88 Botanical Microtechnique 



until the rinse water is colorless. Flood with r)Q% alcohol, in ^vhich 

 destaining of safranin begins to take place. Do not use acetone until 

 the anhydrous stage. After 3 to 5 niin., (hangc to 95% alcohol, in 

 which destaining continues. At first the blue color of the hematoxylin 

 is completely masked by the red safranin, but as destaining proceeds 

 the blue color becomes evident. At intervals of 2 to 5 min. transfer a 

 section to a watch glass of clean 95% alcohol, and examine with a 

 microscope. When there is good contrast between the blue color of 

 nonlignified tissues and the clear, brilliant red of lignified elements, 

 drain, and rinse in five changes of anh\drous alcohol or acetone at 

 2- to 5-min. intervals. Drying of the sections must be avoided in 

 making these changes. Destaining is almost entirely stopped in 

 anhydrous acetone, and the celloidin matrix is dissolved out of the 

 tissues. If absolute alcohol is used, make two changes of ether-alcohol 

 solvent to dissolve the celloidin out of the tissues. Flood with fresh 

 carbol-xylene. There should be practically no destaining action 

 during the 5- to 10-min. interval in carbol-xylene. Rinse in (i\e 

 changes of xylene, and mount in balsam or synthetic resin. 



If the celloidin support is dissohed out of some pathological 

 materials, graft unions, and some fragile subjects, the sections 

 disintegrate or lose important parts. The sections can be cleared by 

 transferring directly from 95% alcohol to terpineol, carbol-xylene, or 

 creosote. The clearing agent nuist be changed se\eral times, and 

 thoroughly rinsed out in xylene before mounting. The suppoiting 

 celloidin is retained by this method. 



lo mount one section on each slide, remo\c a section from xylene 

 with a small brush or section lifter and j)la(i' the section on the center 

 of a dry slide. Stained sections can be selected under a binocidar 

 microscope or a hand lens and the imperfect sections discarded. Keep 

 the section on the slide moistened with xylene. If the section is c urled. 

 straighten with two brushes. kee|)ing the concave side down. Place a 

 drop of resin on the section and lower the cover glass ol)li(]uely, 

 squeezing out air bubbles by gentle pressure or by iap|)ing with the 

 eraser on a pencil. Put a lead weight on the cover glass, ilu' diop ol 

 resin should be of siu h s\/v iluit there is no excess resin around oi 

 over the weighted cover glass. II aii l)ul)bles cannot be expelled oi il 

 too nuuh resin was used, put the slide into a Petri disli ol wlene. 

 The cover glass and section can be slid oil in a lew niinuics and the 

 section remounted. It is much easier to uncover and re-cover than to 

 clean up a messy preparation later. After 1 to 3 days of drying under 

 j)ressure in :i hoii/onial position, the wei«;hi may be removed, and the 



