1916] 
DUGGAR AND DAVIS—NITROGEN FIXATION 425 
tures enabled us to make the nitrogen determinations of both 
inoculated and uninoculated flasks from the entire contents of 
the flasks, therefore to dispense entirely with any transfers of 
culture solution or fungous felt, and to avoid the possibility 
of errors thus ensuing. 
All glassware was cleaned by standard methods; nitrogen- 
free double distilled water was used; and Merck’s reagents. 
Every experiment was set up in triplicate, also with three 
controls; that is, for every series in which a different fungus, 
a different amount or source of nitrogen or of carbon was 
used, there were 6 cultures, 3 of which were inoculated and 
incubated, while the remaining 3 were inoculated, autoclaved 
to kill the spores (since they served as controls), and were 
then incubated with the others. 
The inoculations were made from cultures on potato agar, 
fresh cultures only being employed as a source of spores or 
mycelium. The inoculation procedure was as follows for those 
forms producing spores: Numerous spores were transferred 
to a flask containing 100 ec. sterile НәО. This was agitated 
until there was an evident spore suspension, and this then 
pipetted out with a sterile pipette into a second sterile flask. 
From this last flask Lee, portions were transferred with sterile 
pipettes to each flask in the series. The controls were then 
autoclaved for 15 minutes at 15 pounds pressure. That the 
method was entirely satisfactory is shown by the fact that 
there was only a single case of contamination in all the series 
employed and no case of growth in any of the controls. Sim- 
ilarly, in the inoculation of the series with Azotobacter, loops 
of the organism were diffused in sterile water, then Le, 
portions were placed in each flask by means of a sterile grad- 
uated pipette. All transfers were made with the greatest pre- 
caution in a steamed transfer room. In the case of Phoma 
Betae, where no spores were produced, small masses of hyphae 
of approximately equal size were inserted into each flask. 
Repeated tests have shown that in the incubator rooms for 
the length of time which these experiments were permitted to 
run there is no detectable amount of combined nitrogen ab- 
sorbed by flasks of the culture solution or by flasks of dis- 
