ROOT-NODULES OF LEGUMINOSAE. 119 
They are then rinsed in water and placed in the iodine solution. After washing this off they were 
allowed to remain one or two minutes in the orange G. They were then washed in absolute alcohol 
as long as gentian violet came off abundantly or needed to be removed. ‘They were cleared in clove 
ail, this being preférred to xylol. He states that the bacteria were distinctly differentially stained in 
e tissues. 
In the Bur clover the proportion of root-hairs attacked naturally by the nodule organism was 
estimated to be about oneina thousand. He, however, was able to confirm Miss Dawson’s statement 
respecting the abundance of infected root-hairs when the roots of legumes were subjected to special 
methods of infection, e. g., placed on damp filter paper-under a tumbler, and watered with water 
containing the bacteria in suspension, nearly every hair on a root in some fields of the microscope was 
found to be enlarged and twisted at the end, and showed the beginning of an infection thread. A 
striking characteristic of the infection of the root-hairs is the curvature of the hair about the infected 
portion (fig. 21). It is believed that the organism dissolves or softens the walls of the root-hair and 
thus enters. A wound is not necessary. In fact, wounded root-hairs lose the power of curvature 
displayed so strikingly in the ones showing the infections. The curvature is unquestionably due to 
the presence of the bacteria. : 
“The bending is the evident response to irritation.” * * * ‘Since the majority of infected 
root-hairs show the bending at or near the tip, * * * we may infer that the bacteria enter unin- 
jured hairs which are able by growth-curvatures to respond to mechanical or chemical stimuli.” 
* * * “Having entered the root-hair by softening or dissolving a small portion of the cell-wall, 
and moving or growing through this, the tubercle bacteria multiply rapidly, forming a thread-like 
zoogloez from the infection spot along the hair into the epidermal cell of which the hair is a branch. 
From the epidermis the infecting zoogloez grows fairly straight into the underlying cortical paren- 
chyma.” * * * ‘The direction of the infection thread—which is solid, and is incorrectly termed 
infection ‘tube’—is too regular not to encourage one to suppose that the course of the growing strand 
of bacteria is determined by attraction exerted by the host-cells upon the bacteria. This then is 
chemotropic growth of the strand or, if the bacteria are motile in the cells, chemotactic movement of 
the bacteria. The course of the thread is toward the conducting tissues of the host.” * * * “The 
growth does not extend into the central cylinder and the conducting tissues, so far as I have seen. 
Instead, in the layer of cells just outside the endodermis of the root, division takes place in the cell 
into which the infection thread has penetrated and in the cells adjacent to it. The daughter-cells 
grow, repeated divisions and growth follow, and there arises a conical mass of cells which are some- 
what larger, and which contain more protoplasm than the adjacent cortical parenchyma cells. This 
conical mass is the young tubercle. At first all of its cells are merismatic, but later the divisions 
become more and more limited to the cells near the rounded apex of the blunt cone. Thus a regular 
cambium is differentiated in the tubercle. Thiscambium * * * lies near the tip of the tubercle, 
and forms a bowl-shaped or shallow thimble-shaped layer.” 
“The growing tubercle pushes out the overlying cortical parenchyma and epidermis, forming an 
increasing swelling on the side of the root. Cortical parenchyma and epidermis, at least for a time, 
nearly keep pace with the growth of the tubercle. Thus, although the cortical cells are compressed 
somewhat, the epidermis is not ruptured, and the tubercle does not burst out of the side of the root as 
a lateral root does.” 
Morphologically, in origin the nodules are indistinguishable from the nascent lateral roots. In 
subsequent growth they are more and more dissimilar. 
“Morphologically, then, the root-tubercles are lateral roots.” * * * ‘The bacteria in the 
infection thread, which grows through the root-hair and the cortical parenchyma cells of the root 
to the pericambium layer, multiply, but they multiply most rapidly in the infected cells farthest from 
the surface of the root. New threads form, which grow out into and infect the cells of the mass of 
new cells composing the embryo-tubercle. Thus a majority of the cells in the young tubercle contain 
bacteria.” 
“Though infected cells do divide, they probably divide less often than the uninfected cells.’’ 
* * * “The infection of the daughter-cells composing the embryo-tubercle is accomplished by 
branching infection threads growing in fairly straight lines radiating from the base of the tubercle. 
In this way the cells near the base of the growing tubercle are most infected, those near the tip least.” 
The meristem continues to form new cells between itself and the cells containing bacteria and 
infection threads. When, however, he imprisoned the nodules in plaster of Paris, the meristematic 
cells also were infected. 
The infected cells became enlarged and in their definitive condition are said to be from half as 
large again to twice as large as the uninfected cells. There was less difference in size of normal and 
infected cells in nodules imprisoned in plaster casts. 
The infected cells are thin-walled and contain only one large vacuole. ‘his is not traversed by 
