37 



(a) For general purposes it is customary to use a nuclear and 

 a cytoplasmic stain either in combination or successively. The 

 double staining most employed is hematoxylin and eosin. Triple 

 stains are generally not so satisfactory; hematoxylin and picro- 

 fuchsin is one commonly used. Neutral stains ( 85) are essentially 

 triple stains. Quadruple stains are rarely serviceable; see, however, 

 129. For stains to be employed for special purposes, see Special 

 Methods, 174-. 



(b) In the case of most of the fixers given 11-, there are no 

 restrictions as to the stains that may be employed though some 

 are more satisfactory than others. With Flemming's fluid and 

 Hermann's fluid, however, it is well to employ only such stains 

 as iron hematoxylin (94) and anilin stains, safranin being suggested 

 as a red stain, gentian violet as a blue stain. Dichromate fixers whose 

 action has been prolonged usually require strong stains. Iron hema- 

 toxylin and similar stains may be used after any fixer. The Ehrlich- 

 Biondi-Haidenhain stain ( 111) is most satisfactory after Mercuric 

 chlorid ( 11). 



(c) Celloidin is deeply stained by such stains as iron hema- 

 toxylin, safranin, methylene blue, gentian violet, and basic stains in 

 general. If it is necessary to use these, with celloidin material, the 

 celloidin should be removed from the sections before mounting 

 ( 136). 



85. Neutral Stains. In the case of most of the anilin stains, if an aqueous 

 solution of a basic stain is added to an aqueous solution of an acid stain, there is 

 formed by combination a neutral stain which is usually but slightly soluble in 

 water and hence precipitates out. Neutral stains are however moderately soluble 

 in strong methyl or ethyl alcohol. These facts it is well to keep in mind in using 

 anilin stains. The reaction may be made use of for increasing the selectivity, 

 sharpness and color tones of the original stains. Neutral stains may be used in 

 one of three ways: (a) The neutral stain is often soluble in excess of the acid 

 or basic stain or in an aqueous solution of another acid or basic stain (111). (b) 

 Dissolved in methyl (or ethyl) alcohol and diluted nearly to the precipitation point 

 the activity and selectivity of the component stains seem to become specially 

 marked ( 128, 214). (c) The formation of the neutral stain may be carried out 

 in the staining process itself, "on the slide," by staining first strongly with an 

 aqueous solution of the acid stain, rinsing away the excess stain and staining with 

 the aqueous solution of the basic stain ( 106) and differentiating ( 82) with 

 alcohol, or alcohol and clove oil. 



Of the large number of stains that combine to forrn neutral stains may be 

 mentioned, the acid stain being given first; eosin and methylene blue ( 128); 

 orange G. and gentian violet ( 131), erythrosin and toluidin blue, thiazin red and 

 toluidin blue, coerulein S. and safranin, acid violet and safranin. These may be 

 combined and used in any one of the three ways mentioned above. 



