123 



bine, methyl violet, gentian violet, fuchsin, vesuvin or bismarck brown* 

 The time which is required for the completion of the staining process will 

 vary according to the nature of the dyes. Koughly speaking, 10-15 

 minutes suffices in every case except that of vesuvin or bismarck brown, on 

 which the cover glasses should be left for at least an hour. When the stain- 

 ing process is completed the glasses should be washed with distilled water, 

 and, if the stain is too deep, in a 5-I per cent, solution of acetic acid, then 

 allowed to dry, and mounted in Canada balsam. The best form of balsam 

 is balsam in Xylol, in which the dye does not fade. 



If time is an object, the drying of the cover glasses may be effected by 

 pressing them between folds of blotting paper and then brushing then' sur- 

 faces with a camel's hair brush. 



It will be found that, in solutions of anilin dyes which have been made 

 up some time, various fungi, torula and bacteria, are prone to develop, and 

 their presence, especially on the surface of stained sections, might give rise 

 to an erroneous observation. Their development may be prevented by the 

 addition to the solutions of some antiseptic, e.g., crystals of camphor. In 

 every case the solution should be filtered before it is used. 



For photo-micrographic purposes bacteria are best stained w r ith vesuvin 

 or bismarck brown. 



In the investigation of bacteria in the tissues and organs of the animal 

 body, sections must be made after the specimen has been hardened by one of 

 the numerous hardening processes. A great number of staining reagents 

 have been recommended, of which I shall describe only three, which I find 

 most useful for general purposes. 



1. Bismarck brown. — The sections are allowed to remain in a saturated 

 watery solution of the dye for about one hour, washed in distilled water, 

 and then in a g-1 per cent, solution of acetic acid, dehydrated in absolute 

 alcohol, clarified in oil of cloves or pure anilin, and mounted in Canada 

 balsam. 



2. Alkaline blue. — The formula for the making of this solution is as 

 follows : — To 100 parts of a solution of caustic potash (1-10,000) in distilled 

 water, add 30 parts of a saturated alcoholic solution of methylen blue. 

 The sections should remain in this fluid for about an hour, are then washed 

 in distilled water, and afterwards in a ^-1 per cent, solution of acetic acid, 

 dehydrated in alcohol, clarified in oil of cedar, and mounted in Canada 

 balsam. If they are clarified in oil of cloves it will frequently happen that 

 much of the dye rans out of them, whereas the colour is retained when they 

 are left in the oil of cedar, even for a long time. 



3. Gravis Method. — In this staining process three solutions are used and 

 are as follows : — 



Solution A. — Saturated alcoholic solution of gentian violet, 11 parts. 

 Saturated watery solution of anilin, 100 parts. 



[The anilin solution is made by shaking up pure anilin with distilled 

 water, until no more is dissolved, and filtering.] 



* The best dyes, as far as I know, are those which are supplied by Griibler, of Leipzig, 

 for whom Mr. Baker, 244, fcligh Holborn, is the London agent. 



