20 
diphtheria antitoxic serum, say 0.01 c. c., was injected subcutaneously 
into a guinea pig. Twelve hours later. 0.5 c. c. of a fresh virulent 
culture of the diphtheria bacillus was inoculated into the guinea pig, 
weighing, say, 300 grams. If the guinea pig lived. 0.01 c. c. of 
the serum was considered sufficiently active to protect 300 grams of 
guinea pig, and 1 c. c. of the serum would consequently protect 30,000 
grams of guinea pig. and the strength of the serum was expressed as 
1: 30,000. The strength of a serum according to this method was 
expressed in such figures as 1: 100,000 or 1: 500,000. 
Control animals were inoculated to insure the virulence of the cul- 
ture used. The culture used was considered sufficiently virulent if 
the control guinea pig died within thirty hours. 
A serum with an immunizing strength of 1: 100.000 meant that 1 c. c. 
would protect 100.000 grams of guinea pig; and a serum whose 
strength was 1: 500,000 meant that 1 c. c. of the serum would protect 
500,000 grams of guinea pig against a certainly lethal dose of diph- 
theria culture or later the toxine when injected in accordance with 
the above procedure. 
Madsen^ showed as a result of comparative tests that the German 
method of expressing the strength of antitoxic sera in antitoxin units 
was quicker, cheaper, easier, and more accurate than the French method 
devised by Roux. He showed that with the French method it is often 
impossible to determine diflerences as great as 1: 100, oOO and 1: 200.0U0. 
This method was finalh' abandoned by Roux himself in favor of the 
German procedure. 
In the earlier methods of testing the strength of diphtheria anti- 
toxin, according to both the methods of Behring and Roux, the two 
substances were always inoculated separate! v into a guinea pig. usually 
in different places. Sometimes the toxine and antitoxin were inocu- 
lated at different times in order to make a distinction l)etween the 
immunizing power of a serum and its curative power. 
The method devised by Fhrlich in collaboration with Kossel and 
Wassermann in 1891'^ was based upon an entirely new principle. The 
scientific researches of Behring and Kitasato hadpreviouslv established 
the fact that tetanus poison and its antitoxin neutralize each other in 
a test tube outside of the body. Ehrlich, Kossel, and IVassermann satis- 
fied themselves that this neutralization between the diphtheria poison 
and its antibody’ takes place at once when the two substances are mixed 
in a test tube, and they believed at that time (1891) that it took place 
in accordance with the law of simple proportions. 
« Madsen, Thorvald: Ueber messung der starke des antidiphtherischen serums. 
Zeitschr. f. hyg., Leipzig, y. 24, pp. 425—442. 
& Ueber Gewinnung und Verwendung des Diphtherieheilserums. Deut. med, 
Woch., 1894, no. 16. 
