534 
ADRIAN J. PIETERS 
For the experiments cultures were made in Erlenmeyer flasks of 
Jena or of revsistance glass except in the few cases that will be noted, 
the flasks containing icq or 200 cc. of culture fluid. This medium 
was inoculated by placing in it small bits of pea agar in which the 
fungus was actively growing. Pea agar was used instead of the 
beef gelatin used by Kauffman as it was found more convenient, 
both to make and to handle. It is easily made by putting about 
100 peas in a liter of water together with sufficient agar to make the 
strength desired. This is heated in an autoclave for an hour at about 
15 to 20 pounds pressure and, after filtering, is ready for use. It has 
been found a good culture medium, and has a further advantage over 
gelatin in not becoming soft in warm weather and in not being lique- 
fied by the action of the fungus. When a set of cultures was to be 
inoculated the bits of agar were cut as nearly of the same size as 
possible, about i square millimeter in size. After having grown for 
the necessary length of time the entire mass of mycelium was placed 
in a dish of sterile distilled water and allowed to stand for half or three 
quarters of an hour. It was then cut into pieces of as nearly the same 
size as possible and usually of about one square centimeter in area. 
These pieces were transferred to fresh sterile distilled water and 
were sometimes washed a third time before being placed in the solu- 
tions in which sporangia or oogonia were to be made. For the latter 
solutions glass capsules containing 25 cc. of solution were used. All 
of these cultures were in duplicate, and sometimes in quadruplicate. 
Condition of the mycelium. — For studying the effect of food previ- 
ously consumed, on a mycelium after transfer to haemoglobin, or to 
some other suitable medium for the production of oogonia, or to water 
for the production of sporangia, it is important that the mycelium be 
actively growing, and that, in all the experiments, it shall be as 
nearly as possible in the same physiological condition. If a flask con- 
taining ICQ cc. of pea extract be inoculated, the resulting growth will 
reach the surface after four or more days, depending upon the species. 
When the surface of the liquid is reached, a mat is formed, which, 
after a few days, ma^^ become quite thick and firm. At this stage 
the mycelium is in a very different condition from that in which it is 
just before reaching the surface. In the latter case the hyphae are 
all actively growing, the tips being, of course, the most active; w^hen a 
mat has formed, and especially if the mat has been allowed to become 
thick, the lower parts of the mycelium are dead or dying while the 
