SEROLOGICAL METHODS IN THE STUDY OF PROTOZOA 423 



animal are mixed with the known organism ; in the second case, the 

 experimental organism is mixed with an agglutinating serum pro- 

 duced with a known organism. The materials required are the 

 specific test antigen and antiserum. As various substances, par- 

 ticularly saline, sometimes cause agglutination by themselves, 

 the control tubes are very necessary. 



C. Protocols. The protocol in Table 6 which is set up in 8 by 100 

 millimeter tubes has been used extensively. Dilution, which may 

 be extended indefinitely, should be carried out far enough to rule 

 out natural and group agglutinins. Whether such agglutinins exist 

 should be ascertained by control tests with normal serum and 

 serum from animals either infected or immunized with related 

 organisms. 



The microscopic method for the agglutination reaction has 

 been developed where only a small amount of blood serum or dry 

 blood is obtainable. It is most widely used for the Gruber-Widal 

 reaction for typhoid fever and may be outlined as follows : 



Cover- Homogeneous Moderate 



slip Blood Serum Suspension of Organisms Diluent 



Dilution Amount 



1 1 :20 Platinum loopful Platinum loopful 



2 1:40 



3 Control Platinum loopful 



Mix and invert each cover slip over a vaselined hanging-drop 

 (concave) slide. Place in the dark. At the end of one hour examine 

 with a microscope for clumping and motility, being sure that 

 the control slide shows none. With dried blood the procedure is 

 essentially the same except that the blood can only be approxi- 

 mately diluted. 



In vitro lytic experiments with the protozoa can be carried 

 out in the same manner as hemolysis, as given in Table i. Since 

 physiological sahne frequently causes disintegration of protozoa 

 (especially among the pathogenic trypanosomes) upon incubation, 

 Control Tube 19 is impossible. Dilutions can of course be varied 

 to suit individual requirements. 



In vitro phagocytosis experiments, according to Levaditi and 

 Mutermilch (1910), may be carried out by mixing equal quantities 

 of immune serum and blood containing parasites and leucocytes, 

 and, subsequently, studying microscopically for evidence of phag- 

 ocytosis. In such work, controls have to be used to ascertain how 



