350 THE BACILLUS OF TUBERCULOSIS 



7. Wash well in 95 per cent, alcohol until all fuchsin has been 

 dissolved out. This may require several changes of fresh alcohol. 



8. Anilin oil one-half minute. 



9. Several changes of xylol. 



10. Mount on a slide in Canada balsam. 



Cultural Properties. The tubercle bacillus cannot be readily 

 cultivated directly from sputum or caseous or purulent discharges 

 containing it because it is not present alone, but mixed with other 

 bacteria. Since the tubercle bacillus grows very slowly in artificial 

 culture media the other bacteria multiply much more rapidly, and, 

 in fact, overgrow it, making it difficult if not impossible to find it. It 

 is certain that it can hardly ever be obtained in pure culture by 

 the ordinary method of inoculating tubes directly or pouring plates. 

 The usual method, therefore, of obtaining pure cultures ^jnsists in 

 inoculating the tubercular material either subcutaneoiftlv or intra- 

 peritoneally into guinea-pigs. This highly susceptible animal develops 

 first a local tuberculosis of the regional lymph glands and later a 

 general tuberculosis. If such an infected guinea-pig is killed eight 

 to ten weeks after the infection the proper tubercular material for 

 obtaining pure cultures can be procured. 



The procedure is best carried out as follows: The animal is 

 chloroformed to death and then stretched out on a board with its 

 four legs tied to four nails. The abdomen is shaved and sterilized 

 and a median incision is made from the sternum to the symphysis. 

 This is done with a sterile knife and in such a manner that the peri- 

 toneum is left unopened. The skin is then flapped back on both sides. 

 The peritoneum is now opened in the middle line with fresh sterile 

 instruments (scissors or knife) and held open with sterile forceps on 

 both sides by an assistant or fastened to the side with suitable tacks. 

 The operator now removes, with sterile forceps and scissors, some 

 tubercular abdominal glands and the whole or part of the spleen. 

 These tissues P ^mediately placed in a covered sterile glass dish 

 (a not too .si . Petri dish will answer very well). The material 

 so obtained is then divided, with all aseptic precautions, into small 

 pieces, and portions from the interior of tubercular glands, or from 

 tubercles in the spleen, are brought into tubes containing the proper 

 culture media, where they are left either undisturbed on the surface 

 or rubbed over it with a strong platinum loop. Suitable media for 

 cultivating the tubercle bacillus are sterile coagulated cattle-blood 

 serum (Koch), dog's blood serum obtained sterilly (Smith), eggs in 

 which the white and yellow have been mixed and the mass subse- 

 quently distributed to test-tubes (Dorset), and various other media. 

 After inoculation the tubes must be protected against evaporation, as 

 it is necessary to keep them in the incubator at blood temperature for 

 several weeks. This is done either with rubber caps, ground-glass 

 stoppers, or by closing them with paraffin. If there is enough moisture 

 in the tubes from the beginning the microorganism will grow without 



