514 THE ANAEROBIC BACTERIA 
Tetanus toxin is produced apparently from the protein of culture 
media by the action of the tetanus bacillus. Brieger^ believes that 
toxin cannot be formed from simple nitrogenous compounds, but 
Buchner- seems to have demonstrated small quantities of tetanus toxin 
in cultures of B. tetani developed in a modified Uschinsky medium 
containing only asparagin as a source of nitrogen. Tetanus toxin is 
readily prepared in a slightly alkaline peptone-meat infusion broth. ^ 
It is necessary to heat the medium rapidly to the boiling point of water, 
keep it at that temperature for thirty minutes, and then cool it rapidly 
to incubation heat (37° C.) prior to inoculation to insure the removal of 
free oxygen. Some means for excluding the air during the first few 
days of incubation is necessary. The older method of overlapping 
the broth with a layer of paraffin oil has been shown to be fallacious.^ 
Inoculation should be maintained at 37° C. for at least seven to ten 
days ; a longer period of incubation appears to result in a diminution of 
the potency of the toxin. It is also a matter of common observation 
that the potency of a toxin made from the same organism and under 
apparently similar conditions varies somewdiat in potency from time to 
time. At the end of the period of incubation the toxin is freed from 
bacteria by rapid filtration through unglazed porcelain filters directly 
into dark-colored bottles, which must be completely filled to exclude 
free oxygen after the addition of 0.5 per cent carbolic acid. The filtered 
toxin is stored in a cool, dark place, shielded as far as possible from 
direct contact with the air. 
Properties of Tetanus Toxin.— Tetanus toxin may be obtained in a 
partially purified state by precipitating broth in which it is contained 
with saturated ammonium sulphate solution; the salts are removed 
from the precipitate by dialysis, and the salt-free residue is dehydrated 
in vacuo.^ The dried toxin retains its potency for many months, if it 
is kept in a cool dark place. 
Tetanus toxin is one of the most potent known. As little as 0.0001 cc. 
of the toxic broth frequently suffices to kill a 15 gm. mouse. Puri- 
fied toxin, prepared by precipitation with ammonium sulphate, will kill 
a mouse of the same weight if but 0.000001 gm. is injected. Not all 
toxin, however, is as potent as the latter. Man and the horse are 
very susceptible to the toxin of the tetanus bacillus. Knorr^ estimated 
that gram for gram, a horse is tw^elve times as susceptible as the 
mouse, and three hundred times as susceptible as the hen. The 
reptilia are practically non-susceptible; toxin injected into these animals 
is not bound; it circulates in the blood stream without causing symp- 
toms until it is finally eliminated. Sbarsky and Jermoljewa^ state 
1 Brieger: Ztschr. f. Hyg., 1895, 19, 101. 
2 Miinchen. med. Wchnschr., 189.S, 40, 449. 
' It is said that a small amount of glucose, 0.1 per cent, is advantageously added. 
* See Bayliss: Principles of General Physiology, London, 1915, p. 54. 
6 Brieger and Cohn: Ztschr. f. Hyg., 1893, 15, 1. 
6 Miinchen. med. Wchnschr., 1898, 45, 321. 
' Biochem. Ztschr, 1927, 182, 180. 
