34 PROTEIN POISONS 



the bacterial growth. While it was highly desirable that 

 this should be done at least once in order to demonstrate 

 that the protein reaction given by the bacterial substance 

 was not due to some constituent of the culture medium, 

 ordinarily beef-tea cultures may be employed. As will be 

 seen later, we did grow the colon bacillus once in liquid 

 Uschinsky medium for the purpose of fully satisfying 

 ourselves that the protein material obtained did not come 

 from the culture medium. 



After removal from the tanks the bacterial cellular sub- 

 stance may be washed with various fluids. As a rule, we 

 have washed once or twice with sterile salt solution by 

 decantation and then repeatedly with alcohol, beginning 

 with 50 per cent, and increasing to 95 per cent. The sub- 

 stance is then placed in large soxhlets and extracted first 

 for one or two days with absolute alcohol, and then for 

 three or four days with ether. These extractions with 

 alcohol and ether should be thorough in order to remove 

 all traces of fats and waxes. 



After extraction, the cellular substance is ground, first 

 in porcelain, then in agate mortars, and passed through 

 the finest meshed sieves. If there be bits of agar in the 

 bacterial cellular substance, which is seldom the case, it is 

 separated by the sieve and discarded. The one who grinds 

 the cellular substance should wear a mask in order to pro- 

 tect himself; notwithstanding this precaution, several 

 workers have been acutely poisoned, especially with the 

 typhoid bacillus. Of course, there is no danger of infection, 

 as the material, after the treatment already described, 

 contains no living bacilli. The finely ground cellular sub- 

 stance in the form of an impalpable powder may be kept 

 in wide-mouthed bottles in a dark place, and if so kept 

 it retains its toxicity for years, but when long exposed 

 to the light, even if kept perfectly dry, it becomes less 

 poisonous. 



The yield from the tanks varies with the organism, but 

 generally amounts to from 60 to 80 grams of the purified 

 cellular substance for each tank, and with six tanks in 



