326 STUDIES IN SYMBIOSIS, ii., 



earpineae nodules. Text-fig. 13 shows a large number of bacteria round the 

 cell nucleus. This relation of the intruder to the nucleus is very common. A 

 very interesting observation was ma.de in this connection. In cells free of bac- 

 teria there is generally abundance of starch-grains, but when bacteria enter and 

 multiply in the cell the starch-content decreases. Text-fig. 10 shows a cell filled 

 with bacteria but devoid of starch. The nucleus and cytoplasm retain their 

 normal structure and appearance, and I found no evidence of nuclear division 

 in the infected cells as has been recorded by Spratt (1912) for the infected cells 

 of the nodule of Podocarpus. I have no doubt but that the bacteria give some 

 benefit in return. The individuals of the coccus form differ considerably in 

 size; the smaller form is actively motile and frequently a mass of these may cause 

 the starch-grains to move in the cell. The larger individuals are non-motile and 

 stain more deeply than the smaller. These latter are probably bacteroids which 

 contain proteid and which are digested by the host-cell. 



Vines (1888), Frank (1885), McDougall (1899) and others believe that 

 fungi, growing upon or into the cells of a plant, may aid it in nutritive work, 

 especially by converting free nitrogen or the simpler compounds of nitrogen into 

 more complex forms of nutritive value to the plant. Life (1901) also believes 

 that the tubercles of Cycas revoluta assist the host in nitrogen fixation. Bottom- 

 ley (1909) isolated Pseudomonas radicicola and Asotobacter, which are nitro- 

 gen fixing forms, from Cycas tubercles. Nobbe and Hiltner (1899) have demon- 

 strated that the nodules of Podocarpiis are active agents in the fixing of nitrogen 

 of the atmosphere, by cultivating plants with nodules for five years in quartz 

 sand from which nitrogen was absent, and by demonstrating that it was im- 

 possible to cultivate Podocarpus in the absence of the fungus which caused the 

 nodule formation. Spratt (1912) has since demonstrated that the nodules are 

 formed by bacteria which are apparently identical with the Pseudomonas radi- 

 cicola of the root-nodules of leguminous plants, and of Cycas, etc., and that the 

 bacteria utilize the free nitrogen of the atmosphere during the process of meta- 

 bolism. These results throw light upon those of Hiltner, who cultivated Podo- 

 carpus in quartz-sand containing no nitrogen. 



The disappearance of starch-grains from cells of Macrozamia which have 

 been infected by numerous bacteria might suggest that the carbohydrate is utilised 

 by the bacteria. This is supported by the observed fact that deeply-staining 

 bacteroids are formed in which considerable proteid is present. Nitrogen in the 

 free state, at any rate, must enter the cells of the host — a supply of carbohydrate 

 and nitrogen, therefore, is available to the bacteria if they are concerned in 

 nitrogen fixation. If the bacteria were purely parasitic forms, and gave nothing 

 to the host in return for the carbohydrate, one would expect many cells of the 

 host to show signs of disintegration. It is the absence of this condition, com- 

 bined with the disappearance of starch-grains, and the possibility of free nitrogen 

 entering any cell of the cortex containing bacteria, which suggests to me that 

 the bacteria may be helpful to the plant in the direction of assisting in nitrogen 

 fixation. 



With the object of testing this hypothesis, I carried out a series of experi- 

 ments. The bacteria were first examined in regard to a possible faculty of 

 nitrate formation. A solution containing lOOO c.c. of distilled H2O, 1 gram of 

 ammonium sulphate, 1 gram of potassium phosphate and 4 grams of basic mag- 

 nesium carbonate, was used after filtering. Several flasks each containing 100 

 c.c. of this solution were inoculated from a pure serum-agar culture of the bac- 

 teria, and after several days at laboratory temperature the solution was tested 



