( 72 ) 



in the crude, but also in the pure cultures of Azotobacter, so that 

 it is certainly a product of this species itself. 



In order to ascertain the amount of I he volatile acid and the corre- 

 sponding quantity of decomposed malate, it is supposed in the table to 

 be acetic acid only and produced after the formula: 



26' 4 H* O" Ca + 20 2 = G' 4 H« (> 4 Ca + Ca CO 3 + 3 CO' + H'O 

 Calciummalate Calciumacetate. 



But it may also be formed without access of oxygen. The volatile 

 acid is determined by distillation with sulphuric acid and silver sul- 

 phate and titration the distillate with normal alkali. 



From the table we see that in the crude cultures nitrogen can 

 without doubt be fixed with calcium acetate as carbon source. In 

 truth we have not succeeded in effecting the same in pure cultures, but 

 now that we have the certainty that Azotobacter alone, with malate 

 as carbon food, is able to fix nitrogen, it must be admitted that 

 this also holds good for the acetate cultures, although it is not 

 clear of' what nature is the assistance which other bacteria thereby 

 must necessarily lend. Besides it should be noted that the fixation 

 of nitrogen in the pure cultures, also when malate is used as carbon 

 food, is less considerable than when other bacteria, too, can live on 

 this substance at the same time. 



3. Distribution of Azotobacter in the soil. 



Earlier, already, I showed that it is possible to detect a few 

 Azotobacter colonies among the thousands of those of the other species, 

 when fertile garden soil is sown on mannite-kalium-phosphate plates. 

 The use of calciummalate instead of sugar has proved to be of 

 importance for the examination of the soil in this direction. First 

 it should, however, be observed that no solid or liquid medium x ) 

 could be found on which all the germs of Azotobacter sown out 

 really develop into colonies. Thus, by sowing about 2400 germs 

 (determined by microscopic counting), on various culture plates, 50, 

 12, 1, 30, 8, 20, 10, 20 and 75 colonies developed so that the growth in 

 percents was only 2, 0.6, 0.5, 0.3, 0.3, 0.8, 0.4, 0.8 and 0.3. In 

 another experiment were obtained of 10.000 germs sown on glucose- 

 calcium-malate plates, 20, 25 and 48%. and on calcium-kaliummalate- 

 plates 32.5, 36 and 65%- But in other cases, on agar plates with 

 malate only the results were much better. The germs had 



1 ) The use of thin layers of liquid media for colony culture of microbes has 

 been described in Centralblatt f. Bacteriologie, 2te Abt. Bd 20, 1908, p. 641. 



