i 86 
Journal of Agricultural Research 
Vol. XXIV. No a 
action of his media. The media adopted by Lipman was composed of 
tap water i,ooo cc., mannite 15 gm., K2 HPO4 0.5 gm., Mg.S04 0.2 gm., 
a drop of 10 per cent solution of ferric chlorid, and enough sodium 
hydroxid to make the solution slightly alkaline to phenolphthalein. 
Lipman showed that within certain limits the quantity of nitrogen fixed 
was proportional to the quantity of sodium hydroxid added. He further 
demonstrated that the addition of CaCOg, even to the medium made 
alkaline to phenolphthalein with sodium hydroxid, rendered it more 
favorable for nitrogen fixation. With regard to the influence of cal¬ 
cium carbonate Lipman (4) says: 
It is clear therefore, that the presence of calcium carbonate stimulated growth either 
directly by furnishing calcium, or indirectly by making available more phosphorus, 
sulphur, and magnesium. 
However, Lipman apparently lost sight of the value of calcium car¬ 
bonate, for he did not recommend its use in his laboratory guide (5). 
Ashby (j), apparently following the lead of Lipman, proposed that 
the acidity arising from the phosphate be neutralized with sodium 
hydroxid and in addition an excess of calcium carbonate be added. The 
medium proposed by Ashby has been more widely used than any other. 
It has the following composition: Distilled water 1,000 cc., mannite 12 
or 20 gm., Mg.S04 0.2 gm., KHg PO4 0.2 gm., NaCl 0.2 gm., CaS04 o.i 
gm., and 0.5 gm. of CaCOg to each culture of 75 or 100 c. c. The phos¬ 
phate is dissolved separately in a little water and made neutral to phe¬ 
nolphthalein with sodium hydroxid. Ashby found that the presence of 
calcium carbonate favored nitrogen fixation and that Azotobacter would 
sometimes develop in the presence of calcium carbonate but would not 
form a film if the carbonate were left out. Ashby also found that mag¬ 
nesium carbonate was even more efficacious than calcium carbonate, 
thus showing that calcium was not the essential constituent. Other 
investigators have since shown that other basic compounds can be sub¬ 
stituted for calcium carbonate. 
In addition to the three types of media just mentioned Lohnis and his 
students have made rather extensive use of soil extract to which was 
added K2HPO4 and mannite or some other simple organic source of 
energy. In comparing the fixation of nitrogen in a medium of this type 
with and without the addition of calcium carbonate Lohnis and Pillai 
(7) found, as a rule, slightly greater fixation where the carbonate was 
added. However, Lohnis failed to adopt'the use of calcium carbonate 
generally in his work, or to recommend its use in his laboratory guide (6). 
It remained for Stoklasa {8) to produce the necessary evidence for a 
correct understanding of the function of calcium carbonate in nitrogen- 
fixation experiments by demonstrating quantitatively the formation of 
organic acids in cultures of Azotobacter. A survey of the accumulated 
literature on the subject will show, however, that many investigators 
failed to realize the significance of Stoklasa's results. 
Practically all investigators agree that a neutral or alkaline reaction 
is desirable, if not essential, for the best development of nitrogen-fixing 
organisms. In most work some effort is made to adjust the medium to an 
alkaline reaction before inoculating, but in many cases no effort is made 
to maintain such a reaction. Even the influence of the inoculum upon 
the initial reaction has usually not been taken into consideration. 
So far as the writer is aware no one has ever reported a detrimental 
effect upon nitrogen fixation from the presence of calcium carbonate in 
