IMMUNITY REACTIONS WITH AMCEBJE. 285 



inoculation from solid to liquid media is difficult or impossible. (5) However it 

 was found that some of the ordinary liquid media were very suitable for the 

 cultivation of amoebae, provided that these media were first diluted to such an 

 extent that the growth of bacteria was considerably restricted. Thus, amoebae 

 grew well in a solution of 1 part of peptone in 1000 parts of water and fair 

 growth occurred in ordinary nutrient bouillon when diluted to 1 part in 100, 

 although no growth of amoebae took place in a 0.5 per cent solution of peptone, 

 or in bouillon of the ordinary strength. There was a fairly wide range in the 

 concentrations of peptone which were found suitable, abundant growth taking 

 place in dilutions varying from 1 part in 1,000 to 1 in 5,000. The amoebae could 

 be transferred backward and forward from the solid to the liquid media, a good 

 growth being obtained on the first inoculation, in the same manner as with 

 bacteria. 



Occasionally, after cultivating a species of amoeba for several months on the 

 medium containing agar and beef extract, the composition was altered slightly 

 for one or two generations by the substitutions of 0.1 per cent peptone and 0.05 

 per cent lactose for the beef extract. Sometimes, when the growth on agar became 

 less abundant, a few transplantations were made in liquid media, using either 

 diluted normal rabbit serum or a solution containing 1 gram of peptone and 

 0.5 gram of lactose in 1,000 cubic centimeters of water. As a general rule, , the 

 most aboundant growths of amoebae were obtained at incubator temperature 

 (35° C). 



One well marked exception was found to the apparent necessity of a highly 

 diluted medium for the growth of amoebae. All of the four cultures of amoeboe 

 grew well in normal serum not only when it was highly diluted but also in the 

 lower dilutions. Thus, excellent growth took place not only in dilutions of 1 to 

 500, but also in 1 to 2, and in 1 to 5. This ability to develop in normal serum 

 was utilized later in testing for the production of immune sera. 



Technique for immunization. — The cultures of amoeba on agar slants 

 were used for the inoculation of animals. Living organisms were in- 

 jected in the amoeboid condition, emulsions being prepared in water from 

 24-hour cultures. The amoebae which were used for injection, were always 

 grown with B. prodigiostis. No attempt was made to reduce the quantity 

 of bacteria in the mixed emulsion of amcebse and bacteria, but when care 

 was taken to insure favorable conditions for a vigorous growth of 

 amoebae, the latter always predominated to a considerable extent over the 

 bacteria. 



Rabbits were used for immunization. The first two injections and 

 sometimes the third were made intravenously and the remaining ones 

 intraperitoneally. The injections were made at intervals of ten days to 

 one month according to the condition of the rabbits, and the maximum 

 period of treatment for any one animal was thirteen months. For rabbits 

 weighing about 1.5 kilograms, one-half to one agar slant of the ordinary 

 size, (measuring about 1.5 by 8 centimeters) upon intravenous injection 

 was usually fatal within 12 to 18 hours. Under the same conditions, 2 

 slants were usually fatal upon intra-peritoneal injection. Several rabbits 

 were used for each of the 4 cultures and varying quantities of material 



