TIU-: itAciu.rs /// Ti'H/-:iu.'i-Losis 293 



the bacillus, it is necessary to keep the culture moist by sealing the 

 end in some way. Theobald Smith, who has had a very large experience 

 in growing the tubercle bacillus, gives the following details as to his 

 method: 



"Throughout the work solidified dog's serum was used as being the 

 best medium. 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 clot 

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

 stored with 0.25 to 0.3 per cent, chloroform added. The temperature 

 required to produce a sufficiently firm and yet not too hard and dry 

 serum is, for the dog, 75 to 76 C. The tubes containing the serum 

 were set in a thermostat, into which a dish of water was placed, to 

 forestall any abstraction of moisture from the serum. About three 

 hours suffice for the coagulation. 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 plugged with 

 glass-wool; this plug is not disturbed. The tube is cleaned, filled, and 

 inoculated by removing the cap. With sufficient opportunity for the 

 interchange of air very little evaporation takes place, and contamination 

 of the culture is a very rare occurrence. In inoculating these tubes bits 

 of tissue which include tuberculous foci, especially the most recent, are 

 torn from the organs and transferred to the serum. Very little crushing, 

 if any, is desirable or necessary. I think many failures are due to the 

 often futile attempts to break up firm tubercles. Nor should the bits 

 of tissue be rubbed into the surface, as is sometimes recommended. 

 After a stay of several weeks in the thermostat I usually remove the tubes 

 and stir about the bits of tissue. This frequently is the occasion for a 

 prompt appearance of growth of tiny, dull-grayish colonies within a 

 week, as it seems to put certain still microscopic colonies in or around 

 the tissues into better condition for further development. From this 

 first growth of tubercle bacilli fresh serum tubes are inoculated. From 

 these either serum or glycerin-agar tubes are inoculated. The thermostat 

 should be fairly constant, as urged by Koch in his classic monograph; 

 but I look upon moisture as of more importance. If possible a thermo- 

 stat should be used which is opened only occasionally. Into this a 

 larrje dish of water is placed, which keeps the space saturated. Venti- 

 lation should be restricted to a minimum. As a consequence, moulds 

 grow luxuriantly, and even the gummed labels must be replaced by 

 pieces of stiff manila paper fastened to the tube with a rubber band . 



