Keene—Studies in Zygospore Formation. 1199 
Mixed cultures have been kept growing, and here also the 
zygospore production has been profuse. 
For cytological studies, material ranging from three days 
to six months in age was fixed. Various fixing reagents were 
employed: Flemming’s solution of various strengths, chrom- 
acetic, Bouin’s, and Gilson’s. The younger stages were fixed 
directly on the agar and small portions of the agar were car¬ 
ried through the various stages of washing, dehydrating, and 
infiltration with paraffin. Because the young gametophores are 
much convoluted and stand erect from the surface of the sub¬ 
stratum, it is difficult to obtain satisfactory sections. 
The same difficulties of technic were encountered here which 
were met with in Sporodinia grandis, but in even a more serious 
form. The older zygospores are extremely brittle because of 
the heavy walls and the large amount of reserve material con¬ 
tained within the zygospore. The most satisfactory serial sec¬ 
tions were obtained from material that was fixed in Flemming’s 
solution, thoroughly washed and treated with hydrofluoric acid 
while in 95 per cent alcohol. That the resulting structures are 
normal and not the result of this treatment is evident from the 
fact that in material treated in the usual ways, the same struc¬ 
tures have been found but it is almost impossible to obtain them 
in serial form because of their torn condition. 
For infiltration with paraffin, both the xylol and the cedar 
oil methods have been employed with equal success. The cedar 
oil method is probably better for the older stages. 
The younger zygospores were cut into sections of from 5 to 
10 microns while the older ones were cut into thicker sections 
ranging from 10 to 60 microns. Owing to the size of the zygospore 
inclusions found at maturity, the thick sections were more satis¬ 
factory for all but the nuclear studies. For affixing the sec¬ 
tions to the slides both the albumen-glycerin and the fish-glue 
fixatives were used. 
The Flemming’s triple stain was given the preference al¬ 
though anilin-safranin alone was used in many cases. The 
nuclei stand out quite clearly in such preparations. Gram’s 
anilin-gentian-violet and iodine stain gave some very beautiful 
preparations. They closely resemble preparations made with 
Heidenhain’s iron-alum-haematoxylin combination but are very 
much more quickly made. 
