2 Journal of Agricultural Research voi. xxiv^np. i 
triiici Saito, artocarpi Racib., delemar (Boid.) Wehmer and Hanzawa, 
maydis Bruderl, nodosus Namysl, oryzae Went and Pr. Geerligs, micro- 
sporus V. Tieg., and arrhizus Fischer. As previously explain^ (12) all 
the original cultures except those of nigricans and arrhizus were obtained 
from Mr. E. D. Eddy. Each organism was pure-lined by isolation from 
a single spore. A comparison with the original descriptions of each 
species showed no appreciable disagreement. 
METHODS 
In order to keep the stock cultures in a vigorous state of growth they 
were renewed about once each month by transferring to sweet potato 
agar in small Erlenmeyer flasks and incubated at a temperature of from 
22® to 26® C, Cultures of from 5 to 10 days old were used in the experi¬ 
ments. Preliminary investigations showed that Rhizopus spores 
germinate more readily in some good nutrient medium such as sweet 
potato decoction than in water, hence the former medium made as 
previously described (10) was used. 
In order to have available a solution of uniform composition for all 
the spore germination tests, a large volume of sweet potato decoction was 
prepared and stored in small flasks stoppered with cotton and covered 
with several thicknesses of oiled paper to retard evaporation. Each 
flask contained about 120 cc. of the decoction, which was about the 
amount required for the study of the spore germination of each organism. 
By storing the solution in small flasks the necessity for resterilizing the 
stock solution each time some of it was removed and thereby possibly 
changing its chemical composition was avoided. The loss by evapora¬ 
tion was restored by bringing the solution up to its original volume by 
the addition of distilled water. A loop of a spore suspension made in 
this medium was placed upon a clean cover slip, which was then sealed 
with vaseline in an invert^ position to a glass ring cemented to a slide 
with a mixture of beeswax and vaseline. In the bottom of a cell so con¬ 
structed was placed a small quantity of the same solution. The cells 
used were comparatively large (13 mm. high by 16 mm. in diameter) and 
provided sufficient air for the germinating spores. 
In view of the fact that some investigators have found the hanging- 
drop method of studying spore germination unsatisfactory, the writers 
made several preliminary comparative tests of different methods. 
These showed that so far as these species of Rhizopus are concerned as 
reliable results could be obtained by the hanging-drop method as by 
any of the others, as, for example, in small volumes of solution in test 
tubes. Furthermore, since repeated observations must necessarily be 
made to determine accurately the time of germination the hanging-drop 
method lent itself more readily to manipulation than the test-tube method 
in which a fresh mount must be made for each observation. The hanging- 
drop cultures were placed in the incubators as soon as possible after they 
were prepared. The incubators were manufactured by Paul Altmann, 
were electrically controlled, and ranged in temperature from 1.5® to 
50® C. Usually the temperatures in the individual chambers remained 
almost constant, varying for the most part less than a degree except in the 
lower ones, where there was sometimes a variation of 2° or 2.5° C. Pans 
of water were kept in the bottom of the chambers to keep the air moist. 
At least four and more often eight hanging drops were used at each 
temperature. 
