460 Keene . — Cytological Studies of the 
Methods. 
Sporodinia grandis was used for the most part in this investigation. 
The fungus was first found growing on mushrooms in October, 1911, in the 
vicinity of Columbia, Missouri. Isolations were made and the cultures 
were grown on bread. In the course of 72 to 9 6 hours, the development 
of zygospores occurred in most of the cultures. These were fixed in all 
stages, several different fixing agents being used, as Flemming’s, chrom- 
acetic, Gilson’s, Carnoy’s, and picro-acetic. The best fixation of the earlier 
stages was secured with the weak solution of Flemming. For the older 
stages a strong solution of Flemming, diluted with twice its volume of 
water, gave better results, especially where the outer coat had become 
cutinized. The material was washed in water, dehydrated by the usual 
percentages of alcohol, and infiltration with paraffin was secured by the use 
of xylol. The older stages, however, are very brittle, due to the thick walls 
of the zygospore, and the ordinary methods were not satisfactory for the 
study of serial sections. It was found, after considerable experimenting, 
that if the zygospores were allowed to stand in a weak solution of sodium 
hydroxide for 24 to 48 hours, the outer brown coat became softened and 
somewhat transparent. It was then a relatively simple matter to dissect 
the zygospores from their thick coats and the suspensors. In other cases, 
the material was carried through the killing and fixing process without 
dissection. In all cases after treatment with sodium hydroxide, the material 
was washed in running water for 12 hours in order to counteract any 
tendency to plasmolysis. It was then fixed at once in various reagents, 
here again the best results being obtained with the diluted solution of 
Flemming’s strong. It was necessary to expose the older material to this 
solution for 48 hours, as the coats are very slow of penetration. After 
washing in water, the usual grades of alcohol were used, followed by the 
grades of alcohol and cedar oil, with an exposure of 2-7 days in pure cedar 
oil. The chloroform and xylol methods were also used, but were not as 
satisfactory. The material was then embedded in paraffin and sectioned at 
5 ^ for the younger stages, and 7-30 \x in the case of the older. 
In staining, Flemming’s triple stain of safranin, gentian violet, and 
orange G was used almost entirely, although Haidenhain’s iron-haema- 
toxylin with Bismarck brown and safranin as counter-stains, and Delafield’s 
haematoxylin and eosin, were used in a large number of preparations. These 
all proved highly satisfactory in their differential powers, and brought out 
the same structures in all cases. 
Life-history and Development. 
As has already been noted, Sporodinia grandis readily produces 
zygospores on bread cultures. The ends of certain aerial hyphae enlarge 
and fuse. Externally, there is little or no difference between these two 
