124 APPLIED BACTERIOLOGY 



Another method of obtaining a pure culture would be 

 to use Pastor's method, described on page 125 ; 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 of two distinct methods : (1) Direct 

 staining ; (2) demonstration of the presence of the tubercle 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 ZiehlNeelsen method as follows : 



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

 solution for three minutes. 



2. Decolourise with 25 jer cent, sidphuric acid. 



3. Wash in water. 



4. Counterstain in methylene blue for three minutes. 



5. Wash, dry and mount in xylol balsam. 

 Sections of tissue are stained as under : 



1. The sections are treated ivith hot carbol fuchsine solu- 

 tion for ten minutes. 



2. Decolourise with 25 per cent, sulphuric acid, dipping 

 the sections for about a minute or so into the acid and then 

 water alternately. 



3. Wash well in water. 



