Nitrogen by Azotobacter and the Growth of the Organism . 879 
hydrate. In order to prevent any vitiation of results by the abstraction of 
small quantities of the solution for examination for the presence of carbo- 
hydrate, the following method was adopted. One flask more than was 
required for analysis was included in each series, and since all the flasks had 
been supplied with exactly the same quantity of culture solution, and 
inoculated with the same amount of bacterial suspension, it was assumed 
that growth would take place at the same rate in each, and ’when the 
mannite in the extra flask had been consumed, it would also have dis- 
appeared from the others. Nos. 6 and 12 were used as these extra flasks, 
and at the end of the seventh day Nos. 1 , 2 , 3, 7, 8, and 9 were analysed for 
their nitrogen content, while a drop was extracted from each of Nos. 6 and 
12 , evaporated to dryness on a glass slide, and examined microscopically 
for the crystals of mannite. Since the latter appeared to be present in both 
cases, Nos. 4, 3, 6, 10, 11, and 12 were re-incubated for another day, when 
Nos. 6 and 12 were again examined. This time no crystals appeared in the 
drop extracted from No. 6, so Nos. 4 and 5 were analysed, but No. 12 still 
contained traces of the food-stuff. The remaining three of the series were 
accordingly re-incubated, and this process of testing No. 12 each day for the 
mannite repeated until all the latter was consumed, when Nos. 10 and 11 
were subjected to analysis. In this way some idea was obtained of the 
relation between the fixation in seven days upon 50 c.c. of the solution con- 
taining \ grm.of carbohydrate, and that upon the total J grm. The results 
are shown in the following table : 
Table III. 
Flask 
No. 
Culture Solution. 
Time 
taken . 
2 
3 
4 
5 
7 
8 
9 
10 
1 1 
50 c.c. Bottomley’s solution — sterile . . 
,, with Azotobacter 
>> >> 
> 5 
>7 
50 c.c. Ashby’s solution — sterile . . 
,, with Azotobacter 
7 days 
7 „ 
8 „ 
8 „ 
7 », 
7 » 
18 „ 
Nitrogen 
Gain in 
Content. 
Nitrogen. 
0.41 mg. 
5-67 mg. 
5.26 mg. ) 
5*95 mg. 
5-54 mg. \ 
6-22 mg. 
5 * 8 i mg. ) 
6.31 mg. 
5.90 mg. j 
0.55 mg. 
4'39 mg. 
3.84 mg. ) 
4.12 mg. 
3*57 mg. \ 
4.27 mg. 
3-72 mg. ) 
4.42 mg. 
3.87 mg. [ 
Average 
gain in 
Nitrogen. 
5*4° mg. 
5-85 mg. 
3.70 mg. 
3*79 mg. 
It is obvious that nitrogen-fixation was almost complete at the end of 
seven days in both series. In Bottomley’s solution, however, the presence 
of mannite was manifested for only one day after this period, and the increase 
in nitrogen content for this day was nearly a half-milligramme ; whilst in 
Ashby’s solution, although the mannite was not completely used for eighteen 
days, yet the increase in nitrogen for the last eleven days of that period was 
scarcely beyond the range of experimental error in analysis. It is clear, how- 
