BACTERIA FIXING ATMOSPHERIC NITROGEN 107 



(c) Bad. -pneumoniae, Bad. aerogenes, and other non-spore forming 



bacteria 



(d) Bac. asterosporus group and other spore-forming bacteria. 

 II. Symbiotic nitrogen-fixing bacteria 



1. Bacteria living in the roots of leguminous plants 



2. Bacteria living on and in the roots of non-leguminous plants 



3. Bacteria living in the leaves of certain plants 



Isolation of anaerobic bacteria. For the isolation of bacteria capable 

 of fixing atmospheric nitrogen, Winogradsky 25 used a solution free from 

 combined nitrogen of the following composition : 



Distilled water 1000 cc. NaCl 0.01 gram 



Glucose 20.0 grams FeSO 4 and MnSO 4 . . . Traces 



K 2 HP0 4 1.0 gram CaC0 3 30 grams 



MgS0 4 -7H 2 0.5 gram 



One hundred cubic centimeters of this medium is placed in a flask and 4 grams of 

 chalk added. The medium is sterilized at 106° to 110° for 30 to 45 minutes. A small 

 quantity of soil, preferably first pasteurized is used for inoculation. After a few 

 days' incubation at 25° to 30°, the surface of the liquid becomes covered with a thin 

 pellicle of aerobic bacteria; gas bubbles are formed abundantly, an indication of 

 butyric acid fermentation. Gas formation begins from the lump of soil and spreads 

 all over the flask, so that the whole surface is soon covered with gas, while the chalk 

 is lumped together by bacterial slime. The culture gives off an odor of butyric 

 acid and its esters. On examining the culture microscopically, it is found that 

 the bacteria in the film and in the residue are not alike. The film contains an 

 aerobic organism, while the residue contains Clostridium {Bac. amylobader) in 

 abundance, in the characteristic forms. Transfers are made, by inoculating a 

 piece of chalk from the bottom of the flask into fresh lots of media. When the 

 culture is sufficiently enriched in Clostridia (after three to four transfers), at- 

 tempts are made at isolation of pure cultures. Pieces of potato smeared with 

 chalk are placed in Petri dishes and sterilized. These are inoculated with spore ma- 

 terial and incubated, under anaerobic conditions, at 30° to 35°, in a partial vacuum 

 or hydrogen atmosphere. After 5 to 7 days, there appear upon the surface of the 

 potato elevated, rounded, yellowish colonies filled with gas bubbles. On opening 

 the apparatus, the colonies can be examined for Clostridia and transfers made 

 into liquid media or fresh potato cultures. The organism often occurs on the 

 potato in involution forms, which temporarily lose the capacity of spore forma- 

 tion. The culture can be grown under aerobic conditions in the presence of an 

 aerobic non-spore forming organism, such as Bad. fluorescens or Azot. chroococ- 



25 Winogradsky, S. Sur l'assimilation de l'azote gazeux de l'atmosphere par 

 les microbes. Compt. Rend. Acad. Sci. 116: 1385-1388. 1893; 118: 353. 1894; 

 Recherches sur l'assimilation de l'azote libre de l'atmosphere par les microbes. 

 Arch. Sci. Biol. (St. Petersburg), 3: 297. 1895; Clostridium pasteurianum, seine 

 Morphologie und seine Eigenschaften als Buttersaureferment. Centrbl. Bakt. II, 

 9: 43-62. 1902. 



