70 ENCYSTMENT AND EXCYSTATION OF AMCEBA 



multiplication took place, the amoebae looked more 

 natural, and divisions were watched. The culture 

 was kept going for five days, when it was obvious 

 that reproduction had ceased. The amoebae did 

 not, however, encyst at once, but persisted in their 

 free condition for a fortnight (although many 

 appeared to die from lack of food), when encyst- 

 ment took place, although no living bacteria were 

 present in the tube. We think this may possibly 

 be due to the fact that the choline becomes slowly 

 oxidised to neurine, which is an intensely toxic 

 substance. We should mention that, if there is 

 any bacterial contamination of these preparations, 

 its presence is shown by a visible extension of the 

 bacteria from the part where the tube is inoculated 

 to the edges, together with a cloudiness of the 

 medium, and also by the fact that encystment 

 always occurs rapidly when a tube becomes in- 

 fected. As a further precaution, if there is any 

 doubt about accidental infection, subcultures are 

 made on nutrient agar. 



We were at a loss to explain why the amoebae 

 had ceased to multiply except on the assumption 

 that they were short of something, or that their own 

 products of metabolism were injuring them. A 

 subculture was therefore made on a jelly containing 

 creatine and choline as before, but without the 

 chloroformed solution, which had only been em- 

 ployed in the first case to get the amoebae out of 

 the cysts. Very little growth occurred and some- 

 thing still appeared to be lacking which was 

 necessary for their continued reproduction. A 

 second subculture was then made, this time with 

 the addition of 1 cc. of the chloroformed extract, and 



