SECRETION OF DIASTASE BY PENICILLIUM CAMEMBERTII 243 
pounds pressure for 15 minutes. The culture solutions and vessels 
were weighed before sterilization, and just before analyzing the solu- 
tion, each flask with its contents was brought up to the original weight 
by the addition of water. This was necessary in order to correct the 
change in concentration of the solution which results from the evapora- 
tion of water through the cotton plug of the flask. 
The analysis of the original starch content was made after steriliza- 
tion. After growth had occurred, the fungous mycelium was removed 
by filtering into a Gooch crucible to determine its dry weight after 
the method of Knudson (1913). The filtrate was caught in a 100 cc. 
test tube. Twenty cubic centimeters of this filtrate were removed 
and the undigested starch was determined by the alcoholic precipi- 
tation method described. The acidity or alkalinity of the filtrate to 
phenolphthalein and methyl orange was noted. All determinations 
were made in triplicate except the determinations of the original 
starch content, which in most cases was made in duplicate. 
Method of Inoculating Cultures. — In inoculation the spores from the 
stock culture of the fungus were transferred by means of a sterile 
platinum needle to a second test tube containing sterile redistilled 
water. An equal number of drops of this spore suspension was added 
by means of a sterile pipette to each culture flask. This method of 
inoculation removes the danger of transferring soluble and suspended 
matter derived from the original stock culture to the experimental 
culture medium, as occurs in the method described by Hasselbring 
(1908). This modification was found necessary in view of the extreme 
sensitiveness of the fungus used to traces of inorganic nutrients, and 
of the possibility of introducing fragments of mycelium and organic 
matter. It was found that the same number of drops of the spore 
suspension must be used for each flask in a given series, because, 
within limits, the number of spores used in inoculation influences 
the amount of digestion. This is shown by the following experi- 
ment: 
A set of 24 cultures was prepared, containing 0.8 percent of starch 
in distilled water which had previously been shaken up with cane sugar 
charcoal. One half of these cultures was inoculated with 3 drops of a 
suspension of spores of Penicillium camemhertii in sterile distilled 
water, while to the other half 21 drops of the suspension were added. 
The results summarized in Table III are the average of triplicate 
cultures: 
