3 



Stains and Staining 



The majority of objects made into microscope slides today are "stained"; 

 that is, they are soaked in a solution ot a dye or dyes, either, in the case of 

 wholemounts, to increase their visibility when mounted in transparent media 

 or, in the case of sections, to provide a color differentiation between the vari- 

 ous tissues involved. The staining solutions employed may be divided very 

 broadly into "nuclear stains," which are differentially absorbed by the nucleus 

 and chromosomes, and "plasma stains," which are used to color tissues other 

 than the nucleus. It may be added that, in general, nuclear stains will stain 

 bacteria and are also used in botanical technique for staining xylem. 



There are three general methods of staining employed. The first of these is 

 "direct staining" in which soaking the object or section in the stain is suf- 

 ficient to color the desired structure. This is rarely employed and can be used 

 only with those stains which are entirely specific for the structure which is 

 to be emphasized. It is far more usual to employ a process of "indirect stain- 

 ing" in which the whole object or section is first uniformly stained in the dye 

 selected and then "differentiated" in some solution which removes the dye 

 from all those structures other than the ones which are to be emphasized. 

 The third method of staining is "mordant staining" (from the Latin mordere, 

 meaning "to bite") in which some preliminary solution is used which causes 

 the stain to "bite into" a structure without being absorbed by others. These 

 three great categories are by no means sharply divided, for many of the so- 

 called direct stains actually incorporate mordants, and many of the indirect 

 staining solutions may be used by the direct method if they are first greatly 

 diluted with water. 



The most commonly employed nuclear stains are hematoxylin, carmine, and 

 a few synthetic aniline dyes. Hematoxylin is a natural coloring matter ex- 

 tracted from logwood and depends for its staining action upon its oxidation 

 products, such as hematein, rather than upon itself This dye, therefore, must 

 be "ripened" (actually oxidized) either by being left exposed to the air for some 

 time or by having added to it some oxidizing material. Carmine is a complex 

 lake prepared from a dye extracted from the cochineal insect. 



Mordant Hematoxylin Staining 



The mordant hematoxylin stains are entirely confined to staining nuclei or, 

 more usually, chromosomes. In general, three solutions are required: (1) A 

 mordant, (2) the hematoxylin stain, and (3) a differentiating solution. The two 



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