148 LEVINE: CYTOLOGY OF HYMENOMYCETES 
Malt-beef agar cultures sixty-eight hours old were imbedded 
in paraffin and sectioned. The cultures were made directly by 
sowing spores in Petri dishes or the spores were allowed to germi- 
nate from twelve to twenty-four hours in the Van Tieghem cells 
and were then transferred to Petri dishes. Both methods gave 
satisfactory results. The mycelium reaches a diameter of a 
centimeter on the surface of the agar within three days. Entire 
masses of mycelium of this size, together with the agar, were cut 
out and immersed in fixing solutions so as to disturb the hyphae as 
little as possible. The hardening with alcohols must be very 
carefully done. If dehydration is too rapid, the agar shrivels. 
Considerable difficulty is encountered in staining such sections 
since the agar as well as the fungus takes the stain. In using the 
triple stain very short exposures and dilution of the orange G are 
necessary. Sections of material three days old show both binu- 
cleated and uninucleated cells making up the mycelium. Long 
multinucleated cells are also found, which are the germ tubes of 
spores that germinate late. Cultures of the same age do not all of 
course show the same stage of development. The cytoplasm (PL: 
4, FIG. 3, 4) is less dense in these hyphae and stains better. The 
vacuoles are large and extend across the entire width of the cells. 
The structure of the nuclei in the uninucleated cells appears very 
clearly (PL. 4, FIG. 3). The chromatin is composed of delicate 
strands distributed irregularly through the nuclear cavity. The 
nuclei of the binucleated cells as shown in FIG. 4, PL. 4, are smaller 
but show the same structure as those in the uninucleated cells. In 
cultures three days old, clamp connections and hyphal anastomoses 
were first noticed. Lyman (1907) reports for Corticiwm roseo- 
pallens that clamp connections may be found on germ tubes imme- 
diately after spore germination. Cultures, from spores sown in 
Petri dishes in malt-beef agar seven days old, make a layer of myce- 
lium covering the entire surface of the agar. The mycelium from 
such cultures shows considerable numbers of multinucleated cells. 
Their cytoplasm is dense, the vacuoles are small and they resemble - 
the multinucleated cells in the younger cultures described. Binu- 
cleated cells similar to those observed in cultures three days old 
appear more frequently and uninucleated cells are also observed — 
; 
occasionally. The latter are long and their cytoplasm shows large ‘a 
