EXCYSTATION 59 



of the bacteria present, their rate of growth, and 

 the resistance of the cysts, etc., makes precise figures 

 of no value, but from general observations we feel 

 convinced that a balance exists between the 

 amoebae and accompanying bacteria which is more 

 than that of a mere food supply. Thus, peptone 

 broth has been found useless for ordinary cultural 

 purposes, as the bacterial growth is so rapid that the 

 amoeba* either do not excyst at all or are rapidly 

 killed after they have done so. With 0-1-per-cent. 

 peptone water, however, cultures with a mixture of 

 bacteria can be maintained. 



Age and resistance of cysts and the time factor 

 in excystation. It is most important to recognise 

 that in any culture of amoebae the cysts vary in 

 age. Those amoebae surrounded by a dense bac- 

 terial growth may encyst long before those which 

 are comparatively free from bacteria. For this 

 reason it is of no value to select an individual 

 cyst or even a dozen or so in any attempt to 

 discover the cause of excystation. As pointed out 

 by Musgrave and Clegg, the resistance of cysts 

 increases with their age, and younger cysts will 

 therefore develop more rapidly than old ones. In 

 order to minimise the error of random sampling, it 

 is necessary to select some hundreds of cysts for 

 each experiment, and, by choosing young ones (e.g. 

 from cultures only a few days old), one can avoid 

 the tedious waiting which the choice of older ones 

 involves. Living cysts can be recovered from old 

 cultures which have been exposed to considerable 

 ill-treatment, e.g. a temperature of 56 C. for 

 half an hour, or 4-per-cent. solution of hydrochloric 

 acid (31 '8 per cent. HC1) for three days, or 5-per- 



