PLATING-OUT METHOD 85 



present adopt whenever a culture with a different 

 bacillus is required or when cysts are wanted free 

 from all bacteria. The acid apparently does very 

 little harm to the cysts, as excystation of treated 

 cysts is not noticeably slower than in untreated 

 ones. The acid can be washed away with sterile 

 water, but except for special purposes the dilution 

 in a fresh medium is sufficient, as amoebae will 

 excyst in an acid medium. 



METHOD 2. RESULT : CULTURE OF AMOEBA (IN- 

 FECTED WITH A PARASITE) WITH Two BACILLI 



This method was adopted as a possible means 

 of preparing pure mixed cultures when our ex- 

 perience with germicides seemed so little hopeful. 

 It depends entirely on the mechanical isolation 

 of cysts with a particular bacillus by a succession 

 of platings out in colonies, sterile precautions 

 being taken throughout and no chemical agent 

 being employed. As in Method 1, a most impure 

 culture was used in order to see if this plan 

 would be available under the most adverse circum- 

 stances. One cyst was easily placed on a sterile 

 jelly with a few bacteria only, as described in the 

 beginning of this chapter, and a culture rich in 

 cysts obtained. Areas distant from the site of in- 

 oculation were scraped, and the cysts and bacteria 

 so removed were washed many times in a beaker 

 with large volumes of water. The cysts fall to the 

 bottom more rapidly than the bacteria, and re- 

 peated washings reduce the relative number of the 

 bacteria considerably. This is necessary, as one 

 must deal with a large number of cysts, and how- 

 iv 6* 



