46 THE PRINCIPLES OF IMMUNOLOGY 



more than five days too little strength. It can be seen that the selection of 

 the weight of the pigs and the length of time are arbitrary but universal 

 standards. A strong toxin might give results as follows: 



Guinea-pig Toxin dose Result 



1 0.0036 c.c. Lives 



2 0.0038 c.c. Dead 6 days 



3 0.0040 c.c. Dead 4 days 8 hours 



4 0.0042 c.c. Dead 3 days 20 hours 



5 0.0044 c.c. Dead 2 days 



Guinea-pig No. 3 died at the right time interval and 0.004 is the M.L.D. 

 of this toxin. Experiments with a preliminary series using more widely vary- 

 ing doses of toxin would be necessary before the final experiment could 

 be set up. 



The Lo dose (Limes null) is that amount of toxin which is so thoroughly satu- 

 rated with one unit of antitoxin that neither local nor general symptoms 

 appear following the injection of the mixture. An experiment follows: 



Standard 

 Guinea-pig antitoxin Toxin Result 



1 i unit 0.36 c.c. No reaction 



2 i unit 0.38 c.c. No reaction 



3 i unit 0.40 c.c. Barely visible congestion 



4 i unit 0.42 c.c. Moderate inflammation 



5 i unit 0.44 c.c. Distinct inflammation 



In this experiment the dose of toxin, 0.40 c.c., given pig No. 3, is the L 

 dose. The note as to reaction refers to the shaven site of injection. 



The L + dose (Limes death) indicates the smallest amount of toxin which 

 after mixture with one unit of antitoxin will produce death in four-five days. 

 The plus sign is the mark used in English texts to correspond to the cross 

 mark used in German literature to signify death. An experiment follows : 



Standard 

 Guinea-pig antitoxin Toxin Result 



unit 0.44 c.c. Lives 



unit 0.46 c.c. Dead 6 days 



unit 0.48 c.c. Dead 4 days 



unit 0.50 c.c. Dead 3 days 



unit 0.52 c.c. Dead 2 days 



In this experiment 0.48 c.c. given guinea-pig No. 3 is the L + dose of toxin. 



Titration of Diphtheria Antitoxin. In the actual titration of an 

 antitoxin as practised to-day there must be at hand a standard anti- 

 toxin of known strength as well as a toxin, whose M.L.D. has been 

 at least approximately determined. The antitoxin has been dried 

 in a vacuum and preserved in sealed U-shaped ampoules which con- 

 tain the antitoxin in one arm and P 2 O 5 or some other hygroscopic 

 substance in the other arm, in order to maintain the dryness of the 

 antitoxin. The ampoule is best kept in a light-proof box in the re- 

 frigerator. Against this antitoxin the L + dose of a toxin is deter- 

 mined, and against this toxin the new antitoxin is titrated. The 

 amount of antitoxin which protects against the L + dose for four 

 days is the antitoxin unit of the new serum. In preliminary experi- 

 ments the antitoxin is roughly titrated in dilutions of i : 100, i : 200, 

 1:300, and so on. In each case the antitoxin is used in i.o c.c. 

 amounts and the toxin so diluted that 2.0 c.c. contain the M.L.D., 

 the two being mixed and allowed to stand at room temperature for 



