82 "PURE MIXED CULTURES" OF AMCEBA 



obtained, and they wander from the site of 

 inoculation to a much greater extent than in 

 Petri-dish cultures kept upright to which only 

 a little water has been added. After several days' 

 growth, areas were selected in which the amoebse 

 (which had by this time encysted) looked as free 

 as possible from bacteria, etc., and subcultures 

 were made. Selected loopfuls were again taken, 

 this time with the object of avoiding sporing 

 bacteria, which can occasionally be recognised by 

 their forming long filaments. Subcultures with 

 strictly aseptic precautions were then made, and 

 single cysts isolated on to sterile plates by the 

 method recently described. In this way one is 

 able to obtain cultures with a limited variety of 

 bacteria with the probability of having eliminated 

 sporing forms. To determine this in a particular 

 culture an abundant scraping was taken from all 

 parts of the surface and heated at 80 C. for a 

 quarter of an hour. No growth occurred on 

 inoculation of nutrient agar slopes at room tem- 

 perature and 37 C., and no spores were visible 

 in stained films made from the mixture. 



There now only remained to select a method 

 of removing the few remaining bacteria without 

 killing the cysts, and for this purpose formalin 

 was chosen, as we had previously found that a 

 culture could be exposed even to a 3-per-cent. 

 solution for a week and living cysts could be 

 recovered. Petri-plate cultures were therefore 

 covered with formalin for forty-eight hours, washed 

 thoroughly with sterile water, and loopfuls tested 

 on sterile nutrient agar slopes. They were then 

 found to be free from living bacteria. Cysts thus 



