 and filter the required quantity on using. Immersion from loto 30 minutes 

 usually suffices to stain to the required degree. This solution stains well, 

 although not uniformly so intensely as Ehrlich's, and, if the sections are 

 thoroughly washed, yields permanent results. 



Delafield's Hematoxylin. 



Hematoxylin crystals 4 gm. 



Absolute alcohol ... 25 cc. 



Ammonium alum 52 gm. 



Distilled water 400 cc. 



Glycerine 100 cc. 



Methyl alcohol 100 cc. 



The hematoxylin is dissolved in the absolute alcohol and the alum in 

 the heated distilled water. After the alum solution has cooled, the hema- 

 toxylin is slowly added while stirring and the fluid allowed to stand in 

 a wide open vessel, as a beaker or jar, protected from dust but exposed 

 to light and air, for about 15 days. Filter and add glycerine and methyl 

 alcohol. Expose to the light until the stain acquires a dark purplish tint, 

 then filter and keep tightly stoppered. The advantages of Delafield's 

 hematoxylin are less for coloring sections than for staining tissue in 

 bulk. For mass-staining, the freshly filtered solution is diluted with five 

 to ten volumes of distilled water, in which the pieces of tissue remain 

 from 2,4 to 48 hours, or longer, until uniformly darkly tinted throughout. 

 The stained tissue is rinsed in distilled water and placed in acid 70 alcohol 

 (5 drops hydrochloric acid to 100 cc. alcohol) from 4 to 8 hours. After 

 this differentiation the tissue must be thoroughly washed in running water 

 for 1 2 to 24 hours to remove every trace of acid and to bring out the rich 

 blue color. 



Contrast staining after hematoxylin, especially when the latter 

 has been limited chiefly to the nuclei, adds much to the effectiveness of the 

 preparation. Such double staining may be accomplished by treating the 

 hematoxylin sections with solutions of acid fuchsin, Congo red, or eosin. The 

 latter answers well and is convenient. The most satisfactory results are 



