Methods of Staining Encapsulated Pneumococci 309 



after which a great variety of staining as procedures, difTerential 

 well as simple, may be readily adopted according to the character 

 of the material to be examined, and the purposes of the examination. 

 The simple routine gentian-violet stain may be used, or this, after 

 drying, may be decolorized for a few seconds in saturated bichlo- 

 ride-alcohol and counterstained, or the Gram difTerential method 

 may be employed. Finally bits of diseased tissue may be hardened 

 in 40 per cent formalin, imbedded in celloidin, and sections cut 

 and stained by simple and differential methods to demonstrate the 

 presence of encapsulated organisms. 



Briefly tabulated, the technic for smear preparations is as follows: 



technic for the fixing and staining of smears. 



Simple Stains. 



1 Formalin 40%, 2-5 min.* 



2 Wash in water. 



Differential Stains. 



Gram's method. 



3 10% aq. gentian-violet. 3 Anilin-gentian-violet, a min. 



I I 



4 Wash water. 4 Blot, dry. 4 Iodine solution, 2 min. 



5 Dry, mount balsam. 5 Sat. bichloride-alcohol, s sec* 5 Alcohol, 95% decolori7.e. 



6 Dilute aq. eosin or fuchsin, 5sec.* 6 Eosin alcohol. 6 .\q. (uchsin (diluted 



7 Blot, dry, mount in balsam. 7 Oil of origanum. 7 Wash water. 



8 Balsam mount. 8 Dr)', mount in balsam. 



THE TECHNIC FOR SMEARS OF SPUTUM. 



The demonstration of encapsulated pneumococci in smears of 

 exudates by these formalin methods is thus comparatively simple. 

 The demonstration of encapsulated pneumococci in smears of 

 sputum, however, has proved in my experience another problem. 

 With fresh sputum containing exudate coughed up from the lung 

 good results may often be secured, especially if the more purulent 

 portions of the sputum are selected for examination. But mttrc 

 frequently the organisms fail to show good capsule development, 

 and in the secretions from healthy persons the capsule is even less 

 marked. Apparently saliva and mucus offer poor conditions for 

 capsule formation, and, although the cells coming from the lung 

 exudates may retain their capsules for some time, the organisms 

 growing in the mouth secretions may lose their capsules. To demon- 



♦Thick smears require longer exposure than thin smears of material conlaininf few cells and liiile 



detritus. 



