1891.] MICROSCOPICAL JOURNAL. 19 



hydrate must be added to the i6 c.c. of mordant. The bacillus subtilis requires 

 28 to 30 drops and the bacillus of malignant oedema 36 to 37 drops of the sodium 

 solution. By first determining whether the germ in question is an alkali or acid 

 producing organism the necessary quantity of the acid or alkali solution to be 

 added to the mordant can easily be determined. 



(2) The Stai7ting Fluid. — The staining fluid here recommended is ordinary 

 neutral aniline water in which fuchsin crystals are dissolved to saturation. As 

 the aniline water is very nearly neutral a saturated solution of fuchsin in it is 

 sufficient. Better results may, possibly, be obtained by adding to this as much 

 of a I to 100, or still better i to 1,000 solution of sodium hydrate, as it is neces- 

 sary to bring it almost to a point of precipitation. 



Cover-glass preparations should be prepared from a pure culture (agar or gel- 

 atine) of the germ to be studied in the manner recommended in the first process 

 to eliminate all albuminous material. Sterilized hydrant water is preferred to 

 distilled water for diluting the culture. It is of the utmost importance that the 

 cover-glasses should be free from all grease or other impurities. In order to 

 clean the cover-glasses they are boiled in sulphuric acid, washed in distilled 

 water, immersed in ammoniated alcohol, from which they are dried on clean 

 linen. The film on the cover-glass is fixed by heat, but care must be taken not 

 to overheat the preparation. The desired amount of heat can be obtained by 

 holding the cover between the thumb and index finger over the flame, instead of 

 passing it through the flame by means of forceps. By this method over-heating 

 is avoided. After heating, the film on the cover-glass is covered with the mor- 

 dant and held over a flame until steam is given off". It is then removed from the 

 flame and after ^ to i minute the cover is rinsed in water, then in absolute al- 

 cohol, and again in water until the mordant is completely removed. The film is 

 then covered with a few drops of the staining fluid, and the preparation again 

 heated until the solution begins to vaporize. It is then removed from the flame, 

 and, after allowing the stain to act for about i minute, the cover is washed in a 

 stream of water. The preparation can be examined immediately in water, or 

 allowed to dry and mounted in balsam. 



By this method Prof. Loeffler demonstrated the flagella on a large 

 number of both saprophytic and pathogenic bacteria. On some of 

 these tufts of flagella are observed, while others possess only one fla- 

 gellum at each extremity. His paper is illustrated by eight photographs 

 of different bacteria studied by him, on each of which the flagella can 

 be plainly seen. 



Trenkttiaitn' s Alethod. IL — Loeffler's second process was soon fol- 

 lowed by Trenkmann's modification of his (Trenkmann's) method (9), 

 in which he introduces iodine water to be applied to the preparation 

 immediately after the mordant. This is based upon the same princi- 

 ple as the use of the iodine solution employed in Gram's method of 

 staining bacteria. This method is thought by its author to be superior 

 to the processes previously announced, because it is more simple in its 

 details and more reliable in its results. The method is as follows : 



(i) Tke JlfordafiL — This consists of a 2 per cent, solution of tannin, to which 

 J to :^ per cent, hydrochloric acid is added. 



(2) lod/f/e Water. — This is prepared by adding a small quantity of pure iodine 

 to a few cubic centimeters of distilled water and allowing it to stand, with fre- 

 quent shaking, for twenty-four hours. The iodine solution used in Gram's 

 method, or a drop of the tincture of iodine in 10 c.c. of distilled water, may be 

 used. 



(3) The Staining Fluid. — This is prepared by adding one drop of a satui-ated 

 alcoholic solution of gentian violet to 10 c.c. of distilled water. To this 40 c.c. 

 of aniline water are added. The solution remains clear. It stains the bacilli 

 and flagella very well, while the background is feebly, if at all, colored. 



The cover-glass preparations are prepared with the usual care to avoid greas.e 

 or dirt upon the glass and to secure the necessary dilution. Potato infusion is 

 used for such germs as will grow in it. Cultures in this medium are to be diluted 



