GENERAL TECHNIC 447 



to stand for a few hours in a cool place until cleared. The clear ethereal portion is 

 then carefully decanted off into another clean evaporating dish, and then allowed to 

 become concentrated by evaporating the ether off. The concentrated ethereal 

 solution is now mixed with about 10 volumes of pure acetone. A light yellow precipi- 

 tate forms, which is allowed to settle, and the supernatant fluid is decanted off. Dis- 

 solve each 0.3 gm. of this substance in 1 c.c. of ether and add 9 c.c. of pure methyl 

 alcohol. As a rule, the greater part of the substance goes into solution. This alco- 

 holic solution remains unaltered for a long time, and is kept as a stock solution from 

 which the emulsion for immediate use may be prepared at any time by mixing 1 c.c. 

 with 19 c.c. of saline solution. This solution is then titrated for antigenic, anticom- 

 plementary, and hemolytic action. 



According to Noguchi, if the extract is anticomplementary or hemo- 

 lytic in doses of 0.4 c.c. of a 1 : 10 dilution, it is unsuitable. If it produces 

 complete inhibition of hemolysis with 0.1 c.c. of syphilitic serum in doses 

 of 0.02 c.c. or less of the same dilution ( = 0.2 c.c. of a 1:100 dilution), it 

 is suitable. In making the fixation test, 0.1 c.c. of a 1 :10 emulsion is to 

 be used, thus employing five times the minimal antigenic dose which 

 does not cause non-specific fixation and is not unduly sensitive. 



6. Lecithin and Cholesterin. Browning, Cruickshank, and McKenzie 

 advocate the use of a mixture of ox-liver lecithin and cholesterin as 

 antigen in testing for the syphilitic reaction. They find that the amount 

 of complement fixed by lecithin alone with syphilitic serums is very 

 much increased by the addition of cholesterin; that cholesterin does not 

 increase the anticomplementary action of the antigen; that the binding 

 power or antigenic value of a mixture of lecithin and cholesterin is equal 

 in most cases to crude alcoholic extracts, but is less anticomplementary. 

 These observers use this mixture in their modified method, which con- 

 sists in the accurate estimation of the amount of complement fixed by 

 extract and syphilitic serum. It would seem that this extract should, 

 in the ordinary methods, be controlled with less sensitive antigens, and 

 I have found that ordinary cholesterinized alcoholic extracts of human 

 heart are equally efficient and less difficult to prepare in performing a 

 quantitative Wassermann reaction after the method just outlined. 



Preparation. Minced ox-liver, obtained within three or four hours after death, 

 is digested with four parts of 95 per cent, alcohol for three or four days at room 

 temperature, during which time the mixture is stirred up at least once a" day. The 

 filtrate is evaporated in an open flat porcelain dish on the water-bath at 60 C. for 

 four or five hours until a syrupy mass remains. This is rubbed up with washed and 

 dried quartz sand until a firm mass results. The mixture of dried extract and sand 

 (about 50 grams of sand to the residue of 1000 c.c. of extract) is placed in a spheric 

 flask, closed with a perforated rubber stopper through which runs a short piece of 

 quill tubing drawn out to a capillary point at the end. This tube serves to prevent 

 the vapor in the flask from forcing out the stopper. Ethyl acetate is placed in the 

 flask, which is then stoppered and immersed up to its neck in water at 60 C. The 

 flask is shaken repeatedly, and after ten minutes the ethyl acetate is poured off into 

 a hot-water filter, the funnel jacket being kept at 60 C., and the solution filtered 

 through fat-free filter-paper. Another portion of ethyl-acetate is added to the sand, 

 and the extraction repeated. After a third treatment practically all the soluble matter 

 will have been extracted. In all, about 170 c.c. of ethyl acetate should be used to 



