472 IVAN E. WALLIN 



When my specimens were first examined with a low magnifica- 

 tion of the microscope I was impressed by what appeared to be 

 three distinct regions or areas in the root nodule. On closer 

 examination with oil immersion lens, the three areas were found 

 to contain three distinct forms of organisms. Each form was 

 more or less Umited to a single area. In the part of the nodule 

 next to the plant root, the nodule cells contained no other than 

 the spherical forms. I interpret this part of the nodule to be the 

 older part and on the basis of this interpretation have called the 

 spherical cells "senile" forms. The "bacteroid" forms of the 

 bacillus were likewise limited, almost entirely, to the central 

 portion of the nodule. In what I interpret to be the younger 

 part of the nodule the bacilh were all of the small variety and not 

 nearly so numerous as the other forms. 



My interest in these forms is not "bacteriological" and I have 

 no desire to pursue the investigation any further, at least for the 

 present. However, it does occur to me that the technique that 

 I have used may be valuable in bacteriological research. In a 

 recent publication (Wallin, 1922a), I submitted evidence that 

 mitochondrial methods are not specific, but will also stain 

 bacteria. It has since occurred to me, particularly in connection 

 with my study of root-nodule bacteria, that the mitochondrial 

 technique may, at least in some cases, be superior to the usual 

 bacteriological methods, particularly when deahng with sym- 

 biotic bacteria. I have tested a number of mitochondrial 

 methods on various kinds of bacteriological material: sputum 

 smears, pus smears and sections, tissue smears, bacterial smears, 

 etc. In the majority of instances the differentiation between 

 bacterium and tissue has been decidedly sharp. 



In staining the root nodules, I have used only one method. 

 This consisted in fixation of the entire nodule in a modification 

 of Flemming's fixative: 4 cc. 2 per cent aqueous solution of osmic 

 acid and 6 cc. 1 per cent aqueous solution of chromic acid. (Fix 

 from four to twenty-four hours.) After washing, dehydration, 

 clearing, and embedding in hard paraffin (58°), sections were cut 

 3 micra in thickness. The sections were mounted on slides and 

 stained by Bensley's (1911) anihn fuchsin-methyl green method: 



