SEROLOGICAL METHODS IN THE STUDY OF PROTOZOA 415 



as the technical setting up of the tests has reached a fair degree 

 of standardization, the experimental procedure at the outset of 

 preliminary work pivots upon the satisfactory preparation of a 

 test antigen. The attainment of this in agglutination tests is com- 

 paratively simple but in the complement fixation and precipitin 

 tests depends upon a nicely balanced manipulation of conflicting, 

 complex and often elusive factors, the control of which has taxed 

 individual ingenuity unsparingly. At the outset the first step in 

 the preparation of any test antigen depends upon the isolation of 

 the parasite. This is fairly easy where the form is cultivable or 

 free in the blood stream. Intracellular parasites, however, are not 

 amenable to study by means of agglutination, phagocytosis and 

 lysis but have been used in complement fixation and precipitation 

 where extracts and not whole organisms are required. Even so, 

 it is difiicult to obtain concentrated solutions of the reactive para- 

 sitic material without including too high a percentage of extraneous 

 host proteins. To obviate this, investigators have partially con- 

 centrated the parasites by centrifugation {e.g., large schizonts of 

 Plasmodium falcipanun from minced infected placenta), or where 

 the parasites are resistant to digestion have digested away the 

 tissue, as in Bachman's work on coccidia. 



Once the parasites are isolated, they are simply washed and 

 resuspended in saline or distilled water for agglutination, phagocy- 

 tosis or lysis. Sometimes they are killed for agglutination tests 

 and it is theoretically possible to use dead organisms for phagocy- 

 tosis. For complement fixation and precipitation they may be ex- 

 tracted, either fresh or after drying and pulverizing, in a variety of 

 solvents. Alcohol and ether are common lipoidal solvents. Even 

 when an aqueous solvent is eventually used, a preliminary ex- 

 traction w^ith lipoidal solvents often makes the later aqueous ex- 

 traction more efiicient. The aqueous solvents vary with almost 

 every investigator. The simplest is distilled water or physiological 

 saline. Solution of the protein often seems greatly facilitated by 

 using a slightly alkaline solution, such as Coca's solution (an 

 aqueous solution of 0.5% NaCl, 0.05% NaHCOg, and 0.4% phe- 

 nol) ; by stronger alkalies, such as N/io NaOH or solutions of 

 antiformin with subsequent neutralization with HCl before use ; 

 by 0.1% HCl which also has to be neutralized before use; or by 

 various proportions of glycerin in saline. When necessary, various 

 preservatives such as phenol, thymol, toluol may be added to inhibit 



