498 NITROOEN-FIXINO BACTERIA 



cent, mannitol and 0-02 per cent. K2HPO4. If the medium is spread out in a 

 thin layer in a wide-bottomed flask, Azotobacter chroococcum forms a surface pellicle 

 in 2 or 3 days, which gradually becomes brown, and may later even turn black. 

 Potassium, calcium, or sodium propionate in 0-5 per cent, solution may be sub- 

 stituted for the mannitol. 



Of solid media, one of the best has the following composition : 



Mannitol 2 gm. 



K2HPO4 002 „ 



Washed Agar ....... 2 ,, 



Aq. Dist. . 100 „ 



Azotobacter grows best in solutions containing little or no combined nitrogen ; 

 according to Beijerinck (1901) ammonium salts are not easily assimilated, and 

 peptone can be used only to a slight extent. 



The peculiar property of this group of organisms is to fix atmospheric nitrogen 

 and to convert it into ammonia, nitrites and nitrates (Beijerinck and van Delden 

 1902). To do this they must be supplied with a source of energy in the form of 

 a suitable carbohydrate, such as mannitol. The energy gained from the oxidation 

 of this substance is utilized in the fixation of nitrogen. Gainey (1918) found 

 that in a synthetic mannitol medium inoculated with soil, about 8 mgm. of nitrogen 

 were fixed for 50 ml. of medium. Not all strains of Azotobacter are capable of 

 utilizing mannitol (Smith 1935). Azotobacter is more susceptible to acid than most 

 soil organisms. Its growth limits are about pH 6-5-8-6 in pure culture (Fred and 

 Davenport 1918), but in soil it is probable that growth can occur down to about 

 pH 6-0. Pigment formation is variable in Az. chroococcum ; some variants form a 

 brown pigment, others are achromogenic ; intermediate forms occur (Omeliansky 

 and Ssewerowa 1911). Pigment is formed only in the presence of a free supply 

 of oxygen. We give a description of the type species Az. chroococcum, followed 

 by notes on the lesser known species. 



Azotobacter chroococcum 

 Isolation. — By Beijerinck in 1901. 

 Habitat. — Soil. 



Morphology. — Short, thick rods with rounded ends ; large ovoid forms ; forms like giant 

 diplococci ; pear-shaped rods, and other forms. 4-5 ^u X 1 -5-2 /n. Slowly motile 

 by polar flageUa. Arranged singly, in pairs end-to-end, and in old cultures in 

 packets. Cells often vacuolated ; when grown on mannitol, cells may contain fat 

 droplets. In surface membranes on liquid media cells are surrounded by a thick 

 mucoid capsule. Large involution forms not uncommon. Non-sporing. Gram- 

 negative. Non-acid-fast. 

 Agar Plate. — 3 days at 30° C. Round colonies, 1 mm. in diameter, convex, with smooth, 

 moist, gUstening surface ; edge entire ; structure amorphous ; consistency butyrous, 

 easily emulsifiable ; often differentiated into an opaque brown centre and a trans- 

 lucent lighter periphery. After 6 days the colonies measure up to 3 mm. in diame- 

 ter ; some remain homogeneous, undifferentiated and opaque ; others show an 

 opaque brownish centre, and a clear, shghtly radiate periphery. 

 Agar Slope. — 2 days at 30° C. Moderate, confluent or partly confluent, shghtly raised, 

 translucent, greyish-yellow growth, with glistening, beaten-copper surface ; edge 

 formed of single colonies. 

 Gelatin Stab.^6 days at 20° C. Poor to moderate, greyish-white, fihform growth, con- 

 sisting chiefly of discrete colonies ; extends f way down tube ; surface growth, 

 3-4 mm. in diameter, with a lobate edge and irregular surface. No liquefaction. 



