444 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



They are less stable than alcoholic extracts, and are likely to become 

 highly anticomplementary and lose their power of reacting with syphil- 

 itic serums. Citron is convinced that these changes are brought about 

 by careless handling of the extract, or its exposure to the light. He 

 recommends that the extract be kept constantly in the ice-chest, and 

 that it be kept out only long enough to remove sufficient for the day's 

 work. 



Preparation. The fresh liver taken from a syphilitic fetus, and showing the 

 presence of spirochetes by dark-ground illumination, is weighed and cut into fine 

 pieces. Four times its weight of 0.5 per cent, phenol in physiologic salt solution is 

 added. The mixture is placed in a brown bottle and shaken mechanically at room 

 temperature for twenty-four hours. It is then filtered through gauze, to remove 

 the larger particles, and stored in a brown bottle in an ice-chest. After several days 

 of sedimentation the fluid assumes a yellowish-brown opalescence and is ready for the 

 preliminary titration to determine its anticomplementary and hemoly tic doses. The 

 sediment should not be disturbed, but the supernatant fluid should be carefully re- 

 moved by means of a pipet. According to Citron, extracts that must be used in 

 quantities of less than 0.1 c.c. are, as a general rule, unsatisfactory. Only such extracts 

 should be used as in doses of 0.4 c.c. will not interfere with hemolysis. The method 

 of making these titrations is given on page 452. 



2. Alcoholic Extracts of Syphilitic Livers. These antigens are 

 extensively used. They are not true biologic antigens, for they do not 

 give rise to antibodies (Schatilof and Isabolinsky; Seligman and Pinkus); 

 they are, however, usually better antigens than similar extracts of 

 normal liver, a fact that may be explained, in part at least, by chem- 

 ical changes, namely, fatty changes, autolysis, soaps (Beueker), excess 

 of cholesterin (Piglini), etc., which, while not specifically syphilitic in 

 nature, are often produced to a striking degree in congenital syphilis. 



Preparation. Fetal liver known to contain numerous spirochetes is used in 

 preparing this extract. Fresh organs may be examined at once by dark-field illu- 

 mination, or if this is impossible and the fetus shows signs of syphilis, the liver may be 

 cut into large pieces and preserved in 70 per cent, alcohol. After a few days a section 

 is removed and stained by the Leyaditi method for spirochetes. If these microorgan- 

 isms are numerous, the liver is suitable for preparing the antigen; otherwise it should 

 be discarded. Very fatty livers are to be avoided, and those of still-born fetuses are 

 to be preferred. 



Ten grams of liver are minced, ground with quartz sand, and treated with 100 

 c.c. of absolute ethyl alcohol. The mixture is shaken mechanically with glass beads 

 for twenty-four hours, and extracted in the incubator for ten days. The containing 

 flask or bottle should be well stoppered to prevent undue evaporation, and should be 

 shaken up at least once a day. The extract is then filtered through fat-free paper or 

 paper washed with ether and alcohol to remove the hemolytic substances that may 

 be present. The filtrate is measured, and the loss by evaporation is made up by 

 the addition of more alcohol. If a shaking apparatus is not at hand, extractions may 

 be left in the incubator a few days longer. After standing a few days a sediment 

 forms, which should not be removed or disturbed. 



3. Alcoholic Extracts of Normal Organs. These are used extensively, 

 at present, and apparently yield results equal to those obtained with 

 extracts of luetic liver. It is certainly true that a good extract of a normal 

 organ is superior to a poor one prepared from luetic liver. Many, with 



