50 BACTERIOLOGY 



dip in the acid again. After either acid the preparation is to be 

 washed in alcohol until the last trace of the stain has been removed. 

 An excellent decolorizing agent is a 3 per cent solution of hydro- 

 chloric acid in alcohol, used for about a minute. The contrast 

 stain may be omitted entirely if it is desired. A suitable contrast 

 stain after fuchsin staining is a solution of methylene blue; after 

 gentian-violet staining, safranin. 



Those who have had experience in staining tubercle bacilli 

 so(jn discover that the bacilli exhibit some differences in their 

 resisting power to strong acids. One encounters occasionally 

 bacilli that are perfectly stained side by side with others that are 

 more or less completely decolorized. These facts show the ne- 

 cessity of practice with any method, and of exercising caution 

 and judgment in making a diagnosis where the number of bacilli 

 happens to be scanty. If tubercle bacilli are not found in the 

 first preparation, other preparations should be made. Some- 

 times a large number of cover-glasses must be examined. 



Various expedients have been devised to concentrate tubercle 

 bacilli when only a small number may be present in a sample of 

 sputum. Antiformin (a preparation of chlorinated sodium hydrox- 

 ide) has been employed for this purpose. The following method 

 is that of Williamson.^ The sputum is measured and transferred 

 to a clean flask of resistant 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 albu- 

 men (egg albumen + 0.5 per cent carbohc acid) on a slide and then 



' Williamson, Joiirn. A. M. A., Apr. 6, 1912, Vol. LVIII, p. 1005-07. 



