303 



It is to be remarked that as well in the malate as in the lactate cultures slight 

 amounts occur of a volatile .acid, which will perhaps prove to be acetic acid, although 

 it has not been positively demonstrated by means of B e h r e n s' uranylanatrium- 

 acetate reaction. It is of importance to know that this volatile acid is not only found 

 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 the volatile acid and the corresponding 

 quantity of decomposed malate, it is supposed in the table to be acetic acid only and 

 produced after the formula: 



2 C 4 H* O r > Ca + 20 2 = C 4 H 6 O* Ca + Ca CO 3 + 3 CO 2 + PT-O 

 Calciummalate Calciumacetate. 



But it may also be formed without access of oxygen. The volatile acid is deter- 

 mined by distillation with sulphuric acid and silver sulphate 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 Azoto- 

 bacter 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 Asotobacter in the soil. 



Earlier, already, I showed that it is possible to detect a few Asotobacter 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 *) could be found on 

 which all the germs of Asotobacter sown out really develop into colonies. Thus, by 

 sowing about 2400 germs (determined by microscopic counting), on various culture 

 plates, 50, 12, i, 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 been 

 shaken up in sterile tap-water or in malate solutions, of which i cm 3 was spread over 

 the plate, care being taken that the water was quite taken up into the agar, 

 by its power of imbibition, which is easily effected by softy heating the plate so that 

 the superfluous water evaporates. 



*) 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. 



