IMMUNITY REACTIONS WITH AMCEB^. 283 



room temperature (about 25° to 30° C.) and incubator temperatiire (37° C.) ; 

 either was found to be satisfactory. Within the first 48 hours as many as 10 

 to 20 organisms usually developed from the original parent cell. The drop was 

 then transferred with a comparatively coarse pipette to agar media and the 

 cultures were continued in the usual manner. 



One race (D) had already been isolated from a single cell before it 

 was received at the laboratory. This culture was obtained by Professor 

 M. A. Barber of the University of Kansas from the faeces of a patient 

 with a watery diarrhoea. Individual cysts were selected and isolated 

 directly from the faeces. Upon transferring to nutrient media, some of 

 these cysts developed, thus not only giving a culture consisting of a 

 single species of amceba but also demonstrating that the cyst-like bodies 

 seen in the faeces were true amoebaj. 



The presence of bacteria in the cultures of amoebae - naturally necessi- 

 tates a considerable number of controls in the study of the immune 

 bodies resulting from the injection of these mixtures. To simplify these 

 controls, somewhat, the four different races of amoebae were cultivated 

 with the same species of bacterium. B. prodigiosus was chosen, since 

 amoebae gi'ow well in conjunction with it and its chromogenic properties 

 simplify the detection of contaminations by other bacteria. As a further 

 control, one of the races of amoebae was also cultivated with Vibrio cholera. 

 The separation of amoebae from all of the accompanying bacteria except 

 one species was found to be rather difficult. Even in the encysted stage, 

 the amoebae are in general rather less resistant to physical and chemical 

 agents than the sporeforming bacteria which occur with them. In the 

 motile stage, they are intimately mixed with bacteria, the latter clinging 

 to the surface and also being enveloped in the substance of the amoebae, 

 while the spores may resist any digestive action. 



The method devised by MoutonO) was used for obtaining the amoebES in 

 culture with a single species of bacteria. The center of a Petri plate was 

 inoculated with amoebae, the desired bacterium having been inoculated previously 

 in lines radiating from the center. As the amoebae wandered over the plate 

 they followed the lines of bacterial growth, gradually leaving the contaminating 

 bacteria behind them. After several transplantations had been made in this 

 manner, test tubes were substituted for Petri plates to avoid accidental contamina- 

 tion from the air. The entire surface of an agar slant was inoculated with 

 bacteria, the inoculation of the amoebae being made at the base of the tube- 

 After 24 hours in the incubator with the tubes placed in the erect position, the 

 amoebae grew upward a distance of 3 to 5 centimeters, a sharp line of demarcation 

 frequently appearing at the junction of tlie growths of the bacteria and the 

 amoebae. jSIany repetitions of this process were necessary before all of the con- 



^ Many of the earlier observers have attempted to obtain puie cultures of 

 amoebae, but no successful method, was established whereby development continues 

 indefinitely in the absence of both living and dead bacteria. However, very 

 recently, Williams(2), in a preliminary communication, reports successful cultiva.- 

 tion in the absence of bacteria. 



