IMMUNITY REACTIONS WITH AMCEB.E. 287 



aration, but differences from the control were neither constant nor well 

 defined. In the encysted stage, uniform emulsions for agglutination were 

 readily obtained. The serum from a rabbit which had been treated with 

 cysts for 3 months proved to be entirely inactive. Also, the senim from 

 rabbits treated with organisms in the amoeboid stage showed no agglu- 

 tinative action toward the encysted stage.^ 



Precipitins. — Examination for precipitins was somewhat complicated, 

 partly because of the difficulty of securing a suitable extract of amoebae 

 and also on account of the controls rendered necessary by the possibility of 

 the presence of bacterial precipitins. 



An extract of amoebae grown with B. p-rodigiosus was prepared by emul- 

 sifying the growth from 24-hour agar slants in the water of condensation 

 and grinding this emulsion with sand. The fluid was collected by centri- 

 fugalization and clarified by filtration. A clear filtrate was obtained 

 most readily by the use of a Berkefeld filter, but the precipitation re- 

 actions were more satisfactory when the extract was filtered through mag- 

 nesium oxide. Serum from a rabbit which had been under treatment 

 for five months gave a well marked precipitation in 1 to 3 and 1 to 5 

 dilution with the undiluted extract from the mixture of amoebae and 

 B. prodigiosus. However, this same extract also gave a precipitate with 

 the serum from a rabbit treated with B. prodigiosus alone. In order to 

 control the effect of bacterial precipitins, an extract of amoebaB growing 

 with y. cholercB was tested against the serums of a rabbit immunized to 

 amoebae growing with B. prodigiosus. Only a poorly defined precipitate 

 was obtained in 1 to 2 dilution of serum from animals which had been 

 under treatment for from two to five months. These preliminary results, 

 therefore, did not indicate that precipitin tests would afford a very suit- 

 able reaction for the study of amoebae, both on account of the technical 

 difficulties and also because of the indefinite reactions which were 

 obtained. 



Annphylaxis. — The usual test for anaphylaxis with death of the 

 animal requires rather more material than conveniently can be obtained 

 from cultures of amoebae. However, the intradermal reaction as devised 

 by Knox Moss and Brown, (8) would be especially serviceable for work 

 with amoebae. Eabbits and g-uinea pigs were tested within two to four 

 weeks after the first injection and also at later periods after treatment 

 had been continued for from 1 to 6 months. The injections were made 

 intradermally with amoebae growing with B. prodigiosus. Two prepara- 

 tions of amoebae were used. One consisted of an extract, filtered through 

 magnesium oxide in the same manner as for the precipitin test. The other 



' However, successful agglutination of amoebse by a non-specific serum, has been 

 reported by Zaubitzer(7) who found, that the serum of an animal when immun- 

 ized to V. cholerce, agglutinated amoebse growing with V. cholerce. 



