98 BACTERIA IN RELATION TO PLANT DISEASES. 
growth has, however, been obtained in some instances, it would seem, on soils practically 
destitute of combined nitrogen, and also, I believe, in an atmosphere destitute of all traces 
of combined nitrogen, 7. e., of ammonia and nitric acid. 
Figure 32 shows a pea grown to maturity in a closed space on nitrogen-free sand (on 
which oats and buckwheat starved) by adding soil extract containing the nitrogen root- 
nodule organism. This water extract contained only 0.15 mg. of nitrogen, and the hermeti- 
cally sealed carboy was opened to the date of the photograph (fiftieth day) but three times 
and then for a few moments only to introduce a measured quantity of pure washed carbon 
dioxide (6 liters) necessary for the growth of the plants. The capacity of the carboy was 
44 liters and while the contents of the air 
in combined nitrogen was not deter- 
mined it could not have been over a 
small fraction of a milligram. The pea 
grew for a period of 4 months and 
fruited, yielding a total dry weight of 
10.359 grams, of which 233.5 mgs. were 
nitrogen. Some nitrogen was also re- 
covered from the sand, making (after 
proper deductions for nitrogen in the 
seeds, etc.) 248 mgs. of fixed nitrogen. 
The oats and buckwheat made only a 
very starved growth and finally perished 
without fruiting. 
It will be remembered that Boussin- 
gault got no growth and no nitrogen 
assimilation in a closed space under 
sterile conditions. This is exactly 
Boussingault’s experiment plus the addi- 
tion of soil extract containing root- 
nodule bacteria, the result being entirely 
different. 
The bacteriologist finds that in 
pure cultures the root-nodule organism 
appears to be able to grow on substrata 
in which there is absence of nitrogen 
compounds or at least great paucity of 
et | such compounds. Mazé states that the 
Buchweizen, : Hafer. | organism requires a minimum of com- 
spe ee Sid. .seqnaeinialae bined nitrogen to make a good growth 
fe ii ee | and store nitrogen. He says it can not 
do so if all initial combined nitrogen is 
Fig. 32.* withheld. Others (e. g., Moore) state 
that it can make a decided growth on 
media in which there is no combined nitrogen. According to these observers it makes a 
good growth on media believed to be free from all nitrogen compounds, and hence the 
assumption is that it must be able to obtain its nitrogen from the uncombined nitrogen in 
the air. It is, however, a difficult matter for the ordinary bacteriologist to be assured that 
all traces of nitrogen compounds are absent from a given culture medium and from the air 
— _ 
*Fic. 32.—Hellriegel and Wilfarth’s pea No. 384 grown from germination time in a closed space along with an oat 
plant and a buckwheat plant on nitrogen-free quartz sand. The sand which had been previously heated to redness was 
mixed with nitrogen-free nutrient salts and enough twice-distilled water added to approximate 70 per cent of soil 
saturation. The sand was then inoculated with a little soil extract from earth adapted to peas. 
