138 Immunity 



that the serum contains a maximum antitoxic strength (300 to 1000 

 units per cubic centimeter), the horse is ready to bleed. Some horses 

 can be bled without resistance, but most of them require to be fastened 

 in appropriate stocks. The blood is taken from the jugular vein, 

 which is superficial, of large size, and easily accessible. The skin is 

 carefully shaved* over an area about 9 square inches in extent, thor- 

 oughly disinfected. A small incision is made over the center of the 

 vein, which is made prominent by pressure at the base of the neck, 

 and the point of a small sterile trocar being inserted in the incision 

 through the skin, it is directed obliquely upward into the vein. The 

 blood is allowed to flow through a sterile tube attached to the cannula 

 into sterile bottles prepared to receive it A large horse may furnish 

 7 to 9 liters ; small horses 5 to 7 liters. 



IV. Preparation of the Serum. The blood is stood away in a 

 cool place until the clot retracts after coagulation and the clear serum 

 separates. The serum is then withdrawn under strict aseptic pre- 

 cautions. It is variously prepared for the market. Some manufac- 

 turers bottle it without any added preservative; some add a crystal 

 of thymol; some Pasteurize it; some add carbolic acid; some add 

 trikresol. 



The plain serum would be ideal, but the danger of subsequent con- 

 tamination through careless treatment makes it rather better to have 

 an antiseptic added. Trikresol is probably the most satisfactory of 

 these, though it throws down a precipitate that necessitates the fil- 

 tration of the product, and leaves the serum slightly opalescent. 



V. Determining the Potency of the Serum. The potency of the 

 serum is expressed as so many "immunizing units." Only one method 

 of testing is produced at the present time, though to understand it, 

 it seems wise to mention the original method from which it was derived. 



(A) Behring's Method. Behring's unit was an arbitrary standard 

 chosen in consequence of certain conditions existing at the time it 

 was devised. It is difficult to understand apart from the circum- 

 stances governing its creation, but may be defined as "Ten times the 

 least quantity of antitoxin serum that will protect a standard (300 gram) 

 guinea-pig against ten times the least certainly fatal dose of toxic bouillon." 



The method of determining it is not difficult to those skilled in 

 laboratory technic, and is as follows : 



1. Determine accurately the least certainly fatal dose of a sterile 

 diphtheria toxic bouillon for a standard guinea-pig. 



2. Determine accurately the least quantity of the serum that will 

 protect the guinea-pig against ten times the above determined least 

 fatal dose of toxin. 



3. Express the required dose of antitoxic serum as a fraction of a 

 cubic centimeter and multiply by 10; the result is one unit. 



Example: It is found that 0.01 c.c. of a toxic bouillon kills at least 

 9 out of 10 guinea-pigs, and is therefore the least certainly fatal dose. 

 Guinea-pigs receive ten times this dose of the toxic bouillon plus 

 varying quantities of the serum to be tested, measured by dilution 

 ?ay 2oVo c - c - 2i>W c - c - aoW c-c. The first two live. The fraction sW 

 is now multiplied by 10; ^oW X 10 = ^-Q = 1 unit. So we find 

 that each cubic centimeter of the serum contains 250 units. 



This method would be satisfactory were it not for certain variations 

 in the toxic bouillon by which the strength is worked out. Ehrlich,* 

 in an elaborate investigation of these changes, has clearly proved that 

 an ever-changing toxin cannot be a satisfactory standard, because 

 it does not possess uniform combining affinity for the antitoxin. He 

 shows by a labored scheme that the toxicity of the bouillon is no 



* " Klinisrhes J :hrbuch," 1897. 



