58 JAMES M. SHERMAN 



V. THE EFFECT OF PURE CULTURES OF PROTOZOA UPON THE SOIL 



BACTERIA 



Isolation of protozoa 



The term ''pure culture" as it is here used in connection with 

 protozoa means only an ''animal-pure culture," since the cul- 

 tures of protozoa were not obtained free from bacteria. The 

 soil amoebae, as has been shown by Beijerinck (1896), (1897), 

 Celli (1896), Tsujitani (1908), Frosch (1909), and others, may be 

 cultivated on solid culture media and so can be isolated in the 

 pure state more readily than the other types of protozoa. 

 With the ciliates and flagellates the problem of obtaining cul- 

 tures free of bacteria is more difficult. Berliner (1909) and 

 Nagler (1909) have shown that certain of the flagellates and cili- 

 ates will grow upon agar plates but this method has not given 

 general satisfaction. Many methods have been tried, some of 

 which have been partially successful, but none have proved very 

 practical. 



It was found in this work that animal-pure cultures could be 

 conveniently obtained by the dilution method. As was noted 

 earlier in this paper in high dilutions of soil (1/10,000 gm.) the 

 protozoa present are usually restricted to a few types. By 

 inoculation of a large number of high dilutions of soil (1/10,000 — 

 1/50,000 gm.) into soil extract a few cultures which contain only 

 one type of protozoa will usually be obtained. In order to isolate 

 types which do not occur in soil in very large numbers it is 

 necessary to utilize an enrichment medium which favors the 

 growth of the desired organism. When the desired type has 

 obtained the ascendency, it may be readily isolated by the dilu- 

 tion method. Hay extract, Ashby's solution, Giltay's solution, 

 dilute peptone broth, and a weak beef extract solution have been 

 used successfully for enrichment cultures. 



After the protozoa were obtained in animal-pure cultures it 

 was attempted to simplify the bacterial flora contained in them. 

 This was accomplished by passage through several successive 

 subcultures in Ashby's solution. The types of bacteria capable 

 of development in this medium are somewhat limited. 



