128 PRINCIPLES OF SOIL MICROBIOLOGY 



Nodule formation. The bacteria usually enter the plant through the 

 root hairs, being attracted through the secretion of soluble carbohydrates 

 or organic acids by the plant. On entering the root, the bacteria 

 multiply forming a thread of infection, similar to a fungus hypha, which 

 enters the root and branches out into the parenchymatous cells of the 

 plant. In some cells, the thread breaks up into individual cells 

 which, on multiplication, fill the whole protoplasm of the cell; the 

 bacteria give rise at the same time to branching forms, commonly 

 referred to as "bacteroids." 



The size, form and position of nodules vary with the nature of the 

 plant, soil in which it is grown and virulence of the bacteria, as shown by 

 Hiltner, 123 who explained nodule formation by his theory of immunity 

 discussed elsewhere (p. 589). According to Bryan, ui nodule formation 

 is greatly influenced by the reaction of the soil : alfalfa and clover produce 

 maximum growth and number of nodules at pH 7.8, alsike and red 

 clover at pH 5.6; the critical pH values for nodule formation are 4.0 and 

 9.0 to 10.0. Nodules will be formed at all temperatures at which the 

 plant can make a growth that is at all vigorous. 125 The presence of 

 nitrates or other available nitrogen compounds in the soil depresses 

 nodule formation. 



Isolation of organism from nodules. Harrison and Barlow 126 describe 

 in detail the method of isolation and cultivation of the organism. 



A medium sized nodule, appearing young and sound, is selected. It is cut off 

 so as to leave 2 to 3 mm. of the root on both sides to permit handling it with forceps. 

 The nodule is then washed, rinsed in distilled water and dropped into a sterilizing 

 liquid containing 1 gram HgCl 2 and 2.5 cc. c. p. HC1 in 500 cc. of water. The 

 nodule is well shaken in the solution for 3 to 4 minutes, then washed three times 

 in sterile distilled water. It is then covered with about 1 cc. of sterile distilled 

 water and crushed with a sterile, heavy glass rod. Two or three drops of the 

 cloudy suspension are placed into a test tube of the agar medium, which has 

 previously been liquefied and cooled to 45°C. A second tube of agar is then in- 

 oculated with five drops from the first; a third tube is inoculated from the 



123 Hiltner, L. Die Bindung von freiem Stickstoff durch das Zusammenwirken 

 von Schizomyceten und von Eumyceten mit hoheren Pflanzen. Lafar's Handb. 

 techn. Mykol. 3: 24-70. 1904. 



124 Bryan, O. C. Effect of reaction on growth, nodule formation and calcium 

 content of alfalfa, alsike clover and red clover. Soil Sci. 15: 23-35. 1923. 



126 Jones, F. R., and Tisdale, W. B. Effect of soil temperature upon the de- 

 velopment of nodules on the roots of certain legumes. Jour. Agr. Res. 22: 

 17-31. 1921. 



1,6 Harrison and Barlow, 1907 (p. 127). 



