272 AZOFICATION 



efficiency of the organism. It seems, therefore, more likely that 

 they are reserve protein material. 



Lohnis and Smith have recently observed that Azotobacter, in 

 common with many other bacteria, pass through a life cycle which 

 is not less complicated than those of other microorganisms. Under 

 certain conditions they pass over into an amorphous or " symplastic" 

 stage, appearing under the microscope either as an unstainable or a 

 readily stainable mass without any easily distinguishable organiza- 

 tion, which, if not discarded as dead, later gives rise to new regenera- 

 tive forms. They multiplsAnot only by fission, but by the formation 

 of gonidia. 



Methods. Clostridium pasteurianum grows readily in a vacuum on 

 carrots. The organism also grows on sliced potatoes, but ordinarily 

 is grown in an aqueous solution containing 1 gm. K 3 PO 4 , 0.5 gm. 

 MgSO 4 , 0.1 to 0.02 gm. NaCl, FeSO 4 , and MnSO 4 , and 1.0 gm. 

 CaCO 3 , and 10 to 15 gms. of a suitable carbohydrate in 1 liter of 

 water. One method used by Winogradsky in isolating B. Clostridium 

 pasteurianum was to add garden soil to a non-nitrogenous solution 

 and to allow a stream of nitrogen gas to pass through the solutions, 

 after which several successive transfers were made into similar 

 media. The final culture, after B. Clostridium pasteurianum had 

 formed spores, was heated to 80 C. 



The organism ferments certain carbohydrates with the formation 

 of butyric acid, acetic acid, carbon dioxid, and water. When grown 

 in nutritive solution devoid of combined nitrogen, it assimilates 

 atmospheric nitrogen. Although in pure cultures it is an anaerobe, 

 in impure cultures it may fix nitrogen under aerobic conditions. In 

 nature it occurs in connection with two other bacteria which do not 

 possess the power of fixing nitrogen, and their nitrogen requirements 

 are small. When in conjunction with these organisms, Clostridium 

 pasteurianum has the ability of growing in the upper layers or soil 

 and of assimilating free nitrogen. 



Azotobacter chroococcum grows readily on solid or liquid media, 

 one of the best being: 



Per Cent. 

 Monopotassium phosphate (neutralized to phenolphthalein by 



Sodium hydroxid 0.02 



Magnesium sulphate 0.02 



Sodium chlorid . 02 



Calcium sulphate 0.01 



Ferric chlorid (1 per cent, solution), 2 drops per 100 c.c. mannite 1.00 



The organism is readily isolated by seeding this medium with 

 soil. After the characteristic membrane forms, it is transferred by 

 dilution to a similar medium containing agar in which the charac- 

 teristic brownish black colonies form readily. 



On mannite agar the colonies first appear as milk-white glistening 

 drops, round and convex, which under a low magnification show 



