12J2 



Fiirthermore it is pi'oved that the usiuil laboratory experiments 

 cannot give back to the ))olytrophic form, when it is kept in absence 

 of sohible organic matter and cultivated in a dilute nitrite solution, 

 the power of oxidising nitrites, not even in the course of 10 years. 



Consequently the process of nitratation in pasture ground must 

 take place as follows. 



When the soil contains a great deal of organic matter this need 

 not be exclusively oxidised and destroyed by other species of bacteria 

 in order to make the action of the nitrate germs possible, but it 

 may also be done in part of the germs themselves. It is true that 

 they get lost thereby as they pass into the polytrophic form, but in the 

 soil always places must be present without any considerable quantity of 

 organic substance, where unchanged oligolrophic germs occur. These, 

 after the destruction of the organic matter in their environment, 

 can multiply and again provide the soil witii a new nitrataring flora. 



It is very difficult to obtain pure cultures of the nitrate ferment 

 in the nitratating or oligotropliic condition. The best way is as follows. 

 First a crude nitratation is produced by bringing pasture soil into a 

 liquid of the composition : tapwater 100, sodiumnitrite 0,05 to 0,1, 

 bipotassiumfosfate 0,01. and cultivating at 30° C. 



After about one or two weeks the nitrate ferment of the infection 

 material has strongly increased and all the nitrite may be converted 

 into nitrate. 



A little of this nitratation, diluted with much water, is now sown 

 on the surface of a plate of the composition : tapwater 100, care- 

 fully extracted^) agar 2, sodiumnitrite 0,05, potassium fosfate 0,01, and 

 again cultivated at 30° C. As the nitrate ferment and the other microbes 

 accumulating in the crude nitratations, do not attack the agar, the rate 

 of soluble organic food present is very low. The nitrate ferment can 

 grow upon such a plate without losing its faculty of nitratation and 

 forms very minute colonies of about Vs ^^ J "^i'l- i'l diameter, which, 

 being very transparent and glassy little discs, are hardly visible. 

 With a greater |)ercentage of water in the agar they are denditrically 

 branched, with a smaller percentage they remain unchanged circular, 

 or become somewhat crenate. In such a pure culture the distance 

 between the colonies must be so great, that they do not touch one 

 another and can be separately examined. In consequence their number 

 on the plates must be relatively small, the counteraction of the still 

 remaining soluble organic substances great, and the nitratative powei' 



I) For the extraction the agar is left many days in distilled water which is now 

 and llien renewed. 



