282 SELLARDS. 



teric patients have been examined for changes analogous to those produced 

 by artificial immunization with amoebae. The number of cultures under 

 investigation was limited to four and these were selected, without regard 

 to morphology, from sources which represented fairly distinct conditions. 



The first culture. Race A/ was obtained from the city water supply of Manila. 

 This is an unflltered river water wliich comes, liowever, from a practically un- 

 inhabited watershed. Amoebae are constantly present in this water. Eace B. 

 was cultivated from the stools of a case of amoebic dysentery occurring in 

 Manila. The two remaining cultures were secured from sources outside of the 

 Tropics. Race C. was grown from hay obtained from central Illinois, a region 

 practically free from amoebic dysentery. Race D. was cultivated in Kansas City, 

 from the stools of a patient with slight diarrhoea. 



In the preparation of these cultures for injection into animals, there 

 are two factors which were considered to be of especial importance, namely, 

 (1) the isolation of a single species of amoeba and (2) its cultivation with 

 a single species of bacterium. 



Much of the morphologic work upon amoebae has been carried out upon 

 material which might contain a variety of species but in attempting a 

 differentiation by immunity reactions it seemed especially desirable to 

 isolate single species of amoebaB, i. e., to know that each culture consisted 

 of the progeny of a single organism. 



The most feasible method for the isolation of single cells is that devised by 

 Barber (1) and it is readily applicable to amoebae. In transferring single motile 

 amoebse from cover glasses and pipettes to nutrient media they exhibit some 

 tendency to stick to the glassware in a manner similar to certain cells, such as 

 leucocytes and some of the capsulated bacteria. The cysts of amoebae, do not 

 possess this characteristic and they can be isolated and transferred readily. Ac- 

 cordingly, the isolations were first attempted with the encysted stage. However, 

 it was found that only a small proportion of the isolated cysts would multiply 

 when transferred to agar, prepared for tlie cultivation of amoebae. The substitu- 

 tion of a liquid, nutrient medium for the agar would reduce the manipulations to 

 a minimum, thereby permitting the isolation of organisms in the amoeboid 

 stage without necessitating further transference in order to supply nutrient 

 material. Normal rabbit serum was found to constitute a suitable liquid culture 

 medium. Emulsions of motile amoebae were prepared in serum in dilutions 

 varying from 1 to 5 to 1 to 10 and the isolations were made immediately in 

 hanging drops on cover slips. The entire drop ^^•as not too large to come within 

 the field of the one-sixth or one-twelfth objective. The fluid medium was quite 

 clear and accordingly it could be determined definitely when a single amoeba 

 was obtained. In order to supply an abundance of nutrient material the size 

 of the droplet was increased, after isolation was efi^ected, to a diameter of 1 or 2 

 millimeters. Under these conditions, multiplication took place in about one- 

 fourth of the isolations. In the remaining three-fourth the amoebae disintegrated, 

 encystment rarely taking place. The hanging drop cultures were kept both at 



* These cultures have been designated simj)ly by letters, in as much as the 

 basis for the classification of amoebae into species is incompletely developed and 

 the description of the various species which have been reported is often inadequate 

 for their identification. 



