CHAPTER XXI. 



HISTOLOGICAL METHODS. 



IN the more exact investigations of the tissues, it is often 

 necessary to have recourse to other reagents than those we 

 have used hitherto, in order to bring out plainly the more 

 obscure points of structure. This is especially the case in 

 studies in cell division in the higher plants, where the changes 

 in the dividing nucleus are very complicated. 



For studying these the most favorable examples for ready demonstra- 

 tion are found in the final division of the pollen spores, especially of some 

 monocotyledons. An extremely good subject is offered by the common 

 wild onion (Allium Canadense), which flowers about the last of May. 

 The buds, which are generally partially replaced by small bulbs, are 

 enclosed hi a spathe or sheath which entirely conceals them. Buds two 

 to three millimetres in length should be selected, and these opened so as 

 to expose the anthers. The latter should now be removed to a slide, and 

 carefully crushed in a drop of dilute acetic acid (one-half acid to one-half 

 distilled water). This at once fixes the nuclei, and by examining with a 

 low power, we can determine at once whether or not we have the right 

 stages. The spore mother cells are recognizable by their thick trans- 

 parent walls, and if the desired dividing stages are present, a drop of 

 staining fluid should be added and allowed to act for about a minute, the 

 preparation being covered with a cover glass. After the stain is suffi- 

 ciently deep, it should be carefully withdrawn with blotting paper, and 

 pure water run under the cover glass. 



The best stain for acetic acid preparations is, perhaps, gentian violet. 

 This is an aniline dye readily soluble in water. For our purpose, however, 

 it is best to make a concentrated, alcoholic solution from the dry powder, 

 and dilute this as it is wanted. A drop of the alcoholic solution is diluted 

 with several times its volume of weak acetic acid (about two parts of dis- 

 tilled water to one of the acid), and a drop of this mixture added to the 

 preparation. In this way the nucleus alone is stained and is rendered 

 very distinct, appearing of a beautiful violet-blue color. 



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