248 MEDICAL BACTERIOLOGY 



in that it tends to reduce to the minimum the occurrence of hemolytic and anti- 

 complementary properties of the antigen. It is prepared by drying the ground- 

 up tissue, in vacua, over sulphuric acid, afterward placing it in acetone, 20 cc. 

 for each gram of tissue. This is shaken for 5 minutes and incubated at room 

 temperature, in the dark, for 10 days. It is then filtered through paper and the 

 filtrate evaporated at 37C. To each gram of the residue 100 cc. of methyl 

 alcohol is added and it is stored at room temperature, protected from light. 

 Immediately before use it is mixed with 10 times its volume of normal salt 

 solution. 



Any of the above antigens can be fortified by the addition of 0.25 Gm. of 

 cholesterin to 100 cc.; this procedure seems undesirable as it is adding another 

 reagent, unnecessarily, to a test already too complex. 



Schurmann, Sachs, Rondin and others have employed various chemicals 

 and colloids as antigen, but as yet no chemical antigen has been found to equal 

 the tissue extracts. Varney and Baeslack have recommended an acetone extract 

 of gummata removed from rabbit testicles following the successful inoculation of 

 the rabbit testicles with treponema pallidum. 



STANDARDIZATION OF ANTIGEN 



For the standardization of antigen one requires at least 2 cc. of a known 

 positive, syphilitic serum and a like amount of negative, non-syphilitic serum. 

 Two rows of tubes are placed in a test-tube rack. 



A i : 10 dilution of the alcoholic extract to be standardized is made by mix- 

 ing 2 cc. of it with 1 8 cc. of normal salt solution. 



This is put in the tubes as follows: 



o.i cc. in first tube in top row and first tube in bottom row. 



0.2 cc. in second tube in top row and second tube in bottom row. 



0.3 cc. in third tube in top row and third tube in bottom row. 



0.4 cc. in fourth tube in top row and fourth tube in bottom row. 



0.5 cc. in fifth tube in top row and fifth tube in bottom row. 



0.6 cc. in sixth tube in top row and sixth tube in bottom row. 



0.7 cc. in seventh tube in top row and seventh tube in bottom row. 



0.8 cc. in eighth tube in top row and eighth tube in bottom row. 



0.9 cc. in ninth tube in top row and ninth tube in bottom row. 



1.0 cc. in tenth tube in top row and tenth tube in bottom row. 



1.1 cc. in eleventh tube in top row and eleventh tube in bottom row. 



1.2 cc. in twelfth tube in top row and twelfth tube in bottom row. 



Next put o.i cc. of inactivated syphilitic serum in each tube at the bottom 

 row; put o.i cc. of inactivated non-syphilitic serum in each tube of the top row. 



Put one unit of complement in every tube (both rows). Shake each tube to 

 thoroughly mix its contents and place in incubator. 



When the tubes have been incubated exactly i hour, add one unit of ambo- 

 ceptor (rabbit serum) and i cc. of red cells to every tube, shake to mix contents 

 and again incubate for i hour. 



