140 APPLIED BACTERIOLOGY 



A pure culture is readily obtained by inoculation of a 

 guinea-pig with tuberculous sputum ; after two or three 

 weeks the animal is killed, and its lungs opened with anti- 

 septic precautions : a streak is made from an affected part 

 (in which the bacillus exists as a pure culture) on to media. 

 Another method of obtaining a pure culture would be 

 to use Pastor's method ; this obviates the necessity of 

 employing an animal, and does not require the use of a 

 microscope. 



Bacteriological Examination of Sputum and Sections. 



We may employ one or two distinct methods : (1) Direct 

 staining; (2) demonstration of the p^^esence of the tubercle 

 bacillus by culture. In either case it is advisable to 

 examine the sputum for particles of broken-down lung 

 tissue, which are noticeable as minute yellowish specks of 

 caseous matter, best seen by pouring the sputum into a 

 flat glass dish placed on a piece of black paper. The viscous 

 masses of sputum are then ' teased ' out with a couple of 

 match-ends. To obtain a sample of sputum the patient 

 should be directed first to rinse the mouth well with 

 distilled water, and then to expectorate into a test-glass ; 

 the first expectorations in the morning should be secured 

 if possible. 



Method of Staining the Bacilli in Sputum. — The cover-glass 

 films prepared in the ordinary way after ' fixing ' are stained 

 by the Ziehl-Neelsen method as follows : 



1. The cover-glass is treated with warm carbol-fuchsine 

 solution for three minutes. 



2. Decolourise with 25 per cent, sulphuric acid. 



3. Wash in water. 



4. Gounterstain in Tnethylene blue for three Tninutes. 



5. Wash, dry, and mount in xylol balsam. 



