Mar., 1921] HAENSELER GROWTH OF ASPERGILLUS NIGER I5I 
replaced by sterile distilled water. After cooling, the solutions were forced 
by air pressure through glass tubes into burettes and the proper amount of 
each was carefully measured into the culture flask. All glassware and stop- 
pers were sterilized either in an autoclave or in a hot-air chamber immediately 
before using. In order to reduce the chances of outside contamination, the 
cultures were made up and inoculated in a dust-proof inoculation chamber. 
A number of uninoculated check cultures proved that they had been made 
under perfectly sterile conditions. 
It would have been more convenient to make up the cultures before 
sterilization, but this method was not considered advisable on account of 
the decomposition of the salts which occurs while heating a mixed salt solu- 
tion. Some of the solutions employed, especially those with a relatively 
large amount of KH2PO4, were quite unstable at the boiling point, and even 
at 30° C. a few of the more concentrated cultures contained a slight pre- 
cipitate. The stock solutions were, therefore, sterilized separately and the 
culture solutions were prepared from these under aseptic conditions. 
Baker's analyzed salts were used throughout. On account of the un- 
certainty as to the exact amount of water of crystallization in the calcium 
nitrate, this salt was freed from its water of crystallization by carefully 
fusing and dehydrating at a final temperature of 150° C. A fine grade of 
commercial granulated sugar was used. 
The cultures were well shaken and inoculated heavily with spores from 
a one-week-old agar culture of Aspergilhis niger. Enough spores were 
transferred in the inoculation to produce a very thin, but visible, uniform 
film on the surface of the culture. This heavy inoculation was found neces- 
sary to get a uniform growth. Light inoculations were apt to give ''islands " 
of growth instead of a uniform film over the entire surface. A preliminary 
test to determine the possible error caused by unequal inoculation showed 
that the amount of inoculum may be varied considerably without afTecting 
the yield, provided the sowing is uniform. The amount of inoculum used in 
these experiments could be reduced to one half or doubled without affecting 
the yield. 
The cultures were incubated at 29° to 30° C. for seven days. It has 
been shown by Brenner (2) and others that Aspergillus niger in a good nu- 
trient medium makes its full growth in from three to five days, and that 
there is a gradual loss in dry weight after that time. A seven-day growing 
period was chosen in this work in order to give the poorer cultures a chance 
to get their full development. Under the conditions of these experiments 
it was found that harvesting could be delayed until the seventh day without 
affecting the results appreciably, as was shown by an experiment to determine 
the growth curve in one of the best solutions used in this work (culture 
R1C8, series 3). In this experiment fourteen cultures were inoculated and 
two were harvested each day for seven consecutive days. The graph in 
figure I , which is self explanatory, shows the result of this experiment. From 
