PREPARATION OF CULTURES FROM TISSUES. 301 



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

 tube, and with a heavy, sterilized, looped platinum needle 

 or spatula, rub it carefully over the surface. 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 outside 

 organisms during the manipulation, while a few may 

 give the result desired, viz., a growth of the tubercle 

 bacilli themselves. 



The blood-serum upon which the organism is to be 

 cultivated should be comparatively freshly prepared — 

 that is, should not be dry. 



After inoculating the tubes they should be carefully 

 sealed up to prevent evaporation and consequent dry- 

 ing. This is done by burning off the superfluous over- 

 hanging cotton plug in the gas-flame, and then impreg- 

 nating the upper layers of the cotton with either 

 sealing-wax or paraffiu 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 in the 

 steam sterilizer just before using (Ghriskey). This 

 precaution is necessary because of the slow growth of 

 the organism. Under the most favorable conditions 

 tubercle bacilli directly from the animal body show no 

 evidence of growth for about twelve days after inocu- 

 lation upon blood-serum, and, as they must be retained 

 during this time at the body temperature — 37.5° C. — 

 evaporation would take place very rapidly and the 

 medium would become too dry for their development. 



If these primary efforts result in the appearance of a 

 culture of the bacilli, further cultivations may be made 

 by taking up a bit of the colony, preferably a moder- 

 ately large quantity, and transferring it to fresh serum, 



14 



