root-nodulHs of leguminosae;. 113 



"For the purpose of a close comparison of 'nitragin' with organisms direct from the nodule, 

 grown on gelatin, a double series of tubes (gelatin ioper cent; asparagin 0.25 per cent; saccharose 

 1 per cent ; pea extract) were infected with cultures A and B respectively and kept under the same 

 conditions at ordinary temperatures. From these at intervals of 24 hours, preparations were made, 

 and stained with carbol fuchsin." 



The nodule bacteria grow most rapidly on gelatin at 15 to 18 C. and on agar at 30 to 35 C. 



The microscopic characters of the organisms in both cases were quite similar, but those of A 

 (nitragin), after 24 hours' growth, had enlarged to nearly twice their former size. After 48 hours' 

 growth, the maximum size was reached with a few X and Y forms present. The size gradually 

 diminished until after 5 days the original size was reached. No X and Y forms were seen in the last 

 preparations of either type. 



In drop cultures of the organism from Desmodium, colonies 8 to 10 days old consisted of 

 numerous small rodlets, with some long rods and intermediate stages. 



In all three types, the formation of a typical colony was observed in a hanging drop of 5 or 2.5 

 per cent gelatin. Within 5 days the colony reached its maximum size (28/* in diameter). From this 

 time it slowly disintegrated when it was obvious that many X and Y forms were present, the latter 

 predominating. Some rods were curved, others straight. Several individuals were in turn observed 

 for the formation of bacteroids. The fact that X and Y forms arise as a distinct branching of the 

 rods was repeatedly demonstrated (fig. 37). In 14 days the branched forms had disappeared from 



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F.g. 37.* 



the drop under observation. This now contained a few large rods and large numbers of small ones 

 with intermediate stages. 



"When branching is about to take place, the rod, as a rule, becomes at first curved, and then 

 from the point of greatest curvature on the convex side a lateral branch grows out, giving the resultant 

 Y form. In other cases both arms, here usually very short ones, appear to form simultaneously, 

 suggesting a kind of dichotomous branching of the already swollen head of the rod." 



She also says that in pure cultures on nutrient gelatin large numbers of X and Y forms occurred. 

 The bacteria stain by Gram. On treating with methylene blue or carbol fuchsin deeply stained 

 bodies appeared at the ends of the cell and also one or more in other parts. 



In the branched forms they are seen at the extremities of the arms, at the angles, and sometimes 

 along the course of the main rod. These are doubtless the bodies which have been described as 

 spores by Schneider, and as cocci by Frank. 



She says further: "The daughter cells produced are approximately equal, but there does not 

 appear to be absolute regularity in the sequence of further divisions, most frequently both halves 

 divide again in a regular manner; sometimes, however, only one divides, whilst the other undergoes 

 no further change or takes on the 'bacteroid ' form. Again, both daughter rods resulting from a 

 division may become thus transformed, though rarely simultaneously, into bacteroids." 



These observations support the theory of Beyerinck (1888) whose conclusions respecting branch- 

 ing were drawn not from the continuous study of single rods but from observations upon numerous 

 individuals assumed to represent different stages in the formation of bacteroids. 



*Fig. 37. Stages in formation of branched bacteroids in pure cultures from root-nodules of Desmodium gyrans, 

 observed in hanging drops of nutrient gelatin. After Maria Dawson. 



