Clearing and Staining of Leaves and Stems. 95 



manner make beautiful studies in leaf venation. If the staining 

 is properly done, the tracheal elements will be of a deep trans- 

 parent red color, while all of the rejnaining tissues will be trans- 

 parent and luistained. In some cases of ordinary leaves, it is 

 not necessary to bleach out the stain with chloral hydrate; a 

 half hour or longer soaking in water will suffice. 



If cross sections and tangential sections are wanted, de- 

 hydrate, and embed bits of the cleared leaves in paraffine ac- 

 cording to some of the usual methods. Secti ms may now be 

 cut upon the microtome, fixed to slides in the customary way, 

 but stained in the manner just outlined. 



Stem sections are treated in exactly the same way as are 

 leaves, when a single stain is all that is desired. When stems 

 treated in this way are proper!}^ stained, all lignified, suberized 

 and cutinized cell walls will be of a deep transparent red color, 

 while all cellulose cell walls will be transparent and unstained. 



Leaf and stem sections thus bleached and cleared, should 

 be double- stained as follows: Stain for a few hours or over 

 night, in a concentrated aqueous solution of chloral hydrate 

 deeply colored with safranin; rinse in water, and transfer to 

 concentrated chloral hydrate, where they remain until the stain 

 is removed from all cellulose walls; rinse thoroughly in water, 

 and stain for five to sixty seconds in a saturated aqueous solution 

 of Bismarck brown; rinse quickly in water, and transfer to dilute 

 glycerine, where the sections may remain indefinitely, or be 

 made into permanent mounts in glycerine jelly. 



When the specimens are fixed to the slides, the Bismarck 

 brown is better applied by holding the slide in one hand, while 

 the sections are flooded ^^itll the stain from a dropping bottle 

 pipette in the other hand. The Bismarck brown will readily 

 stain some specimens within five seconds, but will take a longer 

 time for others. The worker will have to experiment in order 

 to adapt the time to his specimens. In using this stain, care 

 must be taken in order not to overstain. If this be done the Bis- 

 marck brown will mask the safranin. 



If the double staining has been properly done, the lignified 

 cell walls will be of a deep transparent red color, the suberized 

 and cutinized cell walls will be transparent and red to a degree 

 in proportion to the extent to which they have been suberized 



