SUGGESTED BEST METHOD 87 



it is only possible to obtain scattered colonies, 

 amoebae do not multiply rapidly enough to 

 make the method of any use ; and, on the other 

 hand, the addition of water tends to make the 

 colonies coalesce. On this account, it is necessary 

 to plate out several times instead of only once or 

 twice. The second point is that amosbse avoid the 

 actual colonies of bacteria, and the cysts are most 

 abundant round their edges and in between them 

 (fig. 27). This method was only partially successful, 

 but it shows that amoebae can be separated by it 

 without resorting to germicides. 



After these experiences, we give the following 

 suggestions as to the steps which might be taken 

 by others in the preparation of pure mixed cultures 

 of amoebae, because we think the principles would 

 be useful in the case of other organisms which form 

 resistant cysts, and much time would be saved if 

 the experimenter had some information to guide 

 him in the subject. No definite plan can be laid 

 down as a hard-and-fast rule for every case, and 

 discretion may be used as to the order of proceed- 

 ing, and as to what stages may be omitted, viz. : 



Separate a single cyst by the method of placing 

 it on a jelly, as described at the beginning of the 

 chapter. Do not waste time in trying to pick 

 one off the culture, but be careful to place only 

 one cyst on to the jelly. Eliminate spore-forming 

 bacteria by selecting areas from cultures as free 

 as possible from all bacteria, especially from those 

 which grow into long filaments. 



Before using germicides, test for the absence of 

 spores. At this stage, treat the cultures with a 

 germicide such as 2-per-cent. HC1 for two or three 



