266 BACTERIAL POISONS. 



demonstrated. It has Dot been found in the braiu and 

 nerve tissue of persons dead from tetanus. A portion of 

 the jelly-hke mass taken from the amputated arm was 

 found to contain tetanus bacilli as well as staphylococci 

 and streptococci, and when planted on beef-broth, tetanine 

 was formed, but no tetanotoxiae or spasmotoxiue. 



KiTASATO and Weyl (1890), employing pure cultures 

 of the tetanus bacillus, obtained from IJ kilogramme beef 

 used as culture medium 1.7118 gramme of tetanine hydro- 

 chloride (0.137 per cent.). Tetanotoxine was also present. 



For its isolation Brieger employed the following 

 method : The cultures were slightly acidulated with 

 hydrochloric acid, heated and filtered; the filtrate was 

 then treated with lead acetate and with alcoholic mercuric 

 chloride in the manner described under mytilotoxine (page 

 255). KiTASATO and Weyl digest the cultures with 0.25 

 per cent, hydrochloric acid for some hours at 60°, then render 

 slightly alkaline, filter, and distil in vacuo at 60°. The 

 residue in the retort is worked for tetanine by Beieger's 

 method, while the distillate contains tetanotoxine, ammo- 

 nia, indol, hydrogen sulphide, phenol and butyric acid. 

 The filtrate from the above mercuric chloride precipitate 

 contains the greater part of the active principle, provided 

 the precipitate has been thoroughly washed. After the 

 removal of the mercury by hydrogen sulphide, it is evap- 

 orated and the residue is repeatedly extracted with absolute 

 alcohol, in which the tetanus poison readily dissolves, and 

 can thus be separated from the insoluble ammonium 

 chloride. The alcoholic solution is treated with alcoholic 

 platinum chloride, which precipitates the ammonium and 

 creatinine platinochlorides, whilst the platinochloride of the 

 poison remains in solution. The filtrate from this precipi- 

 tate gives, on the addition of ether, a flocculent precipitate 

 possessing exceedingly deliquescent properties. The pre- 

 cipitate is, therefore, rapidly filtered off by means of a 

 pump, and dried in vacuo. It can then be recrystallized 

 from hot 96 per cent, alcohol, and the beautiful clear-yellow 

 plates thus obtained, if dried again in vacuo, become 

 rather difficultly soluble in water, from which it can then 



