IMMUNITY REACTIONS WITH AMCEB^. 289 



respect being only relative and not absolute. However, after 24 hours 

 at 35° C. a definite result was readily obtained, notwithstanding the fact 

 that rabbit serum in 1 to 2 dilution furnishes a good culture medium in 

 which a few surviving amoebge might develop. In the mixtures with 

 distilled water and normal serum the amoebae remained actively motile 

 without any encystment before the second or third day and without any 

 significant change in ninnbers. No increase in number was to be expected 

 since the concentrations of the amoebse in the suspension was usually 

 greater than that which occurs in liquid culture media. In the test pre- 

 parations with serum from treated animals the reaction was usually very 

 definite. Either there were many motile amcebse present just as with 

 normal serum, or else there was almost a complete lysis of the amoebae 

 with only a few cysts and an occasional motile organism present. 

 Occasionally, in the preparations with immune serum, many of the 

 amoebffi took on a pink color. This was due apparently to the presence 

 of granules of B. prodigiosus, although no color was visible in the sus- 

 pension of bacteria surrounding the amoebae. 



The reaction in this, form was then adopted for testing the behavior 

 of the 4 races of amoebae. The suspension of amoebae was always prepared 

 in the water of condensation, from an actively growing 34-hour culture 

 in the amoeboid stage. Equal parts of this suspension and the serum 

 to be tested were mixed with a capillary pipitte according to "Wright's 

 technique and a hanging drop preparation was made from a portion 

 of the mixture. In order to obtain constant and well defined reactions, 

 it is necessary that the amoebae in the culture should be abundant and 

 should predominate over the bacteria present. The data which follow 

 represent the conditions of the preparations after standing for eighteen 

 to twenty-four hours at 35° C. The concentrations of serum are ex- 

 pressed in the final concentration after dilution with the suspension of 

 amoebas, for example, 1 part of undiluted serum and 1 part of the sus- 

 pension is recorded as a 1 to 2 dilution of the serum. 



On account of the presence of bacteria throughout the entire procedure 

 there are several interpretations which might be considered with regard 

 to the cause of the destruction of the amoebae by the immrme sera. Three 

 general methods were used for detecting the effect of the bacteria. The 

 serum of rabbits immunized to amoebae in mixed culture with B. prodigio- 

 sus was tested against a culture of amoebae growing with B. prodigiosus 

 and with V. cholerce. Also, the serum of a rabbit immunized to B. pro- 

 digiosus alone was tested against a culture of amoebae growing with B. 

 prodigiosus. These controls were carried out with the culture designated 

 -A and the results of the first and second methods appear in Table I. 



