308 AUGUSTUS WADSWORTH 



manent fixation or coagulation of the capsule. Obviously, success 

 in securing permanent fixation depended upon the composition 

 of the capsule. In the absence of data to the contrary, the results 

 of Guarnieri's chemical studies, and the fact that capsule preser- 

 vation or formation is largely determined by the presence of albu- 

 min, suggested with reasonable probability an albuminous com- 

 position. Attention was therefore directed to the study of the 

 action of bichloride of mercury and formalin, which not only pre- 

 cipitate albumins, but enter into chemical combination with them. 



Bichloride methods. Bichloride of mercury was tested in both 

 aqueous and alcoholic solutions. Alcoholic solutions proved more 

 efficient. Dried smears, fixed in saturated bichloride- alcohol for 

 a few minutes and washed in water, may be stained in aqueous- 

 gentian-violet, and mounted in the usual way, or they may be decol- 

 orized by retreatment in the bichloride- alcohol for a few seconds 

 before washing in water and drying. In these preparations the 

 encapsulated cells are often very sharply demonstrated in a per- 

 fectly clear field. With the Gram differential procedures the cap- 

 sules fixed in bichloride- alcohol rarely* withstand the osmosis 

 required to bring the specimen to a balsam mount, and as applied 

 to the study of tissues in section it was a complete failure. 



Bichloride-alcohol thus proved valuable in coagulating and 

 partially fixing the capsules, and also in clearing the field; but, 

 aside from this, it offered little practical advantage over the simple 

 staining procedures of other observers. 



Formalin methods. The fixation obtained with strong solutions 

 of formalin proved more stable. It was also found that, when 

 desirable, formalin may be effectively used as a wash after simple 

 staining with aqueous-gentian-violet, or 5 to 10 per cent may be 

 added to the stain to shorten the procedure. Although useful and 

 simple, these methods offer no special advantage over the definite 

 fixation secured by the stronger (40 per cent) solutions of formalin, f 



*Excellent balsam preparations for demonstration purposes were readily secured, but for routine 

 work the results were uncertain. With other species of encapsulated bacteria this may prove more reliable' 

 for in sections of tissues hardened in bichloride Wright and Mallory" demonstrated capsules on a Gram 

 negative organism resembling the Friedlander bacillus. 



fThe addition of alkali to the formalin interfered seriously with the fixation, but the addition of 

 acetic acid i to 2 per cent in some instances proved advantageous, especially in securing penetration of the 

 tissues, and also it was thought, by counteracting the alkaline reaction of the body tissues. 



