136 BACTERIA IN RELATION TO PLANT DISEASES. 



In 1907, Gino de Rossi contributed a paper on "The Micro-organism Which Causes the Root- 

 nodules of the Leguminosae," taking a view quite different from the ordinary one. 



He used a large number of plants of Vicia faba (fully 60). From each plant, except the youngest 

 which had no nodules, 1 2 or 15 nodules were removed from different parts of the roots, and repeatedly 

 washed in tap-water. 



They were then placed in a stoppered vessel of sterilized distilled water. The water was changed 

 twenty times in an hour, and each time the vessel was shaken vigorously. Finally, without other 

 attempt at surface sterilization the nodules were placed by means of sterile forceps in a Petri-dish 

 containing a few pieces of filter paper, the whole having been previously sterilized in the dry oven. 



All liquids, instruments, and vessels used in the further handling of the nodules were very 

 carefully sterilized by heat. 



After every trace of water was removed by the filter paper, the nodules were removed to another 

 Petri-dish where they were sectioned, and a small portion of the interior removed with a sharp needle, 

 care being used not to touch the surface layers. This fragment was crushed out in 2 to 3 cc. of water 

 in a stoppered vessel. Three or four such vessels were prepared from each plant. Microscopic 

 preparations and cultures were made from this material. 



The following solid culture-media were used: 



(a) Simple 10 per cent gelatin, with beef-extract (1 per cent), pepton (1 per cent) and sodium chloride (0.5 per 

 cent), the reaction of which was mildly alkaline or mildly acid (natural acidity). 



(b) Ten per cent gelatin prepared with Vicia faba extract (1 part leaves cooked in 8 to 10 parts of tap-water and 



filtered), 1 to 2 per cent cane-sugar or glucose, 1 per cent pepton and 0.5 per cent sodium chloride, with a 

 mildly acid or alkaline reaction. 



(c) Simple agar (1.5 per cent) or agar with Vicia faba extract. Poured plates were made. In some experiments 



all these media were used, in some only one. In every case, however, the somewhat acid gelatin containing 

 Vicia faba extract was used. 



De Rossi examined the contents of the nodules in hanging drops and in stained preparations. 

 He found the contents of very young nodules to consist of non-motile rodlets, constant in breadth 

 but not in length, and easily stained with aniline dyes. He did not find motile bacilli, either large 

 or small. 



In older stages of the nodules a series of transitions was observed, the rodlets showing first one 

 end slightly swollen, then a slight dichotomy at the end, and finally the typical X and Y shapes, 

 which, contrary to current statements, were constant in form and dimensions. 



In this phase the central part of the nodule contains an enormous number of the branched forms; 

 only in a few cases were straight rods seen. De Rossi considers these bacteroids as a stage in the 

 development of the organism, not as degenerate forms. 



He observed the development of what appeared to be vacuoles in the bacteroids, and states that 

 this is a constant feature, belonging to a phase in the development of the organism. The bacteroids 

 at this stage are somewhat swollen. 



In no case did he see any very small infecting bacilli or the development of such bacilli into 

 bacteroids. 



Cultures, made on the solid media by flooding the surface with a dilution from nodules full of 

 bacteroids, developed a small number of tiny, whitish colonies. These appeared in 2 or 3 days, at 

 a temperature of 16 C. As development on the plates proceeded a mixture of very different colonies 

 was often apparent. 



By selecting plates with one dominant form, pure cultures of four sorts of Sehizomycetes were 

 made. This variety of forms together with the presence of numerous non-germinating bacteroids in 

 the surface of the gelatin (as determined by a microscopic examination) suggested to de Rossi that 

 the bacteroids were unable to grow on artificial media, and that the colonies present belonged to 

 other organisms which had penetrated the nodule and multiplied there sparingly [they may have 

 come from the surface], but which had nothing to do with its production. He further supposes that 

 these non-infectious intruders have been commonly mistaken for the root-nodule organism. One 

 white form was non-liquefying and motile by means of a polar flagellum. 



To prove these hypotheses he isolated the bacteroids from the surface of such cultures in the 

 following way: 



After 12 to 15 days in the thermostat (probably at 15 to 20 C), the surface of plates, which had 

 yielded a few colonies only, was moistened, and scrapings from the apparently sterile parts between 

 colonies were used for inoculating various media. The actual presence of numerou c bacteroids on 

 this surface was demonstrated by microscopic examination : The results were all negative. The media 

 used were gelatin with mineral salts or Vicia faba extract, beet roots, and raw and cooked potato. 

 Cultures were also attempted in pure nitrogen without positive result. 



Inoculations were then made on plants of Vicia faba using: (1) Scrapings containing the bac- 

 teroids which would not grow on his media; (2) the fourth or fifth sub-cultures of the colonies which 



