1896 THE MIOROSCOPE. l83 



greatly to be discouraged, since such in jections, and those 

 of attenuated cultures contaiifing dead bacilli, and accom- 

 panied by great destruction of the cellular tissues of the 

 animal wliich is to furnish the antitoxin, its physical 

 strength being greatly lessened by such destructive pro- 

 cesses. 



The method is as follows: As virulent a culture of 

 the bacillus diphtheriae is obtained as possible, it ia 

 grown upon Loeffler's solidified blood mixture consisting 



of :— 



Blood serum 3 parts 



I per cent glucose boullion i part 



and placed in an incubator at a temperature of 45 degrees 

 centigrade. 



After a period of 24 hours the cultures are developed. 

 From this a single colony of the bacilli is transferred in- 

 to small flasks of a two per cent peptone boullion, rend- 

 ered decidedly alkaline to litmus. These small flasks 

 are placed in an incubator, which is kept in a constant 

 temperature of 37 degrees C. for 24 to 48 hours, and 

 afterwards the contents are transferred with a pipette 

 into rounded flat flasks, of a capacity of 500 cc. These 

 large flasks are placed in an incubator, and kept at a con- 

 stant temperature of 37 degrees C. until the bacilli have 

 become very numerous, and have secreted enormous 

 amount of active and powerful toxin in the boullion. 



When this has taken place a microscopic examination 

 is made, to see that no foreign bacilli are present and the 

 diphtheria toxin contaminated. If uncomtaminated, 1 

 per cent trikresol is added to prevent contamination, and 

 to destroy the bacillus diptheria. The boullion, or as we 

 now term it, diptheria toxin, is filtered through a modi- 

 fied Chamberland filter, to separate it from the dead 

 bodies of the diptheria bacilli. No bacilli are therefore 

 injected into the animals to be immunized, and they are 

 not given "diptheria" but the toxin secreted by the bacilli. 



