520 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



of tuberculosis in cattle and not practical for general diagnostic purposes. 

 Lucke 1 found that the wax of tubercle bacilli possessed no antigenic 

 properties; Wood, Bushnell, and Maddux 2 have observed a large per- 

 centage of apparently specific reactions with the partial antigens of 

 Deyke and Much. 3 



Preparation and Standardization of Antigen. It is apparent, there- 

 fore, that the question of proper antigen is of paramount importance in 

 complement fixation in tuberculosis. It would appear that Besredka's 

 and Craig's antigens and suspensions of living tubercle bacilli are of 

 definite value. The main objections to the bacillary emulsion are the 

 small difference between the antigenic and anticomplementary units, 

 the turbidity, and fairly high percentage of non-specific reactions. Eich- 

 horn and Blumberg have given the following directions for the prep- 

 aration of a modified Besredka antigen: 



Twenty c.c. each of the white and the yolk of an egg were thoroughly 

 beaten in an automatic egg-beater, and to the whipped material a solu- 

 tion of Liebig's meat extract (3 : 1000) in distilled water is gradually 

 added while the mixture was continuously beaten. A sufficient meat- 

 extract infusion is used to make up the whole to 1000 c.c. The 

 emulsion is strained through cotton and heated to the boiling-point, 

 then strained again, and after the addition of 0.5 per cent, of sodium 

 chlorid is carefully neutralized, heated again and strained, and neutral- 

 ized if necessary. To the neutral medium sufficient normal sodium 

 hydroxid - solution is added to make the medium of 0.2 alkalinity. 

 This medium, to which neither peptone nor glycerin was added, was 

 then autoclaved at 115 C. for twenty minutes, and after cooling is 

 kept at 37 C. for forty-eight hours for observation as to sterility. 



Whatever antigen is employed it must be carefully titrated for the 

 anticomplementary unit each time before the main tests are conducted. 

 I conduct these titrations in the same manner as described under the 

 gonococcus complement-fixation test and use one-third the anticomple- 

 mentary unit as the dose for the main tests. 



The Test. These may be conducted with a 0.2 c.c. dose of serum 

 or with three graded doses, as 0.05, 0.1, and 0.2 c.c., as described in the 

 gonococcus complement-fixation test. I have generally employed the 

 latter with a primary incubation of six hours or over night in the refrig- 

 erator at 8 C. or one hour in the water-bath at 38 C. 



1 Jour. Immunol., 1916, 1, 457. 2 Jour. Immunol., 1917, 2, 301. 



3 Med. Klinik, 1908, 4, 1540; Munch, med. Wchnschr., 1909, 56, 1895; ibid., 

 1909, 56, 1825; Beitr. z. klin. Tuberk., 1910, 15, 277; ibid., 1911, 20, 343. 



