RELATION OF PROTOZOA TO SOIL BACTERIA 431 



influence on the process of free nitrogen fixation. It is probable 

 that the larger protozoa made use of the Azotobacter as food. 

 In certain cases upon staining a small amount of the film from 

 a liquid culture with Gram's iodine solution Clostridium cells 

 could be very readily distinguished within the protozoan cell. 

 The probable presence of Azotobacter cells within the protozoa 

 cells was also observed but by no means as definitely as in the 

 case of the Clostridium. 



It may be concluded that the protozoa have a slight detri- 

 mental effect on free nitrogen fixation in solution because the 

 individual determinations seem to check closely and to be quite 

 outside the limit of experimental error considering the small 

 amount of nitrogen in the cultures. In the case of the soil cul- 

 tures such a slight difference is not so important because the 

 total nitrogen content here is approximately five times that of 

 the liquid cultures. 



DISCUSSION 



From the results of this study of the influence of the protozoa 

 on ammonification, nitrification and free nitrogen fixation in 

 soil it would seem that their effect can not be considered detri- 

 mental. This is in accord with the work of other investigators, 

 even with that of Cunningham (1915) who claims that his re- 

 sults uphold the theory put forth by Russell and Hutchinson. 

 In the work referred to he states that soil protozoa in solution 

 exercise a decided limitmg effect on the numbers of bacteria and 

 that in an ammonifying solution they show their activity by 

 causing a decrease in the amount of ammonia produced as com- 

 pared with cultures free from protozoa. These results are in 

 accord with those already presented in this paper, in regard to 

 free nitrogen fixation in solution. The protozoa seemed to 

 have a detrimental effect on this process but when experiments 

 were carried out on free nitrogen fixation in soil, the protozoa 

 did not appear to influence the amount of nitrogen fixed. Cun- 

 ningham may well conclude that protozoa have a limiting effect 

 on the number of bacteria in solution, for here an environment 



