306 METHODS IN PLANT HISTOLOGY 



method, try this. Transfer the sections from water to 95 per cent alco- 

 hol and leave them for 30 minutes. Stain in safranin for two hours. 

 Rinse in 50 per cent alcohol until most of the safranin is washed out 

 from cellulose walls. Then in 70, 85, and 95 per cent alcohol 10 

 minutes in each. Stain in light green 1 minute. Rinse in 95 per cent al- 

 cohol; absolute alcohol, 10 minutes; clove oil, 2 minutes; xylol; balsam. 



Here is another method, not so rapid, but better. Fix the sections in 

 formalin-alcohol-acetic acid, for 24 hours. Rinse in 50 per cent alco- 

 hol. Stain in safranin overnight or 24 hours: 50 per cent alcohol until 

 most of the stain is drawn from the cellulose walls: 70, 85, and 95 per 

 cent alcohol, 1 hour each; 100 per cent alcohol, 1 hour; crystal violet 

 in clove oil, 1 minute; xylol, balsam. 



Here is a still better, and slower, method. Fix the sections in 

 chromo-acetic acid (1 g. chromic acid, 3 c.c. acetic acid) overnight or 

 24 hours. Wash in water 24 hours. Run the sections through the alco- 

 hols as slowly as if for imbedding in paraffin. Allow the 85 per cent to 

 act overnight or 24 hours. Then 70 per cent, 30 minutes and 50 per 

 cent, 10 minutes. Stain in safranin and light green. With this very 

 slow method, the protoplasm should not shrink away from the walls. 



However, it is better to imbed in paraffin and cut thinner sections. 

 The rhizome of Pteris aquilina, cut 10 /x and stained in safranin and 

 light green, or in safranin, gentian violet, orange, will show the superi- 

 ority of this method in everything except speed. Sections a short 

 distance back from the apical cell, at the region where lignification is 

 just beginning to take place, will show the protoxylem stained red with 

 safranin, while the rest of the xylem (metaxylem) is still in the cellu- 

 lose condition and staining green. The rhizome of Pteris affords an 

 excellent illustration of a mesarch polystele. 



Dicksonia punctilohula has a small rhizome, often on the surface of 

 the soil or rock, so that it is easy to get good clean pieces. About 2 or 

 3 cm. back of the growing point, the xylem is well lignified, but the 

 material still cuts well in paraffin. One could hardly find a better 

 illustration of a mesarch amphiphloic siphonostele (Fig. 96). 



Botrychium is widely distributed, but individual plants are not 

 abundant. The stem is erect, subterranean, and has an endarch si- 

 phonostele with secondary wood. Trim away the roots, which are 

 very thick and fleshy in B. obliquum, fix in formalin-alcohol-acetic 

 acid, and imbed in paraffin. Even the older parts of old stems can be 

 cut in paraffin if you are sufficiently careful (Fig. 97). Transverse sec- 



