1891.] MICROSCOPICAL JOURNAL. 17 



The Mordant. — To lo c.c. of a 20 per cent, solution of tannin a sufficient quan- 

 tity of an aqueous solution of the sulphate of iron is added to give to the fluid a 

 dark violet color. To this is added 3 to 4 c.c. of a logwood decoction (i part wood, 

 8 parts water). The liquid will now have a blackish violet color. Care must be 

 taken not to add an excess of the logwood as it would interfere with the staining 

 process. When prepared the mordant should be kept in a well-stoppered bottle, 

 and in order to preserve it, 4 to 5 c.c. of a 5 per cent, solution of carbolic acid maj 

 be added. 



The Staining Fluid. — To 100 c.c. of a saturated watery solution of aniline 

 oil is added i c.c. of a i per cent, solution of sodium hydrate to give to it a 

 slightly alkaline reaction. This alkaline aniline water is poured into a flask in 

 which has been placed 4 to 5 grams of powdered methylene blue, methyl violet, 

 or fuchsin. The flask is vigorously shaken and closed with a tightly-fitting rubber 

 cork. This solution can be kept for a considerable length of time. It must 

 always be filtered before using. 



The material to be examined must form a very thin layer upon the cover-glass. 

 If the germ-containing substance is albuminous, a very small quantity of it is 

 added to a drop of sterile distilled water on a cover-glass and thoroughly mixed 

 with it; a small quantity of this is conveyed to a second cover-glass and treated 

 in a like manner; and again from the second a third preparation is made. By 

 this treatment the albuminous substance is sufficiently diluted, and the microbes 

 are isolated in a watery medium. The preparations are allowed to dry in the 

 air, after which the films are fixed by passing the covers, film upward, through a 

 flame in the usual manner. 



A few drops of the mordant are poured over the film and the cover-glass held 

 over a flame until the fluid begins to evaporate. It -is then removed from the 

 action of the flame, and after a very short time the mordant is washed oft" in a 

 stream of distilled water. Care should be taken to remove all traces of the mor- 

 dant from the edges of the cover-glass, as it would form, if present, a very 

 troublesome precipitate with the staining fluid. The next step is to filter a few 

 drops of the staining fluid upon the film. This is allowed to act for a brief 

 time, when the cover-glass is held over a flame and gently heated. Better results 

 are obtained if the staining fluid is only slightly warmed and allowed to act for 

 a longer period. As soon as the film becomes darkened (a blackish red if fuchsin 

 is used) the stain is washed off" in distilled water. The preparation is now ready 

 for microscopical examination. This can be made at once in a drop of distilled 

 water, or the preparation allowed to dry and mounted in balsam. 



The microbes with their flagella should be deeply stained, resting 

 upon a colorless background if the germs are in a purely watery me- 

 dium, but if albumen is present they are surrounded by a uniformly 

 feebly stained medium, the intensity of which depends upon the quan- 

 tity of albumen present. With this process Loeffler succeeded in 

 demonstrating the flagella on a large number of motile bacilli, spirilla, 

 and upon the motile micrococcus recently described by Ali Cohen. 



Tre7tk-rnann''s Method. I. — Soon after the publication of Loeffler's 

 method. Dr. Trenkmann (8) announced his process of staining flagella, 

 which in principle is similar to Loeffler's, but differs from it in the com- 

 position of the mordant and staining fluid used, and in several of the 

 lesser important details in its application. It is as follows : 



The cover-glass preparations are prepared in the same manner as Loeffler pre- 

 pared his. After they have been dried in the air they are placed (without pass- 

 ing them through a flame) in a fluid composed of i per cent, tannin and h per 

 cent, hydrochloric acid and allowed to remain in it for from 2 to 12 hours. They 

 are then washed in water and transferred to the staining fluid. This consists 

 of a weak solution of dahlia (2 drops of a concentrated alcoholic solution of dah- 

 lia to 20 drops of water). Fuchsin, gentian violet, methylene blue, methyl 

 green, Vesuvian, or Victoria blue maybe used. The preparations remain in the 

 staining fluid for from i to 4 hours, when they are i-insed and examined. The 

 flagella are stained with any of the aniline dyes, but more satisfactory results are 



