482 MICROBIOLOGY OF SPECIAL INDUSTRIES. 



a period of about six weeks, the horse is allowed a rest of about two weeks, 

 during which time all the toxin which has been injected is absorbed. 

 The animal is then bled from the jugular vein, under aseptic and anti- 

 septic conditions. As much blood is secured as the horse can conve- 

 niently yield. The blood may be drawn through a sterile canula and 

 rubber tube into tall, sterile glass cylinders. After the blood has clotted 

 the serum separates and at the end of twenty-four to forty-eight hours, 

 the clear, amber-colored fluid is poured from the cylinders into large, 

 sterile glass containers, a preservative is added and the material is trans- 

 ferred to the laboratory. The serum is then filtered through a Berkefeld 

 filter. 



Each lot of antidiphtheritic serum is submitted to rigid tests relative 

 to potency, safety and microbial contamination. In determining the 

 potency, accurate data are obtained. Varying amounts of the serum under 

 test are mixed with the L+ dose of diphtheria toxin and injected into a 

 series of guinea pigs, each weighing 250 g. *The L+ dose of toxin is 

 the least amount of toxin, which when mixed with one unit of standard 

 antitoxin, and injected into a guinea-pig of 250 g. weight, is sufficient to 

 kill the animal in four days. From the results of this test it is possible to 

 determine the smallest amount of the antitoxin which will protect a 

 guinea pig of 250 g. weight, when the animal has received simultaneously 

 the L+ dose of toxin. This minimum amount of antitoxin represents 

 one unit. Thus, if 1/500 c.c. of the given antitoxin represents the smallest 

 amount which is capable of neutralizing the L + dose of toxin, then the 

 antitoxin would possess a potency of 500 units per cubic centimeter. 



In order that the antitoxin may be tested for safety, each of several 

 guinea pigs are subcutaneously injected with about 2 c.c. of the serum. 

 These animals are not released until the observer is satisfied that the serum 

 contains no injurious properties. For the purpose of detection of micro- 

 bial contamination, relatively large amounts -of the antitoxin are placed 

 in culture media and inoculated under both aerobic and anaerobic con- 

 ditions. If, after 72 hours' incubation, growth occurs in the culture 

 media, the given lot of serum is refiltered and reexamined for microbial 

 contamination. 



- 



Diphtheria antitoxin is usually distributed in glass syringe containers 

 ready for immediate use. After the product has been tested relative to 

 potency, safety and microbial contamination, it is put up in sterile glass 



* See Bulletin No. 21, Hygienic Laboratory, Washington, D. C. 



