448 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



extract the residue of 1000 c c. of crude extract. The portion that is precipitated when 

 the ethyl acetate solution is placed in the ice-chest overnight is again dissolved in 

 ethyl acetate at 60 C. and the solution again placed in the ice-chest overnight. 

 Finally the portion insoluble in ethyl acetate in the cold is dissolved in water-free 

 ether (sp. gr., 0.717) at room temperature. To the ethereal solution in a glass cyl- 

 inder four volumes of acetone are added, causing a precipitate to be deposited. 

 Precipitation is aided by shaking the mixture for several minutes. Separation is 

 complete in ten minutes. The supernatant fluid is poured off, and the crude precipi- 

 tate of lecithin is redissolved in ether and the precipitation with acetone repeated 

 twice. The precipitate is finally rubbed up with sand and the soluble portion taken 

 up by extraction with absolute ethyl alcohol for twenty-four hours at room temper- 

 ature. The last traces of lecithin may be removed by extracting the residue with an 

 additional small quantity of alcohol. The ordinary commercially "pure" reagents 

 are satisfactory for this method of preparation of lecithin. 



The lecithin of 1000 c.c. of crude extract should be extracted with about 100 c.c. 

 of alcohol. The strength of the solution is estimated by evaporating 10 c.c. at 57 

 C. and weighing. The alcoholic lecithin solution is kept in a stoppered bottle at room 

 temperature in the dark. After allowing it to stand for a week a 0.75 per cent, 

 solution in alcohol is diluted 1 : 7 with normal salt solution, to secure the maximum 

 turbidity, and titrated. Browning and McKenzie use 0.6 c.c. of this emulsion as the 

 antigenic dose. 



7. Aqueous Extract of Pallidum Culture. This antigen is prepared 

 by Noguchi, who uses pure cultures of Treponema pallidum in ascites 

 kidney agar. Preferably several strains should be used in the prepara- 

 tion, which corresponds quite closely to luetin. Cultures grown seven, 

 fourteen, twenty-one, twenty-eight, thirty-five, and forty-two days are 

 chosen and examined, and those that show the best growths in the agar 

 columns are selected. The oil is poured off, the tubes cut just above the 

 kidney, and the column of ascites agar between the piece of kidney and 

 the oil removed with particular care, so as not to include the kidney or 

 the oil. This substance is ground in a mill until the spirochetes show 

 disintegration. The thick emulsion is then diluted with normal salt 

 solution and heated to 60 C. for one-half hour; 0.4 per cent, phenol is 

 added as a preservative, and the emulsion titrated for its anticomple- 

 mentary dose. In conducting complement-fixation reactions with 

 pallidum antigen one-half of the anticomplementary dose is used, and the 

 serum must be inactivated. 



COMPARATIVE ANTIGENIC VALUES OF VARIOUS EXTRACTS 

 For several years past I have been particularly interested in studying, 

 from a practical standpoint, antigenic values of the extracts most com- 

 monly employed in the serum diagnosis of syphilis, and comparing them 

 with suitable alcoholic extracts of syphilitic liver as a standard antigen. 

 For this purpose antigens were carefully chosen after titration, and 

 only those were employed that were safely free from anticomplementary 

 action and whose antigenic dose was known. A large number of serums 

 and cerebrospinal fluids from syphilitic and non-syphilitic persons were 



