Jan., 1921] 
WANN — FIXATION OF FREE NITROGEN 
3 
involved in its analysis, a solid medium was chosen because of the very 
long-continued, vigorous growth produced on it. So far as has been 
observed, solution cultures, at least when unaerated, do not give a very 
extended or abundant development of these organisms. Since many pre- 
vious experiments have also shown that these forms do not grow in pure 
culture in the complete absence of combined nitrogen, no attempt was 
made to include such cultures in the experiments. 
Two experiments were performed, the first in the winter of 191 7-18 
and the second in the summer of 191 9. In both cases the following mineral 
nutrient solution was employed as a standard for the preparation of the 
media: 
NH4NO3 0.5 gram 
MgS04 0.2 gram 
K2HPO4 0.2 gram 
CaCl2 0.1 gram 
FeS04 trace 
Distilled water 1000 cc. 
With the nitrogen content of this solution as a basis, the NH4NO3 was 
replaced in the various series of 191 7-1 8 by glycocoll, asparagine, 
(NH4)2S04, and CaCNO.Oa, and by urea, (NH4)2S04, and Ca(N03)2 in 
1919, the nitrogen content as such being approximately the same in all 
media. Each of these sources of nitrogen was used in duplicate series, 
to one of which glucose was added. (In 1919 NH4NO3, (NH4)2S04, and 
Ca(N03)!> were also used in series to which mannite was added.) No change 
was made in the other constituents of the full nutrient solution, so that in 
all series these salts were present in the proportions indicated above. 
The 191 7-18 experiment included the following series, arranged according 
to nitrogen sources and presence or absence of glucose : 
Series i. Glycocoll (1.07 gr. per liter) — no glucose. 
Series lA. Same solution, with i percent glucose. 
Series 2. Asparagine (0.942 gr. per liter) — no glucose. 
Series 2A. Same solution, with i percent glucose. 
Series 3. Ammonium sulphate (0.942 gr. per liter) — no glucose. 
Series ^A. Same solution, with i percent glucose. 
Series 4. Ammonium nitrate (0.5 gr. per liter) — no glucose. 
Series 4^. Same solution, with i percent glucose. 
Series 5. Calcium nitrate [Ca(N03)2.4H20, 1.475 gr. per liter]— no glucose. 
Series 5^4. Same solution, with i percent glucose. 
In making up the media for the above series, sufficient nutrient solution 
with any one nitrogen source was prepared to supply both the series without 
glucose and the series with glucose. For these solutions the required 
amounts of the several constituents, with the exception of ferrous sulphate, 
were weighed out individually and dissolved in the proper volume of distilled 
water. (A stock solution of ferrous sulphate was prepared by dissolving 
