324 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 10 



For sterilization the media were autoclaved at 16 pounds steam 

 pressure for 15 minutes. The sterile flasks were carried from the 

 autoclave immediately to a thoroughly steamed culture room 

 and inoculated when cool. 



Three methods of inoculation were used in the course of the 

 work. In the first and second series with Aspergillus niger 

 plantings of the fungus were effected by wetting a platinum loop 

 in the sterile media, placing it on the aerial spores of a potato 

 glucose agar slant culture, and then transferring the loopful of 

 spores to the medium. In the third series with this fungus a 

 spore suspension was made by pouring 10 ml. of sterile distilled 

 water on an agar slant culture, loosening the spores into the HjO 

 by means of a platinum needle and pouring the suspension into 

 100 ml. of H 2 in an Erlenmeyer flask. The flask was thoroughly 

 shaken to obtain a uniform distribution of the dilute suspension 

 and let stand over night to aid in wetting the spores. After 

 shaking again the inoculum was taken up by means of a 25-ml. 

 graduated pipette and 0.5-ml. portions placed into each flask 

 of medium. With the Sphaeropsis and Diplodia, foims which 

 did not sporulate in culture, uniform inoculations were obtained 

 by planting the culture media with small (5 mm.) discs of inoc- 

 ulum from giant petri dish cultures on a thin (2 mm.) layer of 

 potato-glucose (or sucrose) agar. As checks, several flasks of 

 each medium were planted immediately before sterilization with 

 the same amount of inoculum. All cultures in all series were 

 incubated at 28° C. in darkness, a large incubator with double 

 doors and water-jacket being used. 



At the intervals shown in the following tables 5 cultures of 

 each medium were analyzed, determinations being made of the 

 dry weight and percentage N of the fungous crop, and hydrion 

 concentration, sugar, NH, plus NH*.N, total N, NO..N, NO*.N, 

 total amino N, acid amide N, and peptid N of the culture fluid. 

 Near the end of each experiment entire cultures, including the 

 mat and medium, were Kjeldahlized to ascertain the loss or 

 gain of N and the presence or absence of the capacity of the fungi 

 for N fixation under the conditions employed. The 5 culture 

 solutions of each medium were filtered into a 500-ml. volumetric 

 flask and the mats thoroughly washed, the wash water being 



