31 
used. According to Debrand,® the strongest poisons may be obtained 
by symbiosis with B. suhtilis. The best medium is a weak alkaline 
bouillon with 1 per cent peptone and 0.5 per cent salt. The addition 
of glycerin and grape sugar is to be avoided, as the acids produced 
by their decomposition have an injurious effect upon the toxine. 
Tizzoni obtained good results by the addition of gelatin to the culture 
medium. It is most important to have a culture which is a good 
toxine producer, just as in the case of diphtheria. It seems that the 
strongest poisons are obtained after a term of from six to eight days 
in the incubator. By observing the proper conditions it seems 
comparatively easy to obtain cultures that will kill a mouse weighing 
15 grams in a dose of 0.000005 gram. Most of those who have 
worked much in this subject have experienced at times difficulty in 
obtaining strong tetanus toxine; not all of the factors involved are 
understood. 
It is noteworthy that the strength of the toxine produced is greater 
in contaminated cultures than in pure cultures. In the Marburg 
Institute of Hygiene and Experimental Therapy, presided over by 
Behring,^ the strongest tetanus toxines have been obtained from 
mixed cultures. The first work, it will be remembered, upon the 
tetanus toxine by Faber, Brieger, Fraenkel, Nicolaier, and Rosenbach 
was with impure cultures. 
Ilschinsky ^ cultivated tetanus bacilli on a proteid-free medium of > 
the following composition: 
Water 1,000 
Glycerin. 30 to 40 
Sodium chloride •. 5 to 7 
Calcium chloride 0.1 
Magnesium sulphate 0.2 to 0.4 
Dipotassium hydrogen phosphate 2 to 2.5 
Ammonium lactate 6 to 7 
Sodium aspartate ^ 3.4 
preferably with the addition of 1 to 2 per cent of grape sugar. The 
air was excluded by means of liquid paraffin. 
According to Buchner, Tizzoni and Cattani, following the plan of 
Brieger and Frankel, were the first to succeed in obtaining a dried 
poison with the use of ammonium sulphate. 
“Debrand, L.: Sur un nouveau precede de culture du bacille du tetanos. Ann. d. 
Inst. Pasteur, vol. 14, 1900, pp. 757-768. 
& V. Behring, E. : Aetiologie und aetiologische Therapie des Tetanus. Beitr. z. exper. 
Therapie, vol. 7, 1904, pp. 1-72. 
c Ilschinsky, “Ueber eine eiweissfreie Nahrlosung f. pathog. Bakt.” Cantralb. f. 
Bakt., vol. 14, 1893, p. 316. 
Buchner, H.; Erwiderung betreffend das trockene Tetanusgift. Deut. med. 
Woch., vol. 20, 1894, p. 179. 
