416 APPLICATION OF METHODS OF BACTERIOLOGY 



to isolate bacillus tuberculosis from the tissues may cause 

 total failure. It is, therefore, necessary that the injunc 

 tions for obtaining it in pure culture be carefully observed. 



Preparation of Cultures from Tissues. — Under strict aseptic 

 precautions remove from the animal a diseased organ — ^the 

 liver, spleen, or a lymphatic gland being preferable. Place 

 the tissue in a sterilized Petri dish, and dissect out with 

 sterilized scissors and forceps the small tubercular nodules. 

 Place each nodule upon the surface of the blood-serum, one 

 nodule in each tube, and without attempting to break it 

 up or smear it over the surface, leave it for four or five days 

 in the incubator. After this time it may be rubbed over 

 the surface of the serum. The object of this is to give to 

 bacilli in the nodule an opportunity to multiply, under 

 the favorable conditions of temperature and moisture, 

 before an effort is made to distribute them over the surface 

 of the medium. It is best to dissect away twenty to thirty 

 such tubercles and treat each in the same way. Some of 

 the tubes will remain sterile, others may be contaminated 

 by extraneous saprophytic organisms during the manipula- 

 tion, while a few may give the result desired, viz., a growth 

 of the tubercle bacillus itself. 



The blood-serum upon which the organism is to be cul- 

 tivated should be comparatively freshly prepared — that is, 

 should not be dry. 



After inoculating the tubes they should be carefully 

 sealed to prevent evaporation and consequent drying. 

 This is done by burning off the overhanging cotton plug 

 in a gas-flame, and then impregnating the upper layers of 

 the cotton with either sealing-wax or paraffin of a high 

 melting-point; or by inserting over the burned end of the 

 cotton plug a soft, closely fitting cork that has been sterilized 



