Nitrogen by Azotobacter and the Growth of the Organism. 885 
liquid is, however, to be advocated as assisting in the aeration of the culture, 
and thus inhibiting, to some extent, the growth of any contaminating 
Clostridium. 
The use of sand slopes, as a means of increasing the surface, and there- 
fore the supply of oxygen and nitrogen to the organisms, is to be strongly 
recommended, the adoption of this method resulting in a more rapid, 
vigorous, and healthy growth, giving a much greater increase in nitrogen in 
a given space of time, and being much more free from contamination by 
anaerobic organisms than is the method of ordinary liquid cultures. 
The use of the sand-slope method of culture, with Bottomley’s medium 
in volumes of 50 c.c., results in the fixation of 14 mg. of nitrogen per gramme 
of mannite consumed, when the most vigorous organisms are used for 
purposes of inoculation.. 
Another important result of the present investigation is the confirma- 
tion which it gives to the statement of Gerlach and Vogel, that the nitrogen- 
fixing activity of Azotobacters decreases as their age increases, since in the 
latter part of the work, when the organisms had presumably become 
weakened by successive subculture under artificial conditions, they were 
able to fix only between 8 and 9 mg. of nitrogen, their yield in the earlier 
part of the work having been 14 mg. per gramme of carbohydrate consumed. 
In conclusion, my grateful thanks are due to Professor W. B. Bottomley 
for the many helpful suggestions given and the kindly interest maintained 
during the progress of the work. 
Addendum. 
As a result of the unexpected fixation of nitrogen obtained on 1 grm. 
of mannite in a medium containing 1 per cent, of slag, nine solutions were 
made up in order to test the effect of increasing proportions of this neutraliz- 
ing agent. These solutions each contained 1 per cent, mannite, 0-2 per cent, 
di-potassium phosphate, 0-02 per cent, magnesium sulphate, with the addition 
of 1-2, 1*4, 2*0, 2*8, 4 *q, 5*o, 6*o, 8*o, and io-o per cent, of basic slag respectively. 
50 c.c. of the solution containing i-2 per cent, were put into each of three 
flasks, and 5 ° grm. of sand were added to form a slope. These flasks were 
numbered from 16 to 18,. and, similarly, three flasks were used for each of the 
other solutions, the total number being thus twenty-seven. Nos. 16, 19, 22, 
25, 28, 3 1 , 34 , 37 , and 40 were autoclaved to serve as controls, and the whole 
series was incubated for seven days at a temperature of 28° C. They were 
then analysed for their nitrogen content, and the total results obtained with 
the varying proportions of slag are as follows : 
