DopGE : METHODS OF CULTURE OF ASCOBOLACEAE 153 
For the serial sections, Heidenhain’s iron-hematoxylin and 
Flemming’s triple stain were used. If longitudinal sections of 
hyphae were desired the blocks were cut parallel to the original 
upper surface of the culture medium. The ascogonia could also 
be located more easily in sections cut in this plane. The coils 
of the ascogonia do not appear to be oriented in any particular 
plane, and a section parallel to the upper surface of the agar is as 
favorable as any for their study. 
The nutrient medium most frequently used for artificial cul- 
tures was made by soaking 12-14 g. of agar over night in 500 c.c. 
of tap water and then adding to it 500 c.c. of filtered decoction of 
goose dung, obtained by allowing about 100 g. of the dung to 
remain in a liter of warm water for a few minutes. The mixture 
was heated in an autoclave at 120° C. for 30-40 minutes or in a 
steam sterilizer for a longer time. It was then filtered with a hot 
water filter and further sterilized for 30 minutes at 120° C., or 
intermittently for three days at 100° C. Another nutrient decoc- 
tion used with good results when a medium with little color was 
desired, was made by heating about 2 kg. of common garden soil 
man oven at 180° C. for an hour. A liter of a filtrate obtained 
from this soil while still warm was added to 12 g. of agar. 
The decoctions of goose dung were always strongly alkaline. 
During the sterilizing process the ammonia was largely driven off 
so that the medium was only very slightly alkaline to litmus. 
After the ascocarps had ripened on the medium, tests made with 
litmus gave sometimes an acid and sometimes an alkaline reaction, 
the one occurring as often as the other. The medium made with 
an extract of heated soil was very slightly acid. Petri dishes 
S~10 cm. in diameter and 1~2 cm. high, with as thin bottoms as 
could be obtained, were preferred as culture dishes on account of 
the method of observation employed. The plates were poured so 
that the medium was about 3 mm. deep and left to harden without 
disturbing, 
SPORE GERMINATION 
ave  apgadg to obtaining artificial cultures of as large a 
the "Of species as possible, experiments were made to determine 
most favorable conditions for the germination of the spores. 
