80 The Preparation of Microscope Slides 



plication of the mordant and second the application of the dye. The more 

 usual method is known as "indirect staining" in which mordant and dye 

 are applied together from the same "staining solution." The result of this 

 is that the whole tissue becomes colored but the color is fixed only to the 

 nucleus. The tissue is then placed in a "differentiating" solution, more 

 often than not a weak acid, which removes the color from the cytoplasm 

 while leaving it in the nucleus. This process must be carefully controlled 

 since differentiating solutions will ultimately remove all the dye. 



This indirect staining— that is, the staining of everything with subse- 

 quent differentiation of the required structures— may also be applied to 

 wholemounts. For large wholemounts, however, this process is not so 

 satisfactory as "direct staining." In this case the staining solution is very 

 greatly diluted and allowed to act for a long time. The color of the dilute 

 stain is so weak that it does not appreciably affect the cytoplasm but is 

 gradually absorbed by, or built up on, the nuclei. 



The first nuclear stains used by microscopists were "hematoxylin," a 

 reagent extracted from campeche wood, and "carmine," a derivative of an 

 extract of cochineal. These are still extensively used, even though there 

 is today every reason for replacing such natural products with synthetic 

 dyes. Hematoxylin is probably the commonest nuclear stain and descrip- 

 tions of "mordant," "indirect," and "direct" hematoxylin methods will 

 therefore be given first. 



HEMATOXYLIN 



This dye is available as an artificially bleached, whitish powder ob- 

 tained by solvent extraction from the residue of an aqueous extract of 

 campeche wood. The dye is readily soluble in alcohol and water, but 

 these solutions have little staining value except in "mordant" techniques. 

 Staining solutions incorporating mordants require to be "ripened" for 

 some months. The staining qualities of ripe solutions are in part due to the 

 oxidation of hematoxylin to hematein. There must, however, be other 

 factors involved, since neither the substitution of hematein for hema- 

 toxylin nor the addition of oxidizing agents produces a stain of the same 

 quality. 



Mordant Hematoxylin Staining. These techniques give a very intense 

 nuclear stain, of more interest to nuclear cytologists than histologists. 

 Ferric alum is the mordant most commonly used. Simple solutions of 

 mordant and stain were used by Heidenhain , whose name is usually 

 associated with the technique, but better results can nowadays be ob- 

 tained by the technique of Regaud on regular sections or that of Dobell 

 on very thin sections or protozoan smears. 



