APPENDIX 



IV 



CULTIVATION OF BACTERIUM (BACILLUS) TUBERCULOSIS 



The isolation of this organism from tuberculous lesions and get- 

 ting it to multiply readily on artificial media necessitates a very 

 special and careful procedure. When it becomes accustomed to 

 artificial media its continued cultivation is not difficult. Dr. Theo- 

 bald Smith, of Harvard University (Jour, of Exp. Med., Vol. III., 

 1898, p. 451), has the credit of formulating a method by combining 

 details in such a manner that the procuring of cultures is, in most 

 cases, possible. Dog serum is used. The method, as he gives it, 

 is as follows, viz. : 



" The dog was bled under chloroform and the blood drawn from 

 a femoral artery, under aseptic conditions, through sterile tubes 

 directly into sterile flasks. The serum was drawn from the clots 

 with sterile pipettes and either distributed at once into tubes or else 

 stored with 0.25 to 0.3% chloroform added. Discontinued ster- 

 ilization was rendered unnecessary. The temperature required to 

 produce a sufficiently firm and yet not too hard and dry serum is 

 for the dog 75 to 76 C. For horse serum it is from 4 to 5 lower. 

 The serum was set in a thermostat into which a large dish of water 

 was always placed to forestall any abstraction of moisture from the 

 serum. About 3 hours suffice for the coagulation. When serum 

 containing chloroform is to be coagulated, I am in the habit of 

 placing the tubes for an hour or longer in a water bath at 55 to 60 

 C., or under the receiver of an air pump, to drive off the antiseptic. 

 This procedure dispenses with all sterilization excepting that going 

 on during the coagulation of the serum. It prevents the gradual 

 formation of membranes of salts, which, remaining on the surface 

 during coagulation, form a film unsuited for bacteria. Tubes of 

 coagulated serum should be kept in a cold closed space where the 

 opportunities for evaporation are slight. They -should always be 

 kept inclined. 



The ordinary cotton-plugged test tubes I do not use, because of 

 the rapid drying out permitted by them, as well as the opportunities 

 for infection with fungi. Instead, a tube is used which has a ground 

 glass cap fitted over it. This cap contracts into a narrow tube 



