jimeis, I9I5 Azotobacter Chroococcum 229 



standard stainable substance. Since the nature of the components of 

 polymorphonuclear leucocytes is known and since the solutions responded 

 alike on these and on the cells of A. chroococcum, we are justified in 

 comparing these bacterial cells with the blood cells. 



Guignard's method was originated by A. Guignard (6, p. 19), who 

 used it to study the cytology of antherozoids. The organism was fixed 

 with osmic-acid fumes and stained in a mixture of 50 c. c. of a solu- 

 tion of 2 per cent of fuchsin in i per cent of acetic acid, 40 c. c. of a solu- 

 tion of 0.2 per cent of methyl green in i per cent of acetic acid, and i 

 c. c. of acetic acid. This method gave a splendid picture of the 

 structure of the cell. The netw^ork, of which mention will be made 

 later on, was stained violet, the contents of the network were hardly 

 stained at all, and the granules were a deep violet-black. 



The Guignard (6, p. 19) method did not give the color differentiations 

 desired, red and green, perhaps on account of metachromacy of the methyl 

 green used in the solution. On the whole, it proved to be a very satis- 

 factory method. Plate XXXI, figures 2, 3, 4, 6, 8, and Plate XXXII 

 show some very good preparations obtained by it; the network showed 

 plainly and the granulations very distinctly. Plate XXXIII, figure i, 

 shows some cells stained by this method; several granules are to be seen. 

 The cell wall is quite evident in old cells, but in young ones it is to be 

 seen only slightly stained. 



Heidenhain's ordinary ferric hematoxylin stained the network 

 strongly, but gave no differentiation. The fixing was done by passing 

 the glass through a flame. 



The Heidenhain method was used progressively in a great number of 

 cases and regressively only occasionally. When progressive, it stained 

 the cell components black, and it demonstrated the zooglea sheaths, 

 such as Beijerinck (2) and Krzemieniewski (10) found. Plate XXXI, 

 figure 5, shows some cells obtained by the Heidenhain method. Plate 

 XXXIII, figure 3, shows some zooglea and cells escaping from them, and 

 Plate XXXIII, figure 2, shows two of these zooglea nearly empty, all 

 cells having escaped, the same as is shown in Plate XXXI, figure 5. 

 In some groups the walls which formerly separated the different cells in 

 the same mass are to be seen quite distinctly after the cells have escaped 

 (PI. XXXI, fig. 5). 



Smears from a hay-agar culture of A. chroococcum stained by the 

 regressive method showed the network to be made up in many cases of 

 a substance taking a beautiful dark-violet color and of a nearly trans- 

 parent appearance. 



Romanowsky's simple stain is the one used by the originator for 

 the study of the parasitology of malaria. The preparation was fixed by 

 the flame and stained in a mixture of five volumes of eosin in a i per cent 

 aqueous solution and two volumes of a saturated aqueous solution of 



