90 
AMERICAN JOURNAL OF BOTANY 
[Vol. io, 
and carefully agitated to remove the spores. By this method a considerable 
portion though not all of the spores were removed. To insure a complete 
separation of the spores from the mycelium which might have washed off 
into the water, this spore suspension was filtered through two thicknesses of 
a good quality of muslin, of about 20 threads to the centimeter. Most of 
the spores passed through the muslin while the mycelium was held in the 
cloth. At this time a microscopic examination of some of the filtrate was 
made to determine if any of the mycelium passed through the muslin. In 
case any of the mycelium was found mixed with the spores, the entire 
suspension was discarded. It is possible that some short mycelial threads 
escaped notice, but the writers feel certain that these were not sufficiently 
numerous to account for more than a very small part of the hydrolysis of 
the starch which actually took place. The spores were collected on a no. 2 
Whatman chemically prepared filter paper, treated with a large volume of 
acetone for 10 minutes, and collected on a tarred filter paper (no. 2 What¬ 
man). They were then immersed in ether for 3 minutes and dried to 
constant weight at a temperature of about 30° C. in a desiccator over 
calcium chloride. No germination of the spores could be obtained after 
treatment with the acetone and ether. 
The hydrolysis of the starch paste was carried out in 150-cc. pyrex 
flasks for 18 hours. The temperature differed somewhat with the different 
experiments. The amount of spore material varied somewhat, but 100 cc. 
of starch paste were always used. About 3 cc. of toluol were used as an 
antiseptic. At the end of the digestion period the system was heated for 
10 minutes in the autoclave to inactivate the enzym and was then filtered 
through a good grade of filter paper. The reducing sugars formed were 
determined volumetrically according to the method of Clark (1). 
Only in a few preliminary experiments was an attempt made to separate 
the spores from the filter paper before they were used for the hydrolysis of 
starch. This process was attended by many difficulties and was finally 
abandoned. In all subsequent experiments the filter paper on which the 
spores were caught was included with the spores in the system. Before 
the starch paste was added the spores were suspended for 3 hours in 20 cc. 
of distilled water. Tests were made to determine if reducing sugars could 
be obtained from the filter paper, but only in one case, and that within the 
limits of experimental error, were any found. No reducing sugars were 
obtained from the starch when it was suspended in sterile distilled water. 
Experimental Data 
Experiment 1 
The spores were obtained from a culture of Rhizopus tritici incubated 
at a temperature of from 22 0 to 25 0 C. The weight of the spores used in 
this experiment was not determined. Most of the spores were washed 
