50 BACTERIOLOGY 



lowing method is that of Williamson. 1 The sputum is measured 

 and transferred to a clean flask of Jena glass. An equal volume of 

 50 per cent antiformin is added, mixed with the sputum, and the 

 mixture brought to a boil over the flame. This dissolves the 

 sputum promptly. The material is then cooled and to each 10 c.c. 

 of material in the flask 1.5 c.c. of a mixture of chloroform, one part, 

 and alcohol, nine parts, is added. The mixture is thoroughly 

 shaken. As a result the tubercle bacilli imbibe some of the chloro- 

 form and become heavier. The material is next centrifugalized 

 at high speed for 15 minutes, which separates it into three layers, 

 antiformin above and chloroform below with the layer of sediment 

 between the two. This layer is removed and mixed with egg 

 albumen (egg albumen +0.5 per cent carbolic acid) on a slide 

 and then spread into a smear between two slides. The smears 

 are then dried and stained in the usual way. Instead of using 

 albumen to fix the sediment to the slide, it is convenient to save 

 some of the original sputum and mix it with the sediment for 

 this purpose. 



Staining of Spores. The method is applicable to cover- 

 glass preparations which may be prepared in the usual way from 

 material supposed to contain spores. 



(a) After drying the smear on the cover-glass, fix it with heat 

 by passing through the flame three times. 



(b) Float the cover-glass face downward on the surface of 

 steaming hot carbol- fuchsin or aniline- water fuchsin for three to 

 five minutes. 



(c) Wash in 3 per cent hydrochloric acid alcohol one minute, 

 or less. 



(d) Wash in water. 



(e) Stain with watery solution of methylene blue half a minute. 

 (/) Wash. 



(g) Dry. 

 (h) Balsam. 



The spores are intensely stained by the fuchsin. The stain 

 1 Williamson, Journ. A.M. A., Apr. 6, 1912, Vol. LVIII, p. 1005-07. 



