i86 RESEARCH IN PROTOZOOLOGY 



incubation in the water bath and did not become complete until 

 the ice-box incubation was completed. 



The antigenic extracts used in the tests were prepared by ex~ 

 tracting with from seven to ten parts of absolute alcohol the 

 sediment of from sixty to eighty or more cultures of E. histolytica 

 grown upon the Locke-Egg-Serum medium of Boeck and Drbohlav 

 (1925), modified by using inactivated human blood serum in- 

 stead of horse serum. The cultures were forty-eight hours old and 

 the extraction was carried out in the incubator at 37° C. for ten 

 days, the mixture of alcohol and culture being thoroughly shaken 

 several times a day during that time. After extraction the mix- 

 ture was filtered and the filtrate used as an antigen in the tests. 

 Owing to the weak antigenic value of the extracts it was necessary 

 to use them undiluted and in the titration it has usually been 

 found that the workable antigenic amount varied from 0.06 to 

 0.1 of one cubic centimeter. The latter amount was hemolytic if 

 added to a blood suspension which did not contain blood serum 

 but if the usual amount of blood serum was added this amount 

 of antigenic extract was not hemolytic and could be used in the 

 tests. 



Owing to the fact that the antigenic extracts must be employed 

 undiluted the range of the test was markedly restricted and great 

 care must be used to see that all reagents were very accurately 

 measured. Briefly stated, the technique of the test was as follows : 



A double rack was employed, as in the Wassermann test, the 

 anterior row of tubes containing the sera to be tested and the 

 posterior the controls without the antigenic extract. In each tube 

 there is placed 0.9 of one cubic centimeter of normal salt solution, 

 double the amount of complement determined by titration as 

 sufficient to cause hemolysis of the standard amount of blood 

 suspension, and o.i of one cubic centimeter of the serum to be 

 tested. To the anterior tube there is now added the unit of antigenic 

 extract which has been determined by titration and the rack placed 

 in the water bath at 37° C. for one-half hour. At the end of 

 this time there is added to each tube o.i of one cubic centimeter 

 of the blood suspension, and two units of anti-human hemolytic 

 amboceptor, after which the rack is kept in the water bath at 

 37° C. for a full hour and is then kept in the ice-box for two 

 hours longer, when the results of the test are recorded. It has 

 been my practice to read the results again after incubation in the 



