AUTOTROPHIC BACTERIA 75 



The inoculated solutions show no turbidity or pellicle formation. It 

 is only after repeated additions of nitrite that a bluish slime can be 

 distinguished on the bottom and wall of the flask wherever it is in con- 

 tact with liquid. When this slime is examined microscopically, it is 

 found to consist of a layer of minute, spindle-shaped (generally) rods 

 staining with difficulty. After several transfers into fresh flasks con- 

 taining sterile liquid medium, the culture is sufficiently enriched, so that 

 plates can be prepared. 



The following agar medium is used for the isolation and cultivation of the 

 organism: 



NaN0 2 2.0 grams Agar 15.0 grams 



Na 2 C0 3 (anhydride) ... 1.0 gram Tap water 1000 cc . 



K 2 HP0 4 0.5 gram 



The surface of the solidified agar plate is smeared with a drop of the solution 

 in which the organism has developed, and the plates are allowed to incubate 14 

 days at 30°. The streak then appears opaque and rounded numerous small drop- 

 lets are differentiated with the naked eye. The sub-surface colonies are shining, 

 slightly brownish, of various shapes developing in two weeks to a diameter of 

 30 to 50/x. On the surface of the plate, the colonies appear as round homogeneous 

 drops, reaching, in two weeks, a diameter of 100 to 180/*. On slants the growth is 

 dirty white, with a large, semi-fluid drop at the bottom. When a loop of this ma- 

 terial is transferred into a 25 cc. portion of nitrite solution, the nitrite reaction 

 disappears in 3 to 4 days. 



Care should be taken not to mistake other bacteria for the nitrate 

 former, since several soil bacteria grow on this medium. The smallest 

 colonies are selected and carefully checked up with the disappearance 

 of the nitrite reaction, combined with prolonged incubation (3 weeks at 

 30°C). Transfer is then made of characteristic colonies by means of the 

 open capillary glass rods described above. From a series of transfers, 

 some will be found to develop into pure cultures. To ascertain the 

 purity of the culture, several drops of the liquid culture are added 

 to bouillon or agar. 



Nitrobacter is a non-motile, rod-shaped bacterium, obligate aerobic, 

 non-spore forming, gram-negative. The cells are stained with carbol 

 fuchsin, and can then be washed with dilute acidified alcohol. It is 1 by 

 0.3 to 0.4/z in size, with one or both ends pointed; the staining is not uni- 

 form, the central part being stained, while the pointed ends remain almost 

 colorless. In Loeffler's alkaline methylene blue, only the nucleus-like 

 bodies are stained well, but not the surrounding cell. By staining in 

 warm gentian-violet, then washing with a 2 per cent NaCl solution, the 

 cells are found to be 1.2 to 1.5 by 0.6 to 0.7^, surrounded by a capsule, 



