Methods of Staining Encapsulated Pnei-mococci 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 difticulties are often 

 encountered in precise histological work, and are largely eliminated 

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

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

 The lungs are easily injected through the trachea and arc 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, .\fter 

 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 ;t but the Gram method, by virtue 

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

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

 utes, alcohol decolorization, eosin-alcohol counterstain, cosin-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 rectititd 

 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 fi.xed. By 



*Thin sections are readily secured by paiating the surface of (he t)li>rk with dilute cclloidin-rlber 

 between each stroke of the knife. 



tA combination of the Nicollc and Van Gieson methods of slainiiiK hiLS kImii some cxorllcnt prrra ' 

 rations. After staining in Locfflcr alkaline methylene blue, the dye Ls lixcd in the bacterial rrlLs by lo 

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

 in van Gieson 's strong fuchsin-picric acid solution (Freelxjrn, Proc. N. Y. Path. Soc. iRoji, p. r.t>, dif- 

 ferentiation in picric acid alcohol, clearing in picric acidnnl of origanum, and mounting in l>aUam. The 

 Gram stain may be suljstituted for the methylene blue and tannic acid slain, but it i< ap< to decoloricc 

 in the acid alcohol. The picric acid cellular slain is more easily studied than the eo>in slain. 



