ON THE USE OF CALCIUM CARBONATE IN NITROGEN 
FIXATION EXPERIMENTS* 
By P. Gainey 
Associate Professor of Bacteriology, Kansas Agricultural Experiment Station 
In reviewing the literature on nitrogen fixation by soil bacteria one 
is impressed with the great variety of media that have been employed 
by different investigators. Many of these media, while generally satis¬ 
factory, have not proved entirely so when employed by other investi¬ 
gators with slightly different environmental conditions. 
It is not the purpose of this paper to enter into any discussion of the 
relative merits of different media, but rather to call attention to a fre¬ 
quent fundamental difference and its possible bearing upon the success 
attending their use. This difference is the presence or absence of calcium 
carbonate. 
Wino^adsky (py in his original experiments on nitrogen fixation by 
anaerobic bacteria used a dilute solution of the various salts necessary to 
furnish the elements essential to growth. To this was added a simple 
sugar as a source of energy and an excess of calcium carbonate to neutral¬ 
ize the acids formed from the sugar. "Winogradsky's medium has been 
almost universally adopted for anaerobic nitrogen-fixing experiments. 
Beijerinck (2), stud5dng the aerobic Azotobacter group of nitrogen¬ 
fixing bacteria, found that a 0.02 per cent solution of Kj HPO4 in “Lei- 
tungswasser," to which was added a source of energy, furnished the 
necessary conditions for good growth of these organisms. Either the 
water or the inoculum must have furnished the other essential elements 
in sufficient quantity. The reaction of this medium was unaltered, the 
statement being made that— 
Die Nahrlosung reagiert durch das Kj HPO4 schwach alkalisch 
and that— 
Die Alkalisch Reacktion ist fur den versuch giinstig. 
Beijerinck preferred mannite or a salt of propionic acid as a source of 
energy because— 
Mannit kann nnr schwierig und langsam, Propionate durchaus nicht der Butter- 
saureganing anheimfalien. 
Beijerinck further states that— 
Die Produkte die Oxydation sind Kohlensaure und Wasser. 
However, he realized that in impure cultures from soil, organic acids 
might be formed. Beijerinck failed to'secure appreciable fixation of 
nitrogen by pure cultures. 
Lipman (j) began a study of the Azotobacter group of nitrogen-fixing 
organisms shortly after Beijerinck. He demonstrated that Beijerinck’s 
failure to secure fixation in pure cultures was due to the unfavorable re- 
' Accepted for publication June 29, 1922. Contribution No. 46, Department of Bacteriology, Kansas 
Agricultural Experiment Station. 
• Reference is made by number (italic) to "literature cited,” pp. 189-190. 
(185) 
Journal of Agricultural Research, 
Washington, D. C. 
abp 
Vol. XXIV. No. 2 
Apr. 14, 1923 
Key No. Kans.-32 
