COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS 479 



reaction to phenolphthalein, and to which a few drops of sterile hydrocele 

 fluid may be added. After culturing for from twenty-four to forty- 

 eight hours, the growths are washed off with distilled water, and the 

 emulsion is heated in a water-bath for two hours at 56 C. It is then 

 centrifugalized and passed through a Berkefeld filter. A small amount 

 of preservative, as, e. g., 0.1 c.c. of a 1 : 100 dilution of phenol to each 

 cubic centimeter of antigen, may be added. The antigen is then well 

 preserved in small amounts in ampules that are sealed and heated to 

 56 C. for half an hour on, three successive days. Just before being 

 used the antigen is made isotonic by adding 1 part of a 10 per cent, salt 

 solution to 9 parts of antigen. I preserve the antigen in ampules con- 

 taining 1 c.c., and after removing the antigen from the ampule to a large 

 test-tube, add 1 c.c. of 10 per cent, salt solution, and dilute the whole 

 1 : 10 with the addition of 8 c.c. of normal salt solution, after which the 

 anticomplementary titration is made. 



In this method of preparing antigen the endotoxins constitute the 

 main antigenic principle. Kolmer and Brown, after an experimental 

 study of the various antigens, found that a simple suspension of gono- 

 cocci in salt solution yielded slightly better results. The various 

 strains are grown for from forty-eight to seventy-two hours, and are 

 then washed off with sterile saline solution, observing particular care 

 not to include portions of the culture-medium. The suspension is 

 then shaken to break up clumps, and heated to 56 C. for an hour. A 

 small amount of preservative is now added, and the antigen stored in 

 1 c.c. ampules. Before using it is diluted 1 : 10 or 1 : 20, and titrated 

 for the anticomplementary dose. 



Alcoholic extracts of gonococci have very little practical value, as 

 alcohol is not satisfactory for extracting the antigenic principles of 

 bacteria. 



The anticomplementary dose of the antigen should be determined, 

 and one-half or one-quarter this amount should be used in conducting 

 the main test. An antigenic titration may also be conducted with an 

 antigonococcus serum, to determine the antigenic value of the antigen, 

 but in practice it is sufficient to use one-half the anticomplementary 

 dose. This titration should be conducted and the antigen standardized 

 before the main tests are adjusted. 



In the following table the results of an anticomplementary titration 

 of a gonococcus antigen are given, the approximate dose having been 

 ascertained in previous titrations (Fig. 117). 



