GENERAL TECHNIQUES FOR CLASSES OF PLANTS 713 



in which the sporangia and spore walls have become hardened, are 

 troublesome to cut in paratlin. Trim strobili flat on opposite sides 

 to aid infdtration. 



Equisetales. The stems, etc., are heavily impregnated with 

 silica ; they must be desilicified and are best imbedded in celloidin. 

 Growing tips are relatively free from silica and will cut in paraffin 

 without special treatment. 



Filicales. In fixing stem-tips, trim away the ramentum. 

 Mature stems and roots of sclerenchymatous species should be 

 treated as woody specimens. The walls of sporangia, particularly 

 older ones, are resistant to fixatives, and for cytological purposes 

 penetration must be assisted in every way ; Kihara's method (see 

 § 1361) is satisfactory. 



Fern Sorus. Examine fresh material with dark field illumina- 

 tion. Clear portions of leaves in KOH, wash and stain in safranin. 

 Destain with care and lightly eounterstain in dilute light green 

 (or methyl blue) ; dehydrate, clear and mount in balsam. 



Prothallia. Stokey {Bot. Gaz., Ixv, 1918, p. 97) grows fern 

 prothallia on various media : different soil mixtures ; black peat, 

 with and without Knop's solution ; porous clay crock standing in 

 Knop's solution. Costello (Chamberlain, 1932, p. 314) grows 

 them free from soil on a clean flower pot filled with Sphagnum, 

 inverted in a dish of water, and covered with a bell jar. 



See also Steil, Bot. Gaz., lix, 1915, p. 254. 



Flat ones are best fixed in medium Flemming and stained in 

 Heidenhain's haematoxylin. To mount whole see § 1263. The 

 gametophytes of heterosporous species surrounded by a hard 

 impermeable spore wall should be fixed by Kihara's method 

 (§ 1361 Carnoy followed by Flemming). Do not fix female 

 gametophytes by this method after the megaspore wall has 

 ruptured. 



GYMNOSPERM^ 



1405. Careful treatment is required for megasporangial 

 structures, the female gametophyte (endosperm) and the embryo. 

 Segments should be cut as deeply as possible with a razor blade 

 from opposite sides of the ovule to facilitate fixation. The ovule 

 cuts well in paraffin until the stony layer is hardened. Free 

 nuclear stages require special care to prevent shrinkage ; fix in 

 Flemming with a higher proportion of osmic acid. For more 

 accurate fixation, especially in Cycadales and Ginkgo, cut out the 

 upper portion of the nucellus or endosperm. Secure pollen-tubes, 

 with sperm mother-cells or sperms in their tips, in this way. 

 Coulter and Chamberlain {Morphology of Gynuiosperms) and 

 Chamberlain {Methods in Plant Histology) give data on the times 

 of attainment of various stages in development of the different 

 organs in several species. 



The female cones of Conifera; should be dismembered for all 



