284 MANUAL OF BACTERIOLOGY. 



Diphtheria antitoxin.* It is necessary first to obtain the toxin produced 

 by diphtheria bacilli in a concentrated form. For this purpose virulent diph- 

 theria bacilli are cultivated in alkaUne, sugar-free bouillon, in flasks plugged 

 with cotton, exposing a large surface to the air. The bouillon is prepared by 

 leaching out 500 grams of lean chopped beef in 1000 c.c. of water overnight, 

 straining the water off through cheese-cloth, and finally inoculating it with a 

 culture of the colon bacillus. The last-named procedure rids the broth of all 

 muscle-sugar. After adding salt and peptone to the sugar-free beef-juice it is 

 put in wide, flat flasks — Fernbach flasks — and sterilized in the ordinary 

 manner. The cultures are grown in the incubator. After five to ten days 

 they are ready, and are filtered through porcelain.f The filtrate contains the 

 toxin. The toxin is injected into the animal from which the antitoxin is to 

 be obtained in small doses. The dose depends on the strength of the toxin. 

 The animal usually employed is the horse, which should be healthy; the 

 presence of tuberculosis and glanders should have been excluded by testing 

 with tuberculin and mallein; the possible presence of tetanus should also be 

 considered (see page 271). 



The injection is repeated at intervals of about one week, using larger and 

 larger doses, until the animal is able to tolerate a very large dose indeed — , 

 as much as 300 c.c, or even more. If the treatment is successful, the general 

 condition of the animal should not suffer. The injections last over a long 

 period — ^usually about two or three months. The general condition of the 

 animal remaining good, the toleration of these large doses of toxin is presumed 

 to indicate the existence of a concentrated antitoxic substance in the blood. 

 SmaU quantities of blood may be withdrawn from time to time, and the serum 

 tested for its antitoxic strength. When a satisfactory serum has been attained, 

 the animal may be bled and the serum saved for therapeutic purposes. Through 

 an incision in the skin a trocar is inserted into the jugular vein. The blood 

 is drawn into sterilized flasks with every precaution to insure sterility. The 

 blood is allowed to coagulate and is placed for a time in the ice-chest. The 

 serum is then withdrawn with sterilized pipettes. Small amounts of chemical 

 germicides, as carbolic acid or chloroform, are sometimes added to assist in 

 preserving it. This serum is the so-called antitoxin used in medical practice. 



Since antitoxin is not obtained as a pure chemical substance, and conse- 

 quently cannot be weighed and measured as other therapeutic preparations, 

 an arbitrary standard to express the potency of the serum, called an immunity 

 unit, has been devised by Behring and modified by Ehrlich. Formerly this 



* See articles by Park, A. Williams, Atkinson and T. Smith. Journal of 

 Experimental Medicine. Vol. I., p. 164; Vol. III., p. 513; Vol. IV., pp. 373 

 and 649. Journal Medical Research, Vol. IX., p. 173. 



t W. H. Park adds 10 per cent, of a s per cent, solution of carbolic acid to 

 kill the bacilH, and filters through paper on the following day; after adding 

 carbolic acid the Berkefeld filter may be used with advantage instead of filter- 

 paper. 



