316 



METHODS IN PLANT HISTOLOGY 



For sections, use the Chicago formula. If the gradual processes of 

 dehydrating, clearing, and infiltrating at room temperature and on the 

 top of the bath have been observed, about 20 minutes in the bath .may 

 be sufficient; not more than 30 minutes should be needed for any pro- 

 thallia. About 10 /x is a good thickness for such views of the arche- 



gonium as are shown in Figures 

 106, 107, and 108. Safranin, 

 gentian violet, orange is a good 

 stain. 



For the development of the 

 antheridium and sperm, and 

 especially for the blepharo- 

 plasts and their transforma- 

 tion, cut 2 or 3 iu in thickness 

 and use iron-haematoxylin 

 (Fig. 104, D, E, F); for the 

 development of archegonia, cut 

 at 5 M and stain in the safranin, 

 gentian violet, orange combi- 

 nation. 



The gametophyte of Botry- 

 chium is subterranean and tu- 

 berous. It sometimes reaches a 

 length of 7-12 mm. and a thick- 

 ness of 4-5 mm. Usually, it is 

 not more than 5 or 6 mm. long 

 and 2 or 3' mm. thick. Gametophytes showing the development of 

 antheridia and archegonia are not likely to be more than 2 or 3 mm. 

 long and 1 or 2 mm. thick. Near large plants, look for small spore- 

 lings, not more than 1 or 2 cm. in height. Dig very carefully and you 

 may find the gametophytes attached. The soil should be examined 

 for smaller specimens. Most of the gametophytes will be found at 

 a depth of 1-3 cm. Fix in chromo-acetic acid. 



No one has yet succeeded in raising the prothallia from the spores. 

 The prothallia always contain an endophytic fungus, but even when 

 this is present no one has succeeded in raising prothallia. Perhaps 

 there is long-delayed germination and development, as in Lyco- 

 podiuni, and no one has followed cultures for 5 or 6 years. 



The prothallium of Ophioglossum is harder to find or, perhaps it 



Fig. 105.- 

 prothallia. 



-Costello's method of growing fern 



