SEROLOGICAL METHODS IN THE STUDY OF PROTOZOA 417 



themselves, causing a false reaction. Thus, in Table i. Tube 17, 

 by exhibiting no hemolysis, shows that the antiserum is not 

 hemolytic by itself. Tubes 18 and 19 are likewise controls of the 

 guinea-pig serum and saline, respectively. 



Standard laboratory equipment, which is now available from 

 laboratory supply houses, saves time and mistakes. Needless to say, 

 it must be meticulously clean. 



The necessary ingredients, besides the test antigen, are obtained 

 as follows: The experimental, specific, inactivated (to destroy 

 the activity of complement) antiserum is obtained from an animal 

 infected or immunized with the experimental organism by bleeding 

 aseptically, collecting the serum and heating for twenty minutes 

 at 56° C. The complement is obtained by bleeding and collecting 

 the serum aseptically from one or several healthy guinea-pigs. The 

 unhemolysed, thoroughly washed red cells are obtained usually 

 by bleeding a sheep aseptically into a bleeding bottle, containing 

 such anticoagulents as heparin or sodium citrate, or glass beads 

 for defibrination ; and thereafter washing the cells three times in 

 an excess of 0.85% saline (i.e., mixing in saline, centrifuging 

 at low speed, decanting the saline, adding more, etc.). The cor- 

 responding hemolytic antiserum is obtained by aseptically bleed- 

 ing rabbits, which have been immunized by injections of sheep 

 cells at various intervals, collecting the serum and preserving 

 with half glycerin; in this state it will sometimes keep for a 

 year or more. (Commercial, glycerinated antiserum of the needed 

 high titre may now be bought.) It is to be noted that aseptic 

 bleeding is an integral part of the procedure, proficiency in which 

 is secured by practice. Blood may be obtained with a sterile hypo- 

 dermic syringe from human beings by bleeding from the median 

 basilic vein, from sheep from the jugular vein and from smaller 

 animals from the heart; or if quantities of blood are needed from 

 animals, by bleeding to death by cutting the jugular and collecting 

 in sterile Petri dishes. (For more specific details, the reader is 

 referred to Kolmer, 1928.) By preliminary titrations, the precise 

 amounts of these ingredients are ascertained. 



C. Protocols.^ Protocols for the preliminary titrations are given 

 in Tables i through 3. All amounts are given in cubic centi- 

 meters, each test tube having a total of two cubic centimeters. 



* The exact protocol used in each serological test varies with almost every 

 laboratory. In the present review, I have selected only one procedure. 



