METHODS OF STAINING ENCAPSULATED PNEUMOCOCCI 305 



were stained with analin-gentian-violet, then washed and differ- 

 entiated in 2 per cent aqueous sodium chloride, rewashed very 

 quickly in water, dried, and mounted in balsam. Similar methods 

 have since been recommended. Thus, Welch 16 adopted Guar- 

 nieri's method of staining, but mounted the specimen in the salt 

 solution, and suggested a preliminary treatment with glacial acetic 

 acid on the ground that the capsule was composed of mucin.* 



Buerger 2 also adopted Guarnieri's method, but recommended 

 a preliminary fixation of the capsule, first in a solution of chromic 

 acid and bichloride of mercury, then in an alcoholic solution of 

 iodine (U. S. P.). With Gram's method of staining this fixation 

 is essential, but in the simple procedures the advantage of it is less 

 apparent, for practically the same results are secured with Guar- 

 nieri's less complicated method. 



Hiss, 6 however, by substituting the ordinary aqueous-gentian- 

 violet stain for the unstable anilin-gentian- violet, and by using 0.25 

 per cent potassium carbonate solution, which to some extent clears 

 the field, simplified and improved materially the technic of capsule 

 staining. Finally, by using a 20 per cent copper sulphate wash 

 instead of the potassium carbonate he found that the specimen 

 could be dried and mounted in balsam. 



Formerly capsules were found only in the exudates of infected 

 animals, but now they are readily demonstrated in organisms grow- 

 ing in artificial media. Boni found that when cultures of pneu- 

 mococci are smeared in egg albumin, the capsules are easily stained. 

 Hiss secured similar results with blood serum, and by means of 

 his more reliable methods of staining was able to determine more 

 fully the importance of utilizing this principle in the morphological 

 study of the pneumococcus. 



By virtue of these modern procedures it is now a comparatively 

 simple matter to demonstrate capsules on these organisms. It 

 is no longer a question of how encapsulated pneumococci may be 

 stained, but of how they may be most simply and reliably stained. 



*Welch does not state the reasons for this belief; it is therefore difficult to refute the positive, though 

 incomplete, observations of Guarnieri. Mucin is a glycoproteid and reacts to Millon's reagent, but the 

 solubilities differ, and capsules are more constantly noted in albuminous media than in the presence of 

 mucin. In fact, the mucous secretions of the mouth are not a particularly favorable environment for the 

 demonstration of capsules, whereas capsules are readily obtained in simple broth, if only the albumin 

 or even the peptones be sufficiently increased. 



