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agar-potassiumphosphate plates, in absence of purposely added nitrogen compounds, at 

 superficial view make the impression of being quite able to develop, but here too, it is 

 only the formation of much wall substance, as already described above, and not of 

 nitrogen-rich protoplasm, which explains the voluminosity of the colonies 1 ). With 

 other slime-producing bacteria, as B. radiobacter and Aerobacter viscosum, of which 

 it is quite certain that they cannot live on the atmospheric nitrogen, extensive colonies 

 may likewise be grown on the said nitrogen-poor medium with fit carbon food. By 

 a better nitrogen nutrition such colonies may even be greatly reduced in volume, the 

 wall substance then serving as food under a strong increase of the bacterial proto- 

 plasm, which gives rise to very interesting experiments. It is only when being ac- 

 quainted with these facts by personal observation that we can understand how in the 

 literature so many statements can occur on the nitrogen fixation by the nodule bacteria, 

 which does not take place. 



Within the nodules the atmospheric nitrogen is neither fi.red. 



The preceding gives rise to the question, whether the protoplasm of the host plant 

 might bethecatalystthatenablestheinvadingbacteria, in their bacteroidal state, to fix the 

 free nitrogen. However improbable this hypothesis may appear, being in contra- 

 diction with the laws of heredity, still it deserves attention because the rate of albu- 

 minous matter in the nodules is so very high. I myself found about 4% nitrogen, 

 which is about 25% albumen in the dry matter of pease-nodules. Others found 5 to 

 6% nitrogen. It is noteworthy that the bacterial colonies on agar plates, grown out 

 of the plant, contain but i to 2% nitrogen of the dry weight, which consists for the 

 greater part of carbohydrates. So it is certain that the bacterial body is very much 

 modified by its entrance into the plant cell as well morphologically as physiologically. 

 Therefore it was tried gazometrically to state nitrogen absorption in the tubercles. If 

 the hypothesis is founded it must be possible, with a great quantity of tubercles in a 

 closed space and under favourable physiological conditions, easily to observe that ab- 

 sorption. For the number of tubercles, for examples of the woody papilionaceae, being 

 as said very small, while yet these plants are noted in agriculture for their consider- 

 able nitrogen-fixing power, the action of the tubercles must necessarily be very intense. 



To test the hypothesis we acted as follows -). First small, later larger quantities 

 of lupine and serradella tubercles were placed in wide glass tubes which could readily 

 be connected with the gas burettes, then put in thermostats at about 25 C. The 

 tubercles respiring vigorously we had to keep account with a rapid assimilation and 

 supply of the oxygen. Further it was only necessary to determine the quantity of 

 nitrogen still present after deduction of the carbonic acid and the oxygen. The only 

 difficulty we met with was that the nodules, which by their abundant content of al- 

 buminous matter are an excellent food for bacteria, when they touch each other and 

 get moist, easily give rise to fermentations in particular by Bacterium aerogcnes. 



') Likewise for the ordinary saprophytic bacteria the want of nitrogen compounds 

 varies very much: the large-celled Bacillus megatherium requires very little, the small 

 celled Bacterium fiuorescens very much. 



2 ) In some of these experiments I was assisted by Ir. D. C, J. Mink man, formerly 

 assistant to the Laboratory for Microbiology of the Technical High School at Delft. 



