Apr. 14.1923 Calcium Carbonate in Nitrogen Fixation Experiments 187 
the medium, even when present in large excess. This is an important 
consideration, however, since if it has no toxic effect upon the organisms 
it may be added in excess of initial requirements and thereby tend to 
maintain a favorable reaction throughout the experiment. 
There are a number of isolated experiments such as those cited above 
showing the effect of calcium carbonate upon the growth of nitrogen¬ 
fixing organisms. In the course of some experiments, conducted by the 
writer, in which the relative growth of Azotobacter from a large number 
of different soils was compared there was an opportunity to observe the 
effects of CaCOg on nitrogen fixation. 
METHODS 
The medium employed had the following composition: Mannite 20 
gm., Kg HPO4 0.2 gm.. Mg SO4 0.2 gm., NaCl 0.5 gm., FeCLg trace, and 
water 1,000 cc. Two-hundredths gm, of CaClg was sometimes added, 
although in most cases because of the high calcium content of local soils 
the CaClg is not essential and was without effect. In those tests to which 
no CaCOg was added the medium was always rendered slightly alkaline 
to phenolphthalein with sodium hydroxid. When CaCOg was to be 
added the medium was sometimes first rendered slightly alkaline to 
phenolphthalein and at other times the reaction was unaltered prior to 
the addition of the CaCOg. Fifty cc. of the medium were placed in 300-cc. 
Erlenmeyer flasks, and the CaCOg was added in the form of sterile powder 
just prior to inoculation. No superiority is claimed for this medium 
over a score of others that might have been used. Obviously a medium 
with as variable composition as that containing tap water or soil extract 
would be unsuited for comparative work that must extend over a long 
period of time. 
Samples were always set up in duplicate and total nitrogen determina¬ 
tions made on the whole sample. Total nitrogen determinations were 
also made in duplicate upon the inoculum. The inoculum consisted of 
10 cc. of the supernatant suspension prepared by shaking one part of 
soil (50 to 100 gm.) with two parts of water and allowing to settle long 
enough for the larger particles to sink to the bottom. It is believed that 
such an inoculum is more representative of a mass of soil than 5 gm. of 
soil, and at the same time the quantity of solid material added is not 
sufficient to interfere in the least with total nitrogen determinations. 
Incubation was at room temperature for three weeks. In estimating 
the quantity of nitrogen fixed that present in the inoculum was deducted. 
Only the average of check determinations were recorded. 
Frequent examinations of the cultures were made, both macroscopically 
and microscopically to ascertain whether Azotobacter were present. If 
Azotobacter make an appreciable growth it can usually be recognized by 
the appearance of the film. A microscopic examination of an unstained 
mount from such a film will reveal an unmistakable picture. A film is 
sometimes encountered which at certain stages in its development resem¬ 
bles quite closely an Azotobacter film, which, under the mircoscope, is 
found to be composed almost entirely of filimentous fungi, no organisms 
typical of Azotobacter being observed. In other instances nontypical films 
examined under the microscope would be found to be composed largely 
of fruiting fungi, the spores of which often closely resembled individual 
cells of Azotobacter. 
