TUBERCULAR SPUTUM. 31 



DEMONSTRATION XII. 



Pure Cultures of Tubercle Bacillus — Tubercular Sputum. 



1. From the glycerine agar cultures of the Tubercle 

 bacillus prepare cover glass preparations, and stain in 

 the following manner : 



(a) Float in Ziehl's carbol fuchsin for 5 minutes. 

 (6) Wash in water and then rinse quickly through 

 33^% nitric acid solution. 



(c) Wash immediately in'70 % alcohol and then in 

 water. 



(d) Dry and mount in Canada balsam. 



2. Prepare other cover glass films and stain in 

 Sudan III. (Dorset). 



Sudan III staining fluid consists of a saturated 

 solution of Sudan III in 80% alcohol. 



(a) Place the prepared films in the stain for 5 

 minutes. 



(6) Wash the films in 70% alcohol and then in 

 water, dry and mount in Canada balsam. 



Sudan III has a selective affinity for fat, which is 

 present in large amount in the bodies of Tubercle 

 bacilli. 



Examination of Sputum for Tubercle Bacilli. 



Pour a thin layer of sputum into a glass capsule 

 and pick out for examination a yellowish (caseous) 

 particle if present; otherwise choose a purulent part. 



