STAINING THE SECTIONS 265 



clove oil from the drop bottle. This will gradually extract 

 the gentian violet, and the preparation should be watched under 

 the lower power of the microscope so that this action may be 

 stopped as soon as the gentian stain has lost its too great in- 

 tensity and become transparent while yet distinct. Then drain 

 off the clove oil and set the slide in the dish of xylene to thor- 

 oughly rinse away the clove oil. 



7. Remove the slide from the xylene, drain it, place a drop 

 of Canada balsam toward one end of the group of sections 

 and lower a coverglass over it, beginning at the end where 

 the drop of balsam is, by bringing the coverglass into contact 

 with the slide first at that end and gradually lowering it 

 toward the opposite side so as to drive forward any air bub- 

 bles that may become entangled with the balsam. Then set 

 the slide where the balsam can dry for several days at about 

 50° C. 



With this three-color stain the cytoplasm should be gray 

 or brownish, the nucleus violet, the nucleolus red, cellulose 

 walls uncolored or grayish, lignified, cutinized, and suberized 

 walls red. In dividing cells the chromosomes should be red, 

 the spindle fibers violet, and the rest of the cytoplasm gray 

 or brownish. 



Where a fine differentiation of the parts of the protoplasts 

 is not so much sought after as a differentiation of the tissues, 

 other simpler methods of staining may be used to good advan- 

 tage. Double staining with cyanin and erythrosin gives ex- 

 cellent results. For this are needed a saturated solution of 

 cyanin in 95 per cent, alcohol, in a Stender dish or covered 

 tumbler, and a saturated solution of erythrosin in clove oil, in 

 a drop bottle. Set the slide on end in the cyanin for about 

 ten hours, then rinse it in 95 per cent, alcohol until the cyanin 

 no longer comes away in clouds, and this should require only 

 a few moments; then flood the sections for about four seconds 

 with the erythrosin solution, drain and rinse thoroughly with 

 xylene and seal in balsam. If the clove oil is not completely 

 rinsed out in xylene the stains will fade out after a time. The 



