Nitrogen by Azotobacter and the Growth of the Organism. 883 
lapse of about ten days, while at the end of the same period a prolific 
growth was apparent in the vessels containing the sand slopes, the particles 
of the white quartz sand which had been used having become almost black, 
due to the darkening of the film of Azotobacter which thus evidently covered 
each grain. The better aerobic condition maintained by the presence of the 
sand was also effective in assisting to the purity of the cultures, any anae- 
robic Clostridium with which they had become contaminated developing 
here with much greater difficulty than under the less aerated conditions 
found in the ordinary liquid cultures. Clostridium , where present, was also 
seen to develop decidedly better in 100 c.c. cultures than in 50 c.c. ones, the 
deeper layers of the larger volume being obviously more suited to its 
requirements than those of the smaller culture. The presence of sodium 
also seemed to favour the growth of these butyric organisms, and more 
particularly was this evident in the New Jersey medium, where, if the 
feeble growth of Azotobacter became contaminated with Clostridium , the 
latter developed vigorously, while the further development of the former 
was inhibited. This is due to the fact that no excess of the neutralizing 
agent was present in the medium, to correct the acidity of the products 
of vital activity of Clostridium , the accumulation of acid products proving 
fatal to Azotobacter. 
Conclusions. 
The conclusions which have been arrived at as a result of this investiga- 
tion appear to be of some importance in connexion with the production of 
large quantities of vigorous Azotohacters, especially when they are required 
for soil inoculation, in which case the strongest possible cultures are 
desirable. The main points are as follows : 
In the first place, the presence in the medium of an excess of calcium, 
or magnesium carbonate, or basic slag, as a neutralizing agent, is more 
advantageous than that of sodium hydrate ; and the former substances not 
only assist the rapidity of the growth, but also help to maintain its purity. 
The figures obtained show clearly that the presence of sodium salts is 
not only unnecessary, but exerts a depressing influence on the activity 
of Azotobacter as regards the fixation of nitrogen. The beneficial effect of 
the sodium-chloride solution in inoculating agar plates is due to the fact 
that this liquid is isotonic with the cell-contents, a solution of similar 
concentration of many other salts answering the purpose equally well. 
Basic slag is an excellent substitute for precipitated chalk as a 
neutralizing agent in the nutrient medium, and it evidently also contains 
ingredients which exert a tonic effect upon the organism, increasing its 
activity in connexion with nitrogen-fixation by as much as 23 per cent. 
Exactly which of the constituents of the slag have this influence is not 
known, but probably the iron and manganese present are the most important 
3 M 2 
