62 MINNESOTA BOTANICAL STUDIES. 



2. Treated most satisfactorily as follows : Formaline material 

 first washed in a fresh 4 per cent, formaline solution was put 

 on freezing chamber in 4 per cent, formaline solution, and 

 transferred directly into permanent mounting medium of 4 per 

 cent, formaline. 



The thallus, after repeated attempts with xylol and cedar oil 

 as clearing media, proved too loosely constructed to cut by 

 microtome. The Osterhout freezing method was used with the 

 best results. Sections were cut 15 to 45 /J. thick, the first prov- 

 ing best for detailed structure, the second for general outlines 

 of thallus and cells. Hand sectioning proved of value only in 

 a general way. 



The staining of Dictyosph&ria, en masse or in section, proved 

 a difficult matter. The majority of the stains used had little 

 or no value. The loose structure of the thallus could not endure 

 hardening due to alcohol stains therefore, water stains were 

 used in nearly all cases. Owing to the nature of cell wall and 

 mucilaginous contents the alcohol material was not more satis- 

 factory than the formaline. The following stains gave poor 

 results : 



1. Methyl green (saturate solution in H 2 O). 



2. Fuchsin (saturate solution in 50 per cent. Al). 



3. Bismarck brown (saturate solution in H 2 O). 



4. Borax carmine (almost saturate solution in H 2 O). 



5. Ammonia carmine (almost saturate solution in H 2 O). 

 Aniline -water safranine (saturate solution in H 2 O) for four 



minutes' time proved the most satisfactory stain, showing clearly 

 structure of cell wall, needles, haptera, staining the mucilagi- 

 nous cell contents a yellow-brown. 



Sulphuric Acid (very dilute). Twenty-five minutes' time dif- 

 ferentiated clearly starch grains of pyrenoid, a dark brown. 



Nuclear Stains (alcohol material). 



DelafielcFs Hcematoxylin five minutes to twenty hours' time 

 stained the walls a bright purple, but only stained cell contents 

 a muddy brown. 



Gentian Violet (concentrated solution H 2 O), one to three sec- 

 onds' time. Differentiated pyrenoid centers clearly, but proved 

 too strong for all other structures. 



Acid Fuchsin (saturate solution in H 2 O), three hours' time, 

 proved the best nuclear stain. 



