286 SELLARDS. 



were injected. As a rule one-eight to one-fourth of an agar slant was 

 used for the first injection and this was gradually increased to 6 or 8 

 slants. Perhaps the best result was obtained in a rabbit which at the 

 first mjection survived 1 agar slant given intravenousl}-. Pronounced 

 symptoms occurred ; within 6 hours the rabbit became semi-comatose, the 

 temperature rose from 39°. 5 C. to 40°. 1 C. and the respiratory and 

 heart rate were too rapid to count. This rabbit received subsequent in- 

 jections of amcebffi without loss of weight, or other symptoms and was 

 eventually able to withstand 20 agar slants intraperitoneally at a single 

 injection. Five other rabbits receiving a similar injection died within 

 15 hours. 



The preceding technique has been observed as a routine, but other ani- 

 mals and other methods were also tested. Thus one series of guinea pigs 

 was injected with cultures killed by heating to 50° C. for fifteen minutes. 

 Whereas guinea pigs, upon intra-peritoneal injection, were able to survive 

 only a small oese of living amceb« as much as 1 agar slant of a killed 

 culture could generally be used for the first injection and as many as 4 or 

 6 slants could be used in rabbits; however, the formation of irmnune 

 bodies was not active and the subsequent injection of living amoebae. was 

 not borne very much better than when the first injections were made with 

 smaller quantities of the unheated material. 



In one rabbit, the organisms were injected only in the encysted stage, 

 viable cysts being given intravenously for the first two injections and the 

 remaining injections being made intraperitoneally. This rabbit was 

 used only in testing for agglutinins. 



In as much as antibodies form readily upon the injection of vegetable 

 cells, of animal tissues, and of unorganized protein matter, and since they 

 occur in natural infections with protozoa, it would be expected, a priori, 

 that amoebae would also act as antigen. No attempt was made to investi- 

 gate all of the immunity reactions which might be produced with amoebae, 

 but rather I have sought to obtain a reaction which would be suitable for 

 distinguishing cultures from various sources. Accordingly, preliminary 

 tests were carried out with only one of the cultures of amoebae (Bace A). 

 Of the commoner biological reactions those for agglutinins, precipitins, 

 cytolysins, and anaphylaxis have been considered. 



Agglutination. — Agglutination tests were carried out on both the 

 active and the encysted stages of amoebae with serum obtained, first, from 

 guinea pigs and rabbits injected with the motile amoebae and second 

 from a rabbit injected with encysted forms. In the amoeboid stage, some 

 difficulty was encountered in securing a uniform suspension of the organ- 

 isms in the control preparations. The immune sera from rabbits and 

 guinea pigs were tested in varying dilutions up to 1 in 1,000. In hanging 

 drops, irregular clumping sometimes occurred in portions of the prep- 



