As similar methods are used for practically all types of toxins, a description of the preparation of diphtheria antitoxin will be sufficient.

To obtain the necessary toxin virulent diphtheria bacilli are grown in alkaline bouillon containing 0.2 per cent, dextrose at a temperature of 370 C. for five to seven days. The bouillon culture is then passed through a porcelain or Berkefeld filter and stored in sterile containers in an ice-box. On account of general convenience horses are commonly employed. They should be perfectly healthy, free from glanders, tuberculosis, or tetanus. The horse is injected hypodermically with 0.1 c.c. of the toxic filtrate. This is frequently followed by a rise in temperature, local reaction, and some general disturbance. When these disappear, a second dose is given. The doses are cautiously increased in amount and administered every few days until from 500 to 1000 c.c. of the toxin can be given without effect. When the degree of immunity is sufficiently high, blood is drawn from the jugular vein to the amount of from 3 to 9 liters, according to the size of the horse, collected in sterile bottles, then placed on ice for several days until the clear serum separates from the clot. This is then drawn off from the coagulated blood under aseptic precautions, and in it is the antitoxin. It is preserved by the addition of small amounts of phenol, trikresol, etc.; this latter seems to be the most satisfactory. After the serum has been obtained, its strength or potency, as expressed by the term "immunizing units," must be determined. An antitoxic unit may be defined as being ten times the least quantity of antitoxic serum that will protect a standard (300-gm.) guinea-pig against ten times the least certainly fatal dose of toxic bouillon.

To determine the strength of any given serum, the minimum fatal dose of a sterile toxin for a 300-gm. guinea-pig must be ascertained. Then must be determined the least quantity of antitoxic serum that will protect a guinea-pig against ten times the ascertained minimum fatal dose of the toxin. The necessary dose of antitoxic serum is expressed as a fraction of a cubic centimeter and multiplied by 10, the result equaling one unit.

Ehrlich, in determining the unit, makes use of a standard antitoxin (antitoxins not deteriorating or varying as do toxins) by which the antitoxin combining power of the test toxic bouillon is first determined. The toxin unit (the smallest amount of toxin required to kill a guinea-pig weighing 300 gm.), having been found, is then used to determine the antitoxic unit of antitoxins of unknown strength.

The power of antitoxic serums differ greatly: some contain 200 to 300 units per cubic centimeter, while others may contain even 1700 to 2000 per cubic centimeter.

Inasmuch as the antitoxin is only a small portion of the serum, various methods have been sought, by means of which the useless, and sometimes harmful, portions may be eliminated. The method employed at present in order to obtain such a concentrated antitoxin is that elaborated by Gibson - that of globulin precipitation.