189^] MICiROSCOPICAL JOURNAL. 55 



The Preparation of Diphtheria Antitoxic Serum. 



By H. K. MULFORD, Ph. G., 

 PHIIvADEIvPHiA, PA. 



The discovery of diphtheria antitoxin was made by 

 Behring as result of his primary and original investiga- 

 tion in connection with Kitasato upon tetanus antitoxin. 



The method of preparation first proposed was the in- 

 jection into suitable animals of cultures of the diphtheria 

 bacilli in which the bacilli had been killed by heat. 

 When the animal could withstand such injection, mani- 

 festing only a slight irritation or oedema at site of injec- 

 tion, or showing but feeble temperature reaction, highly 

 attenuated living cultures were introduced in increasing 

 amounts, a sufficient immunization or resistance being 

 given by the primary injections to prevent fatal termi- 

 nation. The injection of living cultures, however, is 

 greatly to be discouraged, since such injection and those 

 of attenuated cultures containing dead bacilli are accom- 

 panied by great destruction of cellular tissue of the ani- 

 mal which is to furnish the antitoxin, its physical strength 

 being lessened by such destructive processes. 



The best method is as follows: As virulent a culture as 

 possible of the bacillus diphtheric^, is obtained. It is 

 grown upon Loeffler's solidified blood semm mixture and 

 placed in au incubator at a temperature of 45 degrees C. 



After a period of 24 hours the cultures are developed. 

 From this a single culture or colony of the bacilli is trans- 

 ferred into small flasks of a 2 per cent peptone bouillon 

 rendered decisively alkaline to litmus. These small flasks 

 are placed in an incubator which is kept at a constant 

 temperature of about 37 degrees C. for 24 to 48 hours^ 

 and afterward the contents are transferred with peptone 

 bouillon into rounded flat flasks with a long neck (so that 

 sterilized cotton may be pushed well into the tubulature) 

 of a capacity of 500 ccm. These large flasks are placed 



