8 
METHODS. 
In order to obtain reliable and comparable results, it is necessary 
to take into account all the factors concerned — the composition of 
the poisons, their concentration, the diluting fluid, length of time 
the mixtures are allowed to stand, the site of inoculation, etc. All 
the known factors have been considered in our method of standard- 
izing tetanus antitoxin, so that it is our belief that we now have 
as accurate and satisfactory a standard for this antitoxin as is the 
case with diphtheria. 
Careful regard must be had for the following points in order to 
obtain uniform results : 
The diluting jiuid. — Salt solution containing 0.85 per cent chem- 
ically pure sodium chlorid sterilized by boiling is used for diluting 
both the toxine and the antitoxin. 
Time and temperature. — The mixtures of toxine and antitoxin are 
kept one hour at room temperature in diffused light before injection 
into the guinea pig. 
Amount. — A total of 4 c. c. of the toxine-antitoxin mixture is 
injected into the guinea pig. If the toxine-antitoxin mixture does 
not equal 4 c. c., salt solution equal in amount to the difference 
is used to wash out the syringe, so that the total amount injected 
and the pressure effects are always equal. 
Concentration of the toxine. — One-tenth of a gram of the dried 
tetanus toxine (A) is dissolved in 166.66 c. c. salt solution. One c. c. 
of this dilution equals the test dose (L + dose). 
Concentration of the antitoxin. — The dilution of the antitoxic 
serum should follow the tables on page 50. 
Guinea pigs. — The guinea pigs should weigh 350 grams. Animals 
weighing from 300 to 400 grams are allowable in preliminary rough 
testing. 
Site of injection. — The injection is always given subcutaneously 
into the tissues of the abdomen about the level of the umbilicus.® 
Time of death. — The number of immunity units contained in the 
serum is determined from the amount given the animals that are 
living ninety-six hours after the inoculation of the mixtures. 
An example of a test. — Carefully tare a weighing bottle, then add 
approximately 20 to 50 mg. of the dried poison. Again carefully 
weigh. Dissolve the toxine in the weighing bottle with salt solu- 
tion (0.85) in the proportion of 0.1 gram of the dried poison to 166.66 
c. c. of the salt solution. This proportion is used for the reason 
that each cubic centimeter of this solution will represent 0.000,6 
gm. of the original dried poison (=100 MLD’s). This proportion 
« It is very important to inject the animals in the same place on account of the 
relation to the motor nerve endings and the distance to travel to the central nervous 
system. 
