68 Martin J. Prucha 



EXPERIMENT 13 

 INFLUENCE OF MEDIA 300, 310, 335, AND 400 



The organism used for this experiment was propagated on medium 335, 

 and had been kept in the laboratory for two years and three months, 

 where it was exposed to diffused Hght, and transfers had been made at 

 intervals. When the experiment was started the organism readily produced 

 nodules on the plants, showing that its infecting power had not been lost. 



The plan of the experiment was to propagate the organism on both 

 the nitrogen-free and the nitrogenous media for a period of time, and then 

 to test these cultures for nodule production. The following procedure 

 was adopted: Media 300, 310, 335, and 400 were introduced into test 

 tubes, sterilized, and sloped. Four slopes from each of these media were 

 inoculated with the same culture of the organism. A few days later a 

 second set of four slopes from each medium was inoculated, and so on. 

 Nine such sets of agar slopes from each of the four media were inoculated 

 in one hundred and fifteen days. The four agar slopes of the same medium 

 in each set were inoculated from one of the four slopes of the same medium 

 from the previous set, so that on all the slopes of the same medium in 

 the nine sets the organism was under the influence of the same media for 

 one hundred and fifteen days. The age of the agar slopes, however, 

 differed in the respective sets. When the ninth set was inoculated, four 

 additional tubes of media 300, 310, and 400 were inoculated with a culture 

 of the organism which up to that time had been propagated on nitrogen - 

 free medium 335. These test-tube cultures are designated in table 17 

 as set X. They were fifteen days old and had been under the influence 

 of nitrogenous media for onlj^ fifteen days when tested for infecting power. 

 In order to prevent the effects of drying, melted paraffin was poured on 

 the cotton plugs after a good growth had developed. All the cultures 

 were kept at room temperature. 



The infecting power of these agar-slope cultures was tested by inoculating 

 Canada field peas. The method of growing and examining the plants was 

 the same as in experiment 12. In inoculating the plants, the growth from 

 each agar slope was introduced into 100 cubic centimeters of sterile water, 

 the number of organisms in this was determmed by the plate method, and a 

 definite quantity of this infusion was poured over the seeds in each flowerpot. 



Unfortunately, an accident happened to a large number of the flowerpots 

 in the greenhouse, and consequently the data are not complete. As far 

 as they could be obtained, they are summarized in table 17: 



