FIXATION OF COMPLEMENT 219 



in broth for forty-eight hours, or upon a solid medium and emul- 

 sifying the growth in saline. The culture or emulsion is shaken 

 for an hour to break up clumps and then heated to 60-65 C. for 

 an hour. It is preserved by the addition of 1 per cent, of glycerin 

 and 0-5 per cent, of phenol. 



(&) Cultures are grown on a solid medium, the growth is washed 

 off with a small quantity of saline and the emulsion is centri- 

 fuged so as thoroughly to deposit the organisms. The sediment 

 is collected and dried over sulphuric acid and weighed, after 

 which the dry mass is ground up in an agate mortar with a 

 weighed amount of solid sodium chloride. After thorough 

 grinding sufficient distilled water is added to yield about 0-85 

 per cent, saline, and so that the solution will contain about 

 0-05 gim. of dry bacterial material per c.c. of solution ; this can be 

 arrived at from the weight of dry bacterial material used and an 

 appropriate amount of salt added. Thus, if the bacterial mass 

 weighed 0-5 grm., it should be ground up with about 0-08 grm. 

 of salt and 10 c.c. of distilled water added. The emulsion is 

 then shaken for twenty-four hours, filtered or centrifuged, and 

 the fluid is preserved with 0-5 per cent, of phenol and constitutes 

 the antigen. 



2. The test. -The subsequent procedure may be carried out 

 exactly in the same manner as the Wassermann reaction (see 

 " Syphilis "), fresh guinea-pig serum being used as complement, 

 and will comprise (1) standardisation of the antigen, (2) titration 

 of the hsemolytic serum, (3) titration of the complement, and, if 

 a bacterial species is to be determined, (4) titration of the anti- 

 serum against an antigen prepared from its homologous micro- 

 organism. If a patient's serum is being tested with a known 

 bacterial antigen, after the preliminary titrations (1), (2) and (3) 

 have been completed, two series of tubes are put up, the one con- 

 taining patient's serum, the other normal serum, in descending dilu- 

 tions, e.g. undiluted, and diluted 1 in 2, 1 in 4, 1 in 8, 1 in 16, . . . 

 If specific fixative substances be present in the patient's serum, 

 fixation of complement will take place in dilutions much below 

 that in which it occurs with the normal serum. 



In the titrations and test proper, similar total volumes and 

 volumes of the different reagents should be employed. Any 

 convenient unit volume may be used, e.g. 0-5 c.c. or 0-1 c.c., or 

 less. Thus, we might adopt (as for the Wassermann reaction) 



