METHODS OF STAINING ENCAPSULATED PNEUMOCOCCI 307 



steamed in anilin-gentian-violet and in the iodine solution. After 

 the usual alcohol decolorization and a few seconds' exposure to 

 a mixture of alcohol, 4 parts, ether, 6 parts, the smear is counter- 

 stained, first in aqueous eosin, then in LoefHer's methylene blue. 

 This is followed by slight decolorization in 95 per cent alcohol, 

 dehydration in absolute alcohol, xylol, and balsam. 



Buerger 2 fixes the smears of encapsulated pneumococci in Miil- 

 ler's fluid saturated with bichloride, for one-half minute,* and 

 washes in water. After one minute's exposure to an alcoholic 

 solution of iodine (U. S. P.), and washing in alcohol, the smear 

 is stained in the usual way by the Gram method anilin-gentian- 

 violet, Lugol's solution, alcohol decolorization and washed in 

 water. After counterstaining in aqueous fuchsin, the preparations 

 are washed and examined in 2 per cent salt solution. 



With these methods of Smith and of Buerger the bacterial cells, 

 under favorable conditions, may be stained by the Gram stain and 

 demonstrated with capsules. The procedures, however, are com- 

 plicated, not as accurate and reliable as the simple capsule stains, 

 or give temporary mounts; and none of the methods thus far con- 

 sidered are applicable to the demonstration of encapsulated organisms 

 in sections of diseased tissues. 



THE NEW METHODS. 



In the attempt to secure methods by which the differential stain- 

 ing of encapsulated organisms in tissues as well as films could be 

 easily accomplished with a reasonable degree of certainty, a great 

 variety of procedures were tried without success before satisfactory 

 results were finally obtained. Suffice it to note that all the efforts 

 to keep the capsules from dissolving in the course of hardening, 

 imbedding, and staining, by the addition of various chemicals to 

 the solutions, so successfully adopted in the simple methods of 

 Guarnieri and of Hiss for films, failed to give reliable results. Excep- 

 tionally, a few encapsulated organisms were stained in sections 

 treated in this fashion, but the result of these procedures proved to be 

 wholly beyond control, and was thus of no practical value. It 

 was therefore evident, if the Gram stain was to be used, that the 

 solution of the problem depended primarily upon securing a per- 



* In my experience with this method longer exposures are advisable; in fact, essential. 



