[Vor. 3 
426 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
tilled water. Both from this fact and further from the 
nature of the controls it was unnecessary to place the cultures 
in a chamber arranged to protect against combined nitrogen. 
The data presented in this paper on the determination of 
nitrogen were obtained either with the Kjeldahl-Gunning 
method,—using mercury in addition to potassium sulphate,— 
or, where nitrates were involved, with the Forster modifica- 
tion of the method mentioned. In some preliminary work an 
extended attempt was made to utilize the Folin micro-Kjel- 
dahl apparatus, but that proved inapplieable to the present 
work for the following reason: The amount of culture solution 
which it is possible to use with this method is small, and doubt- 
less would be too small to yield convincing results in view of 
the present confusion regarding the question of nitrogen fixa- 
tion in the fungi. In the light of the results obtained by Purie- 
witsch, Saida, and others, all of whom used from 50 to 100 сс. 
of culture solution, it seemed essential to employ the ‘‘macro’’ 
method and to deal with cultures as large as practicable. 
In the pioneer work of Jodin ('62) gas analysis methods 
were employed for the determination of nitrogen fixation, 
therefore through the indirect method of nitrogen loss in the 
culture chamber. Since that time all the work which may 
claim a right to be considered quantitative has been made 
with the Kjeldahl method, or with some modification of it, 
usually the Gunning. That this method is sufficiently accurate 
to detect any amount of fixation worthy of the name is evident, 
since an experienced analyst can usually secure results 
which often check to within .2 mg. However, if one does not 
observe all possible precautions, errors may creep in which 
will yield widely varying results. Chief among these possi- 
bilities in the problem of nitrogen fixation are the following: 
Impure chemicals. . 
2. Accuracy of standard acid and alkali. 
3. Indicator. 
4. Completeness of digestion and distillation. 
5. Loss of nitrogen in the transfer of the culture material, 
or felt, from one flask to another. 
x 
