542 THE TETANUS BACILLUS 



described on p. 94. Incubate at 38 C. anaerobically for 4 or 5 weeks then 

 filter the culture through a Chamberland bougie. In this way a very toxic 

 liquid is obtained of which a dose of ^5 c.c. is fatal to a mouse when inocu- 

 lated sub-cutaneously. 



Marie advised the addition of a little gelatin to the broth. 



Ch. Nicolle proposes the following medium : 



Water, - - 100 c.c. 



Peptone, - 2 grams. 



Gelatin (extra quality), ... 1 gram. 



Salt, - 0-5 



Grow beneath a layer of vaeline oil (p. 97). This medium yields a toxin in 

 6-10 days which is fatal to mice in doses of j^Joo c * c ' 



The toxicity of broth cultures may also be increased to a remarkable degree by 

 making use of the property possessed by the organism of growing in a medium 

 which has already served for the growth of the bacillus and in which it has 

 elaborated its toxin. 



After incubating tetanus bacilli in broth for 3 weeks filter the culture through 

 a bougie and sow the filtrate with the bacillus. Incubate again for 3 weeks and 

 filter a second time. To the filtrate, add about T T -th of its volume of fresh sterile 

 broth and sow a third time with the bacillus. This third culture when filtered 

 gives a very powerful toxin. 



Action of tetanus toxin on living animals. The inoculation of very small 

 doses of toxin is fatal. The most powerful toxins obtained by Vaillard and 

 Vincent by the methods just described will kill guinea-pigs in doses of O001 c.c. 

 and mice in doses of 0*00001 c.c. 



If a sub-lethal dose of toxin be inoculated a local tetanus results involving 

 only the muscles in the neighbourhood of the site of inoculation. 



Tetanus toxin diffuses very quickly through the tissues. If a fraction of 

 a drop of the toxin be inoculated towards the distal end of a rat's tail, in a 

 part, that is, where absorption is very slow, the tail may be cut off 2 or 3 cm. 

 on the proximal side of the site of inoculation three-quarters of an hour 

 after the operation without in any way affecting the course of the disease ; 

 the animal dies almost as quickly as the control. 



Tetanus toxin inoculated sub-cutaneously or intra-muscularly only passes 

 in minute quantities into the blood but is absorbed by the peripheral expan- 

 sions of the neurones and carried gradually to the cells of the central nervous 

 system, producing in them lesions which are responsible for the characteristic 

 spasms. Tetanus toxin has a special affinity for the nerve cells. This can 

 be demonstrated in vitro. 



Wassermann and Takaki, by mixing an emulsion of cerebral substance in normal 

 saline solution with tetanus toxin and centrifuging the mixture, obtained an 

 opalescent solution containing almost no toxin at all. The toxin had not been 

 destroyed by this treatment but was simply fixed by the nerve substance in the 

 same way as a dye might be fixed to a fabric. The toxin merely combines loosely 

 with the cerebral substance from which it may be again set free ; its nature is not 

 altered (Metchnikoff and Marie, Danysz). Neutral mixtures of brain emulsion and 

 tetanus toxin become toxic on keeping : the toxin diffuses into the surrounding liquid 

 and is again free in solution. On the other hand, toxic mixtures of toxin and anti- 

 toxin become in time less toxic (Knorr). An emulsion of carmine in normal saline 

 solution acts in a similar manner : provided that it has not been sterilized in steam 

 nor macerated it fixes the toxin and renders the filtrate harmless (Stoudensky). 



In these mixtures the toxin is fixed by the particles of cerebral substance or 

 carmine respectively and, if injected into animals, it has no time to diffuse before 

 the leucocytes absorb and destroy it. 



In the living tissues, tetanus toxin induces phenomena similar to those 

 just studied in vitro. Tetanus toxin inoculated sub-cutaneously into guinea- 

 pigs is fixed after the lapse of some hours by the cells of the central nervous 



