APPENDIX. 399 



STAINING. 



The object of staining is to bring out the various structural components 

 of the tissues by taking advantage of their selective affinities for certain 

 dyes. The differentiation usually sought has as its first object, the display 

 of the nucleus; next, the tingeing of the cytoplasm; and thirdly, the exhi- 

 bition of the intercellular substances. Of the large number of staining 

 methods which from time to time have been devised to meet the require- 

 ments of particular lines of work, only a few of the most reliable and gen- 

 erally useful will be here given. For the ordinary needs of the histologist, 

 staining with hematoxylin, followed by eosin, leaves little to be desired, 

 since, when successful, the nuclear differentiation is sharp while the cyto- 

 plasm and intercellular substances are sufficiently tinged to produce clear 

 and instructive pictures. When, as in the case of embryos, it is convenient 

 to stain in bulk before sectioning in paraffin, borax-carmine will be found a 

 most reliable dye, possessing penetration and uniformity of action in a highly 

 satisfactory degree. 



Ehrlich's Hematoxylin. 



Hematoxylin crystals 2 gm. 



Absolute alcohol 100 cc. 



Distilled water 100 cc. 



Ammonium alum in excess 



v Glycerine 100 cc. 



Glacial acetic acid 10 cc. 



A. Dissolve the hematoxylin in the absolute alcohol and let stand in 

 loosely corked bottle for a week, where the sunlight may reach it. 



B. To the distilled water add ammonium alum to excess. Add A to 

 B, while stirring vigorously. After several days add the glycerine and the 

 glacial acetic acid and place the stain, in a corked bottle, in some suitable 

 position insuring exposure to direct sunlight. During the next two weeks 

 uncork for several days at a time. Direct sunlight is important. After three 

 months the stain has ' ' ripened ' ' sufficiently for use. If kept in a well-stop- 

 pered bottle, with an excess of alum, the reagent retains its staining powers 

 for years and, indeed, improves with age. Although requiring a long time 

 until ready for use, Ehrlich's hematoxylin is most satisfactory, staining 

 well after all the usual methods of fixation and, with proper precautions, 

 being permanent. 



This, as all other hematoxylin stains, should be filtered immediately 

 before using. Depending upon the age of the solution and, to some extent, 

 upon the method of fixation, sections require to be left in the stain from 5 to 

 10 minutes. They are then washed thoroughly in running water for one or 

 two hours. If deeply colored they may be placed in a dish of 70 alcohol, 

 acidulated by the addition of 5 drops of hydrochloric acid to each 100 cc. of 

 alcohol. The sections remain in the acid bath, in which they turn reddish, 

 so long as they discharge color, keeping them moving and not allowing 

 them to adhere. During subsequent washing the blue color is restored, 

 becoming brighter as the washing progresses. While the acid alcohol may 

 be omitted with sections not too deeply tinted, full staining followed by the 

 acid alcohol yields sharp nuclear differentiation and brilliant pictures. The 

 employment of the acid bath may be recommended, therefore, as a routine 



