476 ANAEROBIC BACTERIA 



proportions, which may be recognized by their respective physio- 

 logical actions: tetanospasmin, a neurotoxin, which is relatively 

 thermostable and produces the characteristic tonic contractions or 

 spasms which characterize the disease tetanus; and tetanolysin, a 

 relatively thermolabile hemotoxin which dissolves red blood cells. It 

 is doubtful if the tetanolysin is ordinarily of clinical importance. 



Tetanus toxin appears to be produced only in sugar-free media 

 under anaerobic conditions. Buchner 1 seems to have detected 

 small amounts of true tetanus toxin in cultures of tetanus bacilli 

 grown in a modified Uschinsky medium containing asparagin and 

 certain inorganic salts. Brieger, on the contrary, 2 maintains that the 

 toxin is produced only when the organisms are grown in albuminous 

 media. 



Tetanus toxin is best prepared in slightly alkaline peptone-meat 

 infusion broth containing 0.1 per cent, of dextrose. The dextrose is 

 added to insure a large initial development of bacteria which as soon 

 as the sugar is exhausted (within twenty-four hours) attacks the pro- 

 tein constituents of the medium, forming from them the tetanus 

 toxin. 3 It is essential to heat the medium to the boiling point and 

 cool it rapidly immediately before inoculation to drive out all traces 

 of oxygen. Anaerobic conditions are most easily obtained and main- 

 tained by overlaying the broth with pure paraffin oil, according to the 

 method of Park. 4 Incubation should be maintained at 37 C. for seven 

 to ten days. The toxin appears to lose somewhat in potency if incu- 

 bated for a longer period. The potency of the toxin prepared in this 

 manner varies considerably, being influenced by the composition and 

 reaction of the medium and the degree of anaerobiosis. Tetanus 

 bacilli retain their ability to produce toxin with great tenacity and 

 regularity, even after prolonged artificial cultivation. At the end of 

 the period of incubation the broth is rapidly filtered through sterile 

 unglazed porcelain filters into dark-colored bottles, which are com- 

 pletely filled to exclude oxygen after the addition of 0.5 per cent, 

 carbolic acid. It should be kept in a cool, dark place under anaerobic 

 conditions. A small amount of the broth containing toxin thus 

 obtained, freed from bacteria, will liquefy gelatin, thus showing that 

 a peptonizing ferment is present in the filtrate, either inherent in the 

 toxin or in association with it. According to Fermi and Pernossi, 5 



1 Miinchen. med. Wchnschr., 1893, No. 24, 450. 



2 Ztschr. f. Hyg., 1895, xix, 102. 



3 Kendall, Boston Med. and Surg. Jour., 1913, clxviii, 825. 



4 Loc. cit. 5 Centralbl. f. Bakt., 1894, xv, 303. 



