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. 



Through its 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 I2C., 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 i 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, Maze" states that heused a bouillon made by heating white 

 beans in water for half an hour at a temperature of 100 C., being careful not to boil it. This bouillon 

 contained about 0.0005 of nitrogen. To it was added 2 per cent saccharose, i 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. In a 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. They 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 



