EXCYSTATION 69 



place fairly rapidly, and living amoebae were fairly 

 abundant all over the medium, but gradually died 

 out in the course of a week. Nutrient agar sub- 

 cultures of these amoeba* showed the conditions 

 to be bacteriologically sterile in all the tubes except 

 one, and in this the amoebae encysted again in a 

 few days. The effect of the pepsin was striking, 

 as, although not all the cysts excysted, they all 

 exhibited swelling of the ectoplasm, which is the 

 first stage visible in the process, and many of the 

 enclosed amcebas were noticed moving within the 

 cyst walls. This internal movement is comparable 

 to the rotation found by Goodey to take place in 

 the cysts of Colpoda cucullus during excystation. 



Cultures of amoeba with dead bacteria. Hitherto 

 the difficulty had been to maintain the existence 

 of those amoebae which had been made to excyst. 

 Bacteria killed by hydrochloric acid did not appear 

 to be adequate food. No better results were 

 obtained by using boiled bacteria, and recourse 

 was had to other means. A luxurious growth of 

 Bacillus fluorescens liquefaciens on nutrient agar 

 was exposed to ether vapour at 37 C. for twenty- 

 four hours, the ether being then evaporated off until 

 the agar was free from odour. A jelly was prepared 

 as follows : 2-per-cent. agar jelly 5 cc., chloro- 

 formed solution of dead B.f. liq. 3 cc., and creatine 

 added to 0*5 per cent. ; total 8 cc. This was made 

 into a slope, smeared thickly over with etherised 

 bacteria, bacteria-free cysts placed on it, and the 

 tube incubated at 37 C. Excystation took place 

 as usual, but no obvious multiplication. A similar 

 jelly was then made, but with the addition of 

 0'1-per-cent. choline, and on this medium rapid 

 iv 5* 



