12 . Martin J. Prucha 



powder with 140 cubic centimeters of water; after standing for a few 

 iiours, the clear hquid is decanted and is then ready for use. Seed was 

 kept for three hours in this solution. According to Professor Wilson's 

 results, seed treated in this way is completely sterilized. This method 

 of seed sterilization will later be published in detail. 



That the methods of seed sterihzation employed were effective was 

 proved by a large number of experiments, and particularly by experiments 

 with soy beans. The organism producing nodules in soy bean is not 

 present in the soil of this region, and in all the experiments made not a 

 single nodule developed in the cultures in which sterilized seed was used 

 unless the plants were subsequently inoculated. Unsterilized seed oc- 

 casionally produced plants with nodules. 



Sterilization of media and soil 



All the media used for pure cultures, if in test tubes of small volume, 

 were sterilized in an autoclave for fifteen minutes at 120° C. 



In experiments 10, 11, 12, and 13 it was necessary to sterilize the soil 

 in which the plants were grown. Three-inch flowerpots and glass tum- 

 blers, each containing about 300 grams of soil, were used for this purpose. 

 These were sterilized for three hours at 120° C, in a large canner's retort. 

 This retort was found very useful, since several hundred of the flowerpots 

 could be sterilized at one time. 



Method of growing the plants 



In experiments 1, 2, 3, 4, 5, and 6, the plants were grown in water 

 cultures; in experiments 7, 8, 9, 10, 11, 12, and 13 they were grown in soil. 



For the water cultures glass vessels were employed. The vessels were 

 filled with the nutrient solution and the opening was covered with par- 

 affin paper. The seed was then germinated in a moist chamber, and 

 when the radicle was about three centimeters long it was inserted into 

 the solution through a small hole in the paraffin paper, allowing the coty- 

 ledons to rest on top of the paper. 



For the soil cultures flowerpots and glass tumblers were used. These 

 were filled with sandy soil, were covered with paper, and, for experiments 

 10, 11, 12, and 13, were sterilized. The seed was planted directly in the 

 soil, the paper covers being kept on until the seedlmgs began to push 

 them off. 



