320 METHODS IN PLANT HISTOLOGY 



one is to secure a series of stages in the development of the gameto- 

 phytes and embryo. 



For young sporocarps, up to the pale brown stage, try Gourley's 

 basic fuchsin method. Take a small piece of rhizome with a leaf and 

 sporocarps; cut, under the stain, and after the solution has stained all 

 the vascular system of the sporocarp, fix in 95 per cent alcohol, 24 

 hours; absolute alcohol, 24 hours; treat with the xylol mixtures and 

 then with pure xylol. Finally, put it into a very small bottle with 

 equal parts of xylol and carbon disulphide. Such a preparation can be 

 mounted in balsam in a shallow cell. 



For sections showing the development of the antheridium and 

 sperms, it is better to remove the megaspores from the sorus, 

 since they occasion considerable difficulty in cutting. Fix in the 

 Chicago chromo-acetic-osmic solution and stain in iron-alum haema- 

 toxylin. 



The older megaspores, with archegonia or embryos at the apex, are 

 very hard to cut. Fix in formalin-alcohol-acetic acid; wash in 50 per 

 cent alcohol; treat with 10 per cent hydrofluoric acid in 50 per cent 

 alcohol for 2 days; wash thoroughly in 50 per cent alcohol; 70 per cent 

 alcohol, 24 hours; 85, 95, and 100 per cent alcohol, 24 hours each; then 

 the usual xylol series. The need for a good paraffin is more urgent in 

 such a case than for material which is not so hard to cut. A little bay- 

 berry wax will improve a paraffin which is not quite up to grade. li 

 sections come loose from the sHde, use Land's fixative. 



The sperm, which in Marsilia has an unusually large number of 

 turns in the spiral, is easily mounted whole. When the sperms have 

 become numerous, put several megaspores upon a slide and heat gently 

 until dry. Then wet the preparation in any alcohol and stain sharply 

 in acid fuchsin. Dehydrate in absolute alcohol for 2 or 3 hours, chang- 

 ing 2 or 3 times; clear in clove oil, and mount in balsam. Such a prepa- 

 ration will often show a score of sperms in the gelatinous funnel lead- 

 ing down to the neck of the archegonium. 



Azolla is not diflEicult to obtain, and it is easy to get a series of stages 

 in the development of the micro- and megasporangia ; but it is not at 

 all easy to find the gametophytes, since the spores germinate only 

 after they have been set free by the decay of the plant. Azolla does 

 not fix well in any of the chromic-acid series, because it catches so 

 much air that it will not sink. Alcohol-formalin-acetic, or hot alco- 



