292 BACTERIA PATHOGENIC TO MAN 



a single film, which on agitation is easily broken up and then settles 

 on the bottom of the flask, where it ceases to develop further. The 

 liquid remains clear. A practical point of importance, if a quick growth 

 is desired, is to remove for the new cultures a portion of the pellicle 

 of a growing bouillon culture, which is very thin and actively increasing. 



Obtaining of Pure Cultures of the Tubercle Bacillus from Sputum, Infected 

 Tissue and Other Materials. On account of the time required and the 

 difficulties to be overcome this is never desirable except when careful 

 investigations of importance are to be undertaken. The chief point of 

 present interest is the dissemination of bovine bacilli in man. The 

 discovery by Theobald Smith of the greater acid production of the 

 human type of tubercle bacilli in glycerin bouillon over the bovine 

 bacilli has made it a matter of added importance to test as many bacilli 

 as possible for their biochemical characteristics. Pure cultures .can be 

 obtained directly from tuberculous material when mixed infection is 

 not present, and a suitable dog serum or egg culture medium is at hand ; 

 but as it is so difficult to get material free from other bacteria which grow 

 much more rapidly and take possession of the medium before the 

 tubercle bacillus has had time to form visible colonies, it is usually 

 necessary first to inoculate a guinea-pig, both subcutaneously and 

 intraperitoneally, and then obtain cultures from the animal as soon as 

 the tubercle infection has fully developed. From acute tuberculosis 

 in man in other regions than the lungs direct cultures on blood serum 

 or egg may be made with some hope of success. Under the best con- 

 ditions great care and patience are necessary if successful results are 

 to be obtained. 



Animals inoculated usually die at the end of three weeks to four 

 months. It is better, however, not to wait until death of the animals, 

 but at the end of three weeks to kill a guinea-pig, which by its enlarged 

 glands shows evidence of tuberculosis, and to remove, with the greatest 

 care as to cleanliness, one or more nodules from the lungs, spleen, or 

 lymphatic glands. Animals which develop tuberculosis acutely are apt 

 to have abundant tubercle bacilli and give successful cultures, while the 

 chronic cases usually have few bacilli and frequently give unsuccessful 

 cultures. The animals after being killed are placed in trays, and after 

 washing with a 5 per cent, solution of carbolic acid, immediately autop- 

 sied. The skin over the anterior portion of the body having been 

 carefully turned back, an opening is cut with a fresh set of sterile instru- 

 ments into the thoracic or abdominal cavity; then with a sterile forceps 

 the lymph-gland, portion of spleen or other part which it is desired to 

 examine is removed to a sterile covered beaker. This tissue, if suitable, 

 is sliced in thin sections and conveyed directly to the surface of the 

 solid culture medium. It is allowed to remain in contact with the 

 moist serum or egg at 37 C. for three to ten days, and then by means 

 of a platinum wire gently rubbed and drawn over the surface of the 

 media. The tubes are then replaced in the incubator for ten days to 

 three weeks, when a visible culture should be obtained. Owing to the 

 liability of the blood serum to become too dry for the development of 



