PREPARATION OF BACTERIAL ANTIGENS 499 



same microorganism have particular and more or less individual patho- 

 genic and sometimes biologic characteristics, and it is reasonable to 

 assume that the antibody will likewise show individual properties and a 

 special affinity for its particular antigen. When, therefore, one antigen 

 is being used in complement-fixation work, the results are more likely 

 to be satisfactory if a large number of different strains are included in 

 the antigen, with the hope that at least one of them will show a par- 

 ticular affinity for the antibody in the patient's serum. 



Bacterial antigens may be prepared in various ways. 



First Method. Cultures are grown in a suitable fluid medium, such 

 as plain bouillon, for forty-eight hours, or upon a solid medium, and 

 washed off with a suitable quantity of normal saline solution. The 

 culture or emulsion is shaken for an hour or so to break up the clumps, 

 and then heated to 60 C. for an hour. It is preserved by the addition 

 of 1 per cent of glycerin and 0.5 per cent, of phenol. 



This constitutes the simplest bacterial antigen. It is composed of 

 both bacterial cells and the products of bacterial activity, and frequently 

 yields uniform and satisfactory results. 



Second Method. Cultures are grown on a suitable solid medium for 

 from twenty-four to forty-eight hours. Growths are removed by adding 

 sufficient distilled water or normal saline solution to yield a milky sus- 

 pension. The emulsion is heated to 56 C. in a water-bath for one hour, 

 followed by one hour at 80 C., and shaken mechanically with glass 

 beads for twenty-four hours, to facilitate disintegration. It is then 

 filtered through paper pulp and a sterile Berkefeld filter with a neutral 

 reaction, or thoroughly centrifugalized; the filtrate is then heated at 

 56 C. for one-half hour on each of three successive days to sterilize, and 

 preserved with 0.5 per cent, phenol and used as antigen after being di- 

 luted and made isotonic with 1 part of 9 per cent, salt solution to 9 

 parts of the antigen. 



This antigen is composed essentially of endotoxic substances, 

 and is the one usually employed in the preparation of gonococcus 

 antigen. 



Third Method. Cultures are grown on a solid medium, washed off 

 with normal saline solution, and the emulsion centrifuged thoroughly. 

 The sediment is dried over sulphuric acid or calcium chlorid, and the 

 dried material thoroughly ground with crystals of sodium chlorid. Suf- 

 ficient distilled water is then added to render the solution isotonic, and 

 so that it will contain about 0.05 gram of dried material in each cubic 



