299 



If some of this film is inoculated into another jar containing the same medium, 

 corresponding phenomena are seen when the culture conditions are alike. At first 

 sight already, there can be no doubt but under these circumstances fixation of con- 

 siderable quantities of nitrogen must take place, and chemical analysis proves that 

 this is really the case. 



The microscopic image of the Asotobacter growth in the malate commonly 

 shows smaller individuals of greater motility than the formerly described forms which 

 are obtained in the mannite solutions. They keep about the middle between A. chroo- 

 coccuni and A. agilis, and remind strongly of a variety found in America, which has 

 received the name of A. vinlandi. The plate cultures of such a malate accumulation 

 again prove not to be pure but to consist of the usual mixture of non spore-forming 

 species. They are best grown on a medium of the composition: 100 tap water, i cal- 

 ciummalate, 0.05 K 2 HPO 4 , i to 2 agar, on which the Asotobacter colonies become 

 already visible after 12 hours at 30 C., which is not the case with any other species 

 of microbes known to me. As these plates are somewhat cloudy by the produced cal- 

 ciumphosphate and the imperfectly dissolved malate, it is desirable to mix the ingre- 

 dients in the way as follows. Into a culture tube are first introduced some drops of a 

 neutral, concentrated solution of kaliummalate and herein are dissolved both the cal- 

 ciummalate and the kaliumphosphate, with a little water to dilute, but the smallest 

 quantity possible, as the dissolving power of the kaliummalate is much stronger in 

 the concentrated than in the dilute solution. Then the contents of the tube are mixed 

 with the agar solution. 



The malate plates prepared in this way have proved to be better for the growth 

 of Asotobacter germs than the mannite and glucose plates, so that of a definite 

 number of germs there develop more to colonies on the former than on the latter. 

 Hence it has become possible more exactly to compute the number of individuals of 

 our species present in a sample of soil than after the old method, to which circum- 

 stance we return below. 



Before going further I wish to notice the following concerning other salts of 

 organic acids as carbon food for Asotobacter. 



Except with calciummalate there could be obtained an abundant or moderate 

 growth with calciumlactate, calciumacetate and calciumpropionate, particularly when 

 using canal water for the first infection. It was remarkable that the transport of a malate 

 culture into lactate appeared to succeed nearly as well as of malate into malate, while 

 even relatively rich crude cultures in propionate- or acetatesolutions, obtained directly 

 from soil or water when inoculated into corresponding media, hardly grow on, if at 

 all. This fact is the more remarkable when we consider that by inoculation of a 

 malate film into propionate or acetate as abundant cultures are obtained as in the 

 said crude cultures in these media. But if it is tried to continue such cultures by 

 re-inoculating anew into propionate or acetate they also soon lose their power of 

 growth. From this we see that the preceding culture conditions to which the inocu- 

 lation material has been subjected, are by no means indifferent to the vitality of the 

 following generations, which are evidently very easily weakened and then nearly 

 quite lose the faculty of fixing nitrogen. The importance of this fact cannot be 

 denied and certainly deserves a nearer examination. 



Calciumcitrate, calciumtartrate and calciumsuccinate, with either garden soil or 



