146 LABORATORY BACTERIOLOGY 



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 little evaporation takes place, 

 and contamination of the culture is of very rare occurrence. In inoc- 

 ulating 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 tubercules. 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 within a week, as it seems to put certain still 

 microscopic colonies in or around the tissue into better condition for 

 further development. The thermostat should be fairly constant, as 

 urged by Koch in his classic monograph, but I look upon moisture 

 as more important. If possible, a thermostat should be used which 

 is opened only occasionally. Into this a large dish of water is 

 placed, which keeps the space saturated. Ventilation should be 

 restricted to a minimum. As a consequence, moulds grow luxuri- 

 antly and even the gummed labels must be replaced by pieces of 

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

 keeping the tubes inclined, no undue amount of condensation water 

 can collect in the bottom, and the upper portion of the serum 

 remains moist. The only precaution to be applied to prevent infec- 

 tion with moulds is to thoroughly flame the joint between tube and 

 cap as well as the plugged end, before opening the tube. When 

 test tubes are employed it is well to dip the lower end of the plug 

 into sterile molten paraffin and to cover the tube with a sterilized 

 paper cap. The white bottle caps of the druggist are very service- 

 able." 



Unless the tuberculous material is perfectly fresh (uncontami- 

 nated), and in the early stages of the disease, it is safer to inoculate a 

 guinea pig, and after the lesions begin to develop to chloroform it 

 and make the cultures from the recently affected liver or spleen. 



