648 ACTIVE IMMUNIZATION 



fleas of these rodents. Aside from sanitary measures, plague vaccine, 

 especially that of Haffkine, has now been used extensively, with encour- 

 aging results. 



Preparation of Plague Vaccine. The Haffkine prophylactic is pre- 

 pared by growing pure cultures of Bacillus pestis in flasks of neutral 

 bouillon to which a few drops of sterile olive oil or butter-fat have been 

 added, to serve as floats from which the surface growth of the bacilli 

 can take place. The flasks are cultivated at from 25 to 30 C. for five 

 to six weeks, and are shaken every two or three days, by which the hang- 

 ing, stalactite-like colonies are thrown down, so that a new crop of the 

 bacilli can develop in contact with the ah-. 



After growing for six weeks the purity of the culture in each flask is 

 tested by subcultures on agar and by direct smears. The masses of 

 bacilli are broken up by shaking, and the material is sterilized by heating 

 at 65 C. for from one to three hours. Phenol is added to the point of 

 0.5 per cent., and the fluid is tested for sterility by culture. If it is 

 found sterile, it is finally poured into small vials of from 10 to 30 c.c. 

 capacity. 



Kolle prepares a vaccine by cultivating the bacillus for two days in 

 flasks of agar measuring 10 by 9.5 cm. Each surface of agar equals 

 about 15 ordinary agar slant cultures, and an agar slant holds about 

 15 loopsful of culture (4 mm. loop). A loop of this size holds about 2 

 mg. of organisms, and, accordingly, a flask of agar contains about 225 

 loopsful of culture, or 225 doses of 2 mg. each. Kolle prepares the 

 vaccine in amounts of 0.5 c.c. per dose (2 mg. of bacilli), and the growths 

 in each flask are removed with 112.5 c.c. of sterile normal salt solution. 

 The emulsion is shaken to break up clumps, heated for one hour at 70 

 C., and tested for sterility. It is then preserved with phenol or tricresol 

 and placed in ampules containing 0.5 c.c. each. 



The German Plague Commission strongly recommended the use of 

 twenty-four- to forty-eight-hour-old agar cultures instead of the old 

 bouillon cultures employed in the preparation of Haffkine's vaccine. 



Kolle and Strong have also employed living cultures of greatly re- 

 duced virulence for the immunization of man. 



Lustig and Galeotti prepare a vaccine of the toxic precipitate pro- 

 duced by dissolving the bacilli in a 1 per cent, solution of caustic soda 

 and neutralizing with 1 per cent, of acetic acid. This precipitate is 

 dried in vacuo and redissolved in a weak solution of sodium bicarbonate, 

 the dose for adults being 0.0133 gm. of solid substance. 



Terni and Bandi inoculate rabbits or guinea-pigs intraperitoneally 



