288 SELLARDS. . 



was merely an emulsion of a 24-hour culture in physiologic salt solution. 

 ISTo-Jvell defined differences were obtained between the injections in normal 

 animals and those injected with amcebas and B. prodigiosios. The bleb 

 produced by the injection was usually absorbed within the course of an 

 hour. After 12 to 18 hours some oedema and reddening appeared at the 

 site of injection. This was distinctly more marked in the eases where an 

 unfiltered extract of amoebae was used, suppuration eventually resulting in 

 some: instances. The differences between the normal animals and the 

 treated ones were slight and inconstant. However, the general tendency 

 of such differences as were noted, was not in the direction of a greater 

 reaction in the treated animals, but on the contrary these animals some- 

 times showed signs of less inflammation at the site of injection; than the 

 controls. The differences, however, were altogether inadequate for any 

 satisfactory test and attention was next given to cytolysins. 



Cytolysins. — Tests for the cytolytic action of the sera were carried 

 out microscopically. Ordinary hanging-drop preparations were made 

 from equal parts of a suspension of amoebse and immune serum. The 

 suspension of amoebaj was prepared in the water of condensation from 

 the agar culture media, in preference to normal physiologic salt solution, 

 since the latter is hypertonic for amoebae. A certain amount . of salt 

 could be used to advantage, perhaps, imitating the conditions which occur 

 in surface waters, the natural habitat of some of these amoebae, but in 

 attempting to determine the most desirable amount of salt it was found 

 that the amoebae when transferred from agar to distilled water showed no 

 defihiie morphologic change and remained in the amoeboid stage for 

 at least several days. The serum dilutions also were prepared; with 

 water, and not with salt solution. - . 



Investing for cytolysins, control preparations were made with normal 

 serum, with physiologic salt solution, and with distilled water. Slight 

 changes were noticeable immediately after preparing the mixtures. Some 

 of the amoebae lost their motility and assumed a spherical form, both in 

 the preparations with immune serum and also to a lesser extent in those 

 with' normal senxm and with physiologic salt solution, while in those 

 vsath distilled water, no change was observed. With immune serum of 

 a high grade cylotysis of the amoebae could be observed microscopically 

 and in the course of one to two hours there was a well marked diminution 

 in the number present in the hanging drop. Sera which were sufficiently 

 active to cause immediate cytolysis were obtained only with some difficulty 

 and after a comparatively long period of immunization. Furthermore, 

 none of the preparations showed complete cytolysis of all of the amoebae, 

 present, the differences between the normal and immune serum in this 



