106 BACTERIA IN RELATION TO PLANT DISEASES. 
those by which the protoplasm of one cell is connected with that of another. The infection is an 
active and not a passive one. 
The nodules are not to be considered as normal organs of the plant. There is no doubt, however, 
that the plant takes advantage of the presence of the bacteria. 
“The papillionaceous tubercles are bacterial galls, useful to the host plant in so far as the normal 
bacteroids function as providers of albumen—useful to the bacteria in so far as the numerous tuber- 
cles filled with bacteria capable of growth function by their decay as centers for the distribution of 
their occupants.” 
Beyerinck found no formation of nitrates or nitrites and could not establish the assimilation of 
free nitrogen in flask cultures. He says that in 14 days the nitrogen increased according to the find- 
ings of the chemists but only within the limits of error. 
Throughits ability to use asparagin and grape-sugar, B. radicicola has the same nutrition demands 
as the protoplasm of the host. Beyerinck evidently believed that the B. radicicola gets its nitrogen 
from asparagin stored in the plant. 
In his Dutch paper published in 1891, Beyerinck states that by additional experiments he 
succeeded in showing that his B. radicicola from Vicia faba is able to obtain nitrogen from.the air. 
A very great number of active bacteria were used. The increase of nitrogen was, however, so very 
slight (only about 12 milligrams per 100 cc. on an average in 6 cultures 2 to 3 months old) that he 
suggests the possibility of its having come from some nitrogen compound present in the air rather 
than from the free nitrogen. The cultures were kept at 2° to 12°C., higher temperatures being 
thought to induce loss of function; the weakened cultures take their food more readily from am- 
monia salts and nitrates than the unweakened ones. By the diffusion method in gelatin he could 
not be certain of his results, and with the bacillus from the root-nodules of Robinia he could not 
obtain any increase of nitrogen in 8 weeks. 
The cultures were made in Kjeldahl flasks in extract of bean stems (100 gr. germinating sprouts 
to 1 liter of tap water), with 2 per cent cane-sugar and with one-tenth to one-thirtieth gram of 
monopotassium phosphate. Sometimes without the latter. 
Nitrites in all dilutions are said to be injurious to the growth of the organism. Cane-sugar is 
a much better source of carbon than asparagin. The earlier statements of Beyerinck respecting 
the value of asparagin should be rejected as they were obtained from an associated organism confused 
at that time with B. radicicola. Peptone is a better source of nitrogen than asparagin, ammonium 
sulphate, or nitrate of soda or potassa. The growth of B. radicicola is greatly favored by extract 
of papillionaceous plants or dilute must extract. 
Another point insisted upon is that the concentration of the food stuff should be low, especially 
the nitrogen compounds and the phosphates. The negative result of his earlier attempts to prove 
storage of nitrogen is attributed to neglect of this point and to growth at too high temperatures. 
In 1897, in his first paper on thesubject, Mazé states that he used a bouillon made by heating white 
beans in water for half an hour at a temperature of 100° C., being careful not to boii it. This bouillon 
contained about 0.0005 of nitrogen. To it was added 2 per cent saccharose, 1 per cent sodium chloride 
and traces of bicarbonate of soda. This was solidified by the addition of agar and spread in a thin 
layer (o to 4 mm.) on the bottom of large flasks having a side opening by means of which air freed 
from nitrogen compounds could be introduced into the flasks. Into these tubes he introduced air 
which had been passed through an asbestos plug, then over copper turnings warmed to just below 
redness, then through pumice stone saturated with sulphuric acid to remove the free ammonia, and 
afterwards through pure water. ‘The flasks were set up in series, the last one in connection with the 
aspirator. This removed 20 liters in 24 hours, not counting the more rapid movement of the atmos- 
phere every morning to remove the gaseous products of respiration accumulated during the latter 
part of the night. At the end of 15 days the experiment was broken off. Microscopic examination 
indicated the cultures to be pure and transfers to sterile media also indicated the same thing. An 
analysis showed that there was a gain in the 15 days of 40.8 mgr. of nitrogen, the initial amount 
being 62.1 mgr. Ina second experiment which lasted also 15 days the gain in nitrogen was 47.5 mgr., 
as shown by analysis of a mixture of the contents of the two flasks, the initial nitrogen being 70.7 
mgr. In a third experiment, using bean bouillon without the agar the experiment was broken off 
on the sixteenth day, and the results of the analysis of two flasks united showed a gain of 32.4 mgr., 
the initial amount of nitrogen being 22.4. He concludes that symbiosis is not necessary to explain 
the fixation of atmospheric nitrogen by the nodule-forming bacteria. This is a property belonging 
to the organism independent of any influence exercised upon it by the plant. The lack of success 
experienced by former experimenters he thinks due principally to a defect in the method of experi- 
mentation. ‘hey placed too little value on the energy necessary to enable the nodule-forming 
bacilli to convert the nitrogen of the air into an endothermic combination. To place this organism 
