CHEMICAL STIMULATION OF GROWTH OF ASPERGILLUS NIGER 337 
The addition of zinc, etc., where indicated was to the entire solution 
used in the preparation of cultures having the same concentration of this 
heavy metal, etc., and not to the individual flasks. Exceptions to this 
rule, however, were frequently made when only two or three duplicate 
cultures were prepared. Zinc was always added from one or two stock 
solutions (ZnS04.7H20, Baker's Analysed "). One contained 2.5 mg. 
zinc per cubic centimeter {i.e., 11. o mg. ZnS04.7H20 per cc.) ; the other 
0.025 mg. zinc per cubic centimeter. 
The flasks, each containing 50 cc. of nutrient solution, measured in a 
50 cc. graduate, were sterilized at 14^ lbs. for 20 minutes. 
Inoculations of the flasks were always from bread cultures in jelly glasses 
having tinned covers. In order to maintain uniform moisture conditions, 
the bread (a portion of a slice of fresh bread one inch thick) was supported 
in the center of the glass by means of a horizontally placed microscope 
slide so that it did not come in contact with the water in the bottom of the 
glass. In their preparation the water should be omitted until after steriliza- 
tion (i4>^ lbs. for 30 minutes). Immediately after sterilization and while 
still hot, the proper amount (50 cc.) of sterile water (it should be auto- 
claved at the same time as are the cultures) was added. In this manner a 
large surface of growth for the organism was obtained under fairly uniform 
moisture conditions, and the cultures were ready for inoculation even 
after several months. Enough spores^ were added from the bread culture 
by means of a platinum loop to make a visible and apparently almost 
continuous layer on the surface of the solution in the flask. Immediately 
after inoculation the flasks were placed in an incubator (with exclusion of 
light) at 30-31° C. for seven days. 
When harvested, the membrane, together with the solution, was thrown 
on a washed and weighed filter, washed twice with distilled water and dried 
at 103-105° C. for four days. 
The yields, while given to three places, are probably reliable to the 
second place only. 
It will be noted that the experimental conditions are not entirely uniform. 
Particularly is this the case with the A. niger cultures used. The A. niger 
culture used in these experiments was obtained originally from the "Inter- 
nationalstelle fiir Pilz-Kulturen," Amsterdam. Later the two one-spore 
cultures referred to above as W and Y were isolated from this culture in 
the manner described in an earlier paper (65). 
The two strains thus isolated were kept on agar slants (i % each of pep- 
tone, sucrose, and agar), in the incubator at 30-31° C. Sub-cultures (on 
bread) for the inoculation of the flasks were also kept in the incubator at 
30-31° C. The stock cultures were carried in duplicate, transfers being 
at irregular intervals. A duplicate stock culture once having been used, for 
the preparation either of new stock cultures or of a bread culture, was 
^ Approximately four milligrams dry weight. 
