128 BACTERIA IN RELATION TO PLANT DISEASES. 
outgrowths like those which had been observed repeatedly in nutrient solutions. These outgrowths 
stained strongly with carbol fuchsin. However, when these plants actually became green there was 
no longer a trace of the outgrowths on the bacteroids. The outgrowths, therefore, formed when the 
plants began to hunger and disappeared as the nitrogen hunger stage passed away. From this it 
appears that nitrogen assimilation must stand in some sort of relation to the absorption of the out- 
growths of the bacteroids which in active tubercles are continually formed but not easily demon- 
strated. Nitrogen assimilation is made possible not by the bacteroid formation per se, but by the 
process of plasma differentiation therein, in that from the nuclear plasma or through its action a 
nitrogenous substance is formed, the nitrogen for which is drawn from the atmosphere. 
Bacteroid formation and nitrogen assimilation do not always go hand in hand. This showed 
plainly in the cultures of pea bacteria in saltpeter solutions previously referred to, where, though 
bacteroid formation took place, plasma outgrowths did not appear, no staining was obtained with 
Gram’s stain, with iodine the color became yellow, not red-brown, and no nitrogen assimilation 
occurred. ‘These circumstances explain the fact previously mentioned by Nobbe and Hiltner that 
nitrogen assimilation in Leguminosae begins only when the available nitrogen in the soil has been 
exhausted. This is true also in Alnus. 
Hartleb’s idea of the sporangial nature of the bacteroid is not new but originated with Brunchorst 
and Moeller and was confirmed by Hiltner prior to the appearance of Hartleb’s paper. Later experi- 
ments by the authors have not altered this view. The bacteroids of the alder bacteria are even more 
strikingly sporangia-like. All observations point to the conclusion that in certain solutions and under 
certain circumstances the outgrowths from the bacteroids break up into pieces of varying size which 
directly or after further division may grow into bacteria or bacteroids. Some of Hiltner’s figures 
suggest certain figures published by Gino de Rossi. The bacteroids within the nodules show less 
plainly their sporangial nature. 
According to these observations the relation between host-plant and bacteroids is much more 
intimate than was previously supposed. In sharp contrast to the opinion that the nitrogen assimi- 
lation is concerned with an absorption of the bacteroids, Hiltner claims that his observations support 
the view that under normal circumstances this absorption does not take place, but rather that some 
substance produced by the nitrogen assimilation which goes on within the bacteroid is absorbed. 
This opinion is based on the fact that within the root-nodules the bacteria have a tendency to form 
sporangia to protect themselves against the influence of the host-plant, but that this does not succeed 
so long as the plant is active, because the indispensable building material obtained by nitrogen 
assimilation is constantly drawn away from them by the plant. 
Beyerinck also has found that soil bacteria known as true nitrogen fixers (his Granulobacter, 
Radiobacter, Aerobacter) are able to use very little of the product of their assimilation, but that this 
is used chiefly by a species living with them (Azotobacter chroococcum), the nitrogen fixers (other 
then Granulobacter) being stimulated to action only by the symbiotic life. 
The effectiveness of pure cultures of nodule bacteria depends on a number of factors, viz., 
genuineness, nutrition, virulence, and nitrogen-fixing power. 
That true nodule bacteria should be used is self evident, yet that extreme care is required to 
obtain them is not fully realized. Nodules in which decay has begun may contain all manner of 
strange species and it is not out of the question that completely sound nodules may contain intruders. 
Beyerinck showed this to be the case. Though Hiltner did not find the same intruding species 
except in decaying nodules, he did find in 1902 another contaminating species in cultures from 
several distinct sources. This intruding species developed very slowly on gelatin compared with the 
true nodule bacteria, and differed from it quite materially in the appearance of its colonies. On the 
other hand it formed bacteroids and behaved very similarly to the nodule bacteria in grape-sugar 
solutions. ‘This organism was not able to produce nodules and seemed able to penetrate the nodules 
only when the real nodule producer had prepared the way. An especially characteristic peculiarity 
of this species, which does not liquefy gelatin, is that the individuals of a colony are held together 
by such an extraordinarily viscid slime that even when they are allowed to lie for a week in water 
they do not separate. Hiltner suspects that they are able to fix nitrogen, but there is no possibility 
of confusing them with the true nodule bacteria. However, there are, as Beyerinck observed, several 
species of bacteria resembling the true nodule bacteria in the appearance of their colonies and in their 
general behavior which can give much trouble in the securing of pure cultures because of their fre- 
quent appearance in the nodules. Only a test of the nodule producing ability of a culture can here 
prevent mistakes. Additional certainty is obtained by determining under what circumstances bac- 
teroids are formed. ‘ 
But even when it is known that a culture consists of the true nodule bacteria, one does not 
know with any certainty whether he has the desired adaptation form. It seems indeed at first sight 
as if it would be sufficient to know the immediate source of the cultures. Any one who has not 
