ANATOMICAL TECHNIQUE 457 



tissues. The sections are then cut thin by appropriate manipu- 

 lation of the micrometer screw and floated instantaneously on the 

 edge of the knife in alcohol. Thence they are transferred to a dish 

 of water and show all their natural organization and color prac- 

 tically unchanged. Sections as thin as five and ten micra are 

 easily obtained in this manner and are very striking. The leaves 

 of gymnosperms such as cycads and conifers lend themselves to 

 the same treatment. 



METHODS OF STAINING 



The thin sections prepared by the methods above indicated 

 are frequently so tenuous as to present insufficient detail on micro- 

 scopic examination. This inconvenience is ordinarily overcome 

 by the use of stains to bring out contrasts in structure. The sub- 

 ject of staining has been so recently and admirably discussed for 

 American students in Professor Chamberlain's Methods in Plant 

 Histology (3d ed., The University of Chicago Press, 1915) that it 

 need only be elucidated in the present connection in regard to the 

 special conditions presented by the hard tissues in plants. 



A few stains give the best results in most anatomical investi- 

 gations, since the protoplasmic structures are of less importance 

 and the cellulosic and lignified conditions of the cell wall are of the 

 greatest significance. In most instances staining with hematoxy- 

 lin and counterstaining with the anilin dye known as safranin give 

 the best results. In the case of hematoxylin the Haidenhain 

 method on the whole answers best. The sections are washed in 

 distilled water and then immersed in a 3 per cent solution of 

 ammonia-iron alum. The alum should be bluish in color and free 

 from efflorescence. Ten to fifteen minutes in the alum solution 

 are sufficient. Careful washing in distilled water follows. The 

 first change of washing water should remain only a short time and 

 is followed by a second and a third change, so as to remove all 

 alum which has not become fixed in the sections. In material 

 containing a great deal of tannin special precautions must be taken 

 to insure thorough washing by repeated changes of distilled water. 

 When the sections are cleared of excess of alum, they are ready 

 for the hematoxylin solution, which in some cases is employed in 



