WASSERMANN AND OTHER COMPLEMENT FIXATION TESTS 227 



of six, all other tubes are plugged with cotton, flasks are plugged with cotton, 

 and flasks and bottles are also capped. Normal salt and sodium citrate solu- 

 tions are autoclaved at 15 pounds pressure for 20 minutes or heated 20 minutes 

 each day for 3 consecutive days in a steam sterilizer. Needles are kept in 50 

 per cent, alcohol. They are flamed just before use. 



The form of test-tube rack is a matter of personal selection; the author pre- 

 fers a heavy, rigid, metal rack built to carry three tiers of 12 tubes each, in align- 

 ment (Fig. i). 



INCUBATOR 



Biochemical reactions, such as complement fixation, proceed best at tem- 

 peratures that approximate normal body temperature -37C.- consequently 

 an incubator that will constantly maintain this temperature is necessary. 



NORMAL SALT AND SODIUM CITRATE SOLUTIONS 



When red cells (corpuscles) of the blood are removed from their natural 

 vehicle (blood serum) and placed in another fluid, their normal properties are 

 retained, altered or destroyed according as to whether the fluid in which they 

 are placed is isotonic, hypertonic or hypotonic. 



If red cells are placed in water they immediately burst, liberate their hemo- 

 globin and dissolve; if they are placed in normal salt solution they can remain 

 viable and retain all their normal properties for days. Red cells, separated 

 from their native serum, are required in the Wassermann and other Comple- 

 ment Fixation tests; hence normal salt solution is the sole vehicle and dilutent 

 used in this work. 



Normal salt solution is made by dissolving 8.5 Gm. of sodium chloride in 

 1000 cc. of distilled water. To preclude undesirable changes normal salt solu- 

 tion should be sterilized as soon as it is made and stored in sterile containers 

 having a capacity of not more than 200 cc. 



If blood is withdrawn from an animal and placed in a container it will clot 

 in a few minutes; the corpuscles are embedded in the clot and cannot be extri- 

 cated. Since red blood cells are essential in making complement fixation tests, 

 it is necessary to collect the blood in a manner that will prevent coagulation. 

 Sodium citrate precipitates the calcium salts of the blood and so prevents its 

 coagulation; hence sodium citrate solution is used in collecting blood from which 

 to obtain red cells for complement fixation tests. 



Sodium citrate solution is made by dissolving 10 Gm. of sodium citrate in 

 1000 cc. of normal salt solution. Sodium citrate solution should be sterilized 

 when made and stored in sterile containers. 



BLOOD CELLS AND SENSITIZATION OF RABBITS 



After procuring the necessary apparatus and preparing normal salt and 

 sodium citrate solutions, the next step in preparing for complement fixation 

 tests is the sensitization of a rabbit against red blood cells. The rabbit must 

 be sensitized to the. same kind of red blood cells that will subsequently be used 



