METHODS OF STAINING ENCAPSULATED PNEUMOCOCCI 311 



protects many of the bacterial cells from the action of the formalin, 

 and the capsules of these cells are not properly fixed, and are thus 

 dissolved by subsequent treatment. These difficulties are often 

 encountered in precise histological work, and are largely eliminated 

 by injecting the tissue with the hardening fluid, or, when this is 

 not feasible, by cutting the material into small bits or thin slices. 

 The lungs are easily injected through the trachea and are quickly 

 hardened in the distended condition. The lesions may then be 

 cut into small pieces and the fixation completed in fresh, strong 

 formalin, the whole process taking from three to five hours. After 

 alcohol dehydration the material is imbedded in celloidin and cut 

 in the usual way. It is important to have thin sections* for micro- 

 scopical examination; otherwise the encapsulated bacteria lying in 

 the exudate among the cells cannot be easily distinguished. After 

 alcohol dehydration these sections may be fixed on the slide by 

 partially dissolving the celloidin with ether, or alcohol and ether, 

 equal parts. A few seconds in alcohol will then harden the thin 

 film of celloidin covering the section, and after washing in water 

 the preparation is ready to be stained. A variety of staining 

 procedures may be employed;! but the Gram method, by virtue 

 of its differentiation, has proved the most useful. Anilin-gentian- 

 violet stain for two to five minutes, iodine solution one to two min- 

 utes, alcohol decolorization, eosin-alcohol counterstain, eosin-oil 

 of origanum to clear, and balsam mounting, are the several steps 

 of the technic. This has rarely failed to give good results, but occa- 

 sionally the pneumococci, after prolonged exposure, or after expo- 

 sure to weak or impure formalin solutions, decolorize partially in 

 the alcohol. This technical error, when it occurred, was rectified 

 by using a 5 per cent bichloride-alcohol for the decolorization fol- 

 lowing the iodine solution, so that the material could be studied, 

 though not so accurately as when the cells were properly fixed. By 



*Thin sections are readily secured by painting the surface of the block with dilute celloidin-ether 

 between each stroke of the knife. 



t A combination of the Nicolle and Van Gieson methods of staining has given some excellent prepa - 

 rations. After staining in Loeffler alkaline methylene blue, the dye is fixed in the bacterial cells by 10 

 per cent aqueous tannic acid. This is followed by a partial decolorization in alcohol, counterstaining 

 in van Gieson 's strong fuchsin-picric acid solution (Freeborn, Proc. N. Y. Path. Soc., 1893, p. 73), dif- 

 ferentiation in picric acid alcohol, clearing in picric acid-oil of origanum, and mounting in balsam. The 

 Gram stain may be substituted for the methylene blue and tannic acid stain, but it is apt to decolorize 

 in the acid alcohol. The picric acid cellular stain is more easily studied than the eosin stain. 



