Nitrogen by A zotobacter and the Growth of the Organism. 877 
which contains basic slag as the neutralizing agent, has since been used in 
the Botanical Laboratory of King’s College for isolation and growth of 
Azotobacters. This nutrient medium is one of the simplest which have 
been used, and by the addition of 2 per cent, of agar-agar, answers equally 
well for plate cultures. 
One further point in connexion with the nutrient solution remained 
to be determined ; i. e. whether, after complete neutralization of the acidity 
of the solution due to the phosphate, any further addition of basic slag pro- 
duced a beneficial effect. Hoffmann and Hammer have shown that, so long 
as the liquid is neutral, increase in nitrogen-fixation does not follow upon 
increase of calcium carbonate in the medium ; but it seemed probable, since 
the presence of the slag appeared to give a distinct impetus to the fixation 
of nitrogen, that one or more of the constituents of the slag other than lime 
might have a tonic effect on the organism, which effect might be increased 
by the addition of a greater proportion of slag to the medium. The 
presence of o-2 per cent, of the slag was sufficient to render the liquid 
neutral to litmus-paper, so five solutions were made up, each containing 
1 per cent, mannite, 0-2 per cent, di-potassium phosphate, 0-02 per cent, 
magnesium sulphate, with the addition of 0-2, 0-4, o-6, o-8, and i-o per cent, 
of basic slag respectively. 50 c.c. of the solution containing o-2 per cent, of 
slag were put into each of three flasks, which were provided with sand slopes 
and otherwise treated exactly as in the foregoing experiment. These flasks 
were numbered from 1 to 3, and, similarly, three flasks were used for each 
of the other four liquids, the total number being thus fifteen. Each one was 
then inoculated with the suspension of Azotobacter , and Nos. 1, 4, 7, 10, and 
13 were sterilized to serve as controls. The whole series was incubated 
for seven days at a temperature of 28° C., and at the expiration of this time 
the contents of each flask were transferred to a Kjeldahl flask and analysed 
for their nitrogen content, with the following results : 
Table II. 
Flask 
Proportion 
Nitrogen 
Fixation on 
Fixation on 
Average 
No . 
of Slag . 
Content . 
| grm . Mannite . 
1 grm . Mannite . 
Fixation . 
1. 
0.2 % 
0.55 mg. 
11.98 mg. ) 
12-62 mg. ) 
2. 
3 - 
0-2 % 
0*2 % 
6-54 mg. 
6*86 mg. 
5.99 mg. 
6*31 mg. 
12.30 mg. 
4 * 
°*4 % 
0.70 mg. 
6-86 mg. 
5 - 
0 - 4 % 
7.56 mg. 
13.72 mg. ) 
14.28 mg. 
6. 
o -4 % 
8*12 mg. 
7.42 mg. 
14.84 mg. \ 
7 - 
o-6 % 
°-77 mg. 
8. 
9 - 
o-6% 
o-6% 
6.58 mg. 
6 30 mg. 
5.81 mg. 
5*53 m g. 
11.62 mg. ) 
11.06 mg. i 
1 1 .34 mg. 
10. 
0.8 % 
0-84 mg. 
11. 
12. 
o-8% 
0-8% 
6*02 mg. 
6-44 mg. 
5-i 8 mg. 
5.60 mg. 
10.36 mg. ) 
1 1.20 mg. | 
10.78 mg. 
13. 
i-o % 
0*98 mg. 
14. 
15 - 
1-0% 
1*0% 
7.56 mg. 
7.42 mg. 
6-58 mg. 
6.44 mg 
13.16 mg. ) 
12.88 mg. ) 
13.02 mg. 
