86 "PURE MIXED CULTURES" OF AMOEBA 



ever carefully they may be picked off from a plate, 

 the excessive number of bacteria present does not 

 allow of plating out colonies sufficiently far apart 

 to enable a single colony of bacteria to be picked off 

 along with adherent cysts. The washing requires 

 care, the upper layers being siphoned off, leaving 

 most of the cysts at the bottom of the vessel. The 

 washed cysts are centrifugalised, Petri-dish cultures 

 made, and after a few days colonies are found with 

 abundant cysts either on them or more often 

 collected round their edges. One suitable colony 

 was picked off with a platinum loop, cultures pre- 

 pared as before, and the whole process of washing 

 and plating out repeated. Finally an agar slope 

 culture was submitted to analysis, and found to be 

 almost pure B. f. liq. with a slight admixture of 

 B. proteus vulgaris. In the preparation of this 

 slope for analysis it was observed that the amcebse 

 were in the free-living condition, and, while the 

 analysis was in progress, they were expected to 

 encyst, but they did not do so, 1 although every 

 effort was made to bring this about. It was clear 

 that no useful object would be attained if the 

 separation was continued, because a pure mixed 

 culture of amcebae which do not encyst is of no 

 value in the preparation of cultures with other 

 bacteria. 



However, the method is shown to be a prac- 

 ticable one, as spores had been eliminated ; the two 

 bacteria present are easily killed ; and the culture 

 was almost pure. In connection with this plating- 

 out method, two points must be borne in mind : 

 Under the comparatively dry conditions in which 



1 Because of the presence of an amoeba parasite. 



