352 BOTANICAL GAZETTE [NOVEMBER 
this theory we may thus conceive that the layer in which the hyphae 
were present would be permeated with their excretions, which in 
this paper have been termed the “‘staling substance or substances”’ 
(cf. BALLS 1, pp. 559, 576 ff.). Toward the region of the holes 
the concentration of this staling substance would be less and less, 
since here it is diffusing through to the other layer. This decreasing 
gradient of concentration would therefore act as a directive stimulus 
to the hyphae, causing them to grow toward the regions of less and 
less concentration, and thus through the holes. 
In order to prove this. hypothesis, the problem before us was 
to obtain, if possible, this staling substance free from mycelium, 
and determine whether, if placed in a sporeless layer, it would 
prevent the hyphae from growing into that layer. Although cane 
sugar and glucose were tried in order to obtain vigorous cultures of 
Rhizopus mycelium, and hence a strongly staled solution, the results 
were unsatisfactory. No medium tried gave anywhere near the 
vigorous growth that developed in turnip juice, prepared by pressing 
the juice from autoclaved white turnips. 
Several experiments were performed, with unsatisfactory results, 
as regards a turning away from the medium containing the staled 
juice, the failure probably being due to the fact that the staling 
substances were not of the requisite strength. It was eventually 
found that the desired effect could be produced by allowing the 
juice to stale for from 3 to 4 weeks, that is, to grow the fungus in 
it for that length of time. For this purpose sterilized, conical 
flasks containing 10-15 cc. of sterile turnip papi were inoculated 
with Rhizopus and kept in an incubator at 25°C. In a few days 
a white weft of mycelium developed, and later sporangia appeared. 
By the end of 3 weeks the mycelium had taken on a brownish color, 
and growth had apparently ceased. The liquid remaining was 
now poured out of the flask. It gave an acid reaction to litmus 
and had a slightly sour odor of malt. Chemical tests showed the 
entire absence of oxalic acid or oxalates. The staled solution was 
now evaporated to one-half its volume, that is, to double strength, 
at laboratory temperature under reduced pressure. For making 
up the medium for the films, either one part of 6 per cent agar to 
3 parts of the staled solution, or equal volumes of 3 per cent 
