TETANUS TOXIN 543 



system and it is then that the symptoms of tetanus show themselves. Direct 

 proof of this fact is afforded by the severity of the symptoms produced by the 

 inoculation of the toxin into the intact nervous tissue. For example, rabbits 

 are very resistant to sub-cutaneous or intra-venous inoculation of tetanus 

 toxin ; to produce a fatal result in 4 days a dose of 2*5 c.c. must be used. 

 But a dose of Ol c.c. of the same toxin inoculated intra-cerebrally is fatal 

 in less than 20 hours. In this case the disease runs a special course the 

 cerebral tetanus of Roux and Borrel. The animal exhibits an extraordinary 

 degree of excitability, is subject to hallucinations, to sudden fears, and, 

 in short, to mania. Later, intermittent convulsive crises, motor troubles 

 and polyuria appear ; these symptoms terminate in the death of the animal. 

 Guinea-pigs and rats suffer similarly from cerebral tetanus after the inocula- 

 tion of very small doses of toxin into the brain. 



The resistance of rabbits to sub-cutaneous and intra-venous inoculation 

 is not, therefore, due to a relative insusceptibility of the nerve cells but to 

 the fact that much of the toxin injected does not reach the cells, being destroyed 

 (probably by the phagocytes) in some part of the body as yet undetermined 

 (Roux and Borrel). 



Technique of intra-cerebral injection. After incising the soft parts make a hole 

 in the skull with a gimlet being careful to protect the dura mater from injury : 

 then plunge the needle of the syringe to the desired depth previously determined 

 by a probe and inject the toxin. The animals stand these intra-cerebral inocula- 

 tions very well and it is possible to inject 8 drops of sterile broth in 2 stabs into a 

 guinea-pig's brain without producing symptoms. In a rabbit 0'5 c.c. can be simi- 

 larly inoculated. 



Nature of tetanus toxin. The product of the tetanus bacillus (or, tetano- 

 spasmin) is extraordinarily toxic. 



Evaporated in vacuo, 1 c.c. of a toxin which is fatal to mice in doses of 0*00001 c.c. 

 gives a constant residue of 0'04 gram. On calcining, this residue loses 0'025 gram 

 which represents the organic matter. This organic matter consists largely of 

 inactive substances such as peptone, etc. (Vaillard and Vincent) : but assuming 

 for the moment that the whole of the organic matter consists of toxin it follows 

 that these 25 mg. of toxin are sufficient to kill one hundred thousand mice ; on this 

 assumption the lethal dose of the active principle for a mouse is '000,000,25 gram. 

 And the premise of the argument shows that this is a very conservative estimate. 



Tetanus toxin has all the characteristics of enzymes or diastases. Chemi- 

 cally, it is very similar to diphtheria toxin. It undergoes considerable 

 change if heated to 65 C. for half an hour and is completely destroyed by 

 heating for 3 hours at 80 C. 



Stored in sealed vessels in the dark and away from air, the toxin retains 

 its virulence for a long time. The toxin is rapidly weakened by exposure 

 to the air and diffused light and entirely loses its virulence in a few days 

 if exposed to direct sunlight and air. 



Tetanus toxin has the property of adhering to amorphous precipitates 

 produced in liquids in which it is dissolved. 



The addition of calcium chloride to the toxin precipitates calcium phosphate and 

 a part of the toxin is carried down with the precipitate. A minute portion of this 

 precipitate as large as a pin's head if carefully washed and inserted beneath the 

 skin of a guinea-pig, will cause the death of the animal from tetanus in 30 hours. 

 After precipitation a large amount of toxin still remains in solution in the liquid. 



If a little tetanus toxin obtained by nitration be added to a tube of sterile 

 gelatin the medium will be liquefied in a few days. The phenomenon is due 

 to the presence in the toxin of a peptonizing diastatic ferment. This ferment 

 does not appear to be identical with the toxic principle. 



Evaporated at 25 C. in vacuv over sulphuric acid the toxin leaves a brown, 



