4 THE AMEEICAN MONTHLY [January, 



The importance of Loeffler's method in the acquisition of cm- knowl- 

 edge of the flagella of bacteria has prompted me to present it here, in 

 a form as condensed as possible, both for its own value and as a basis 

 for my subsequent remarks. The formulae for the preparation of the 

 mordant and staining tiuid. together with the details in their applica- 

 tion, are as follows : 



(i) The Mordant. — To lo cc. of a 20 per cent, aqueous solution ot tannin 5 

 cc. of a cold saturated solution of the sulphate of iron and i cc. of an aqueous or 

 alcoholic solution of fuchsin, methyl-violet, or " Wollschwarzlusung" are added. 

 The fuchsin is especially recommended. 



The foregoing solution is to be regarded as the standard or stock solution to 

 be used, and one which is successfully employed in staining the flagella of cer- 

 tain microbes, but for others the addition of an acid or alkali is necessary. Thus 

 for the comma bacillus it is necessary to add to the 16 cc. of mordant i to i drop 

 of a solution of sulphuric acid, equivalent to a i per cent, solution of sodium 

 hydrate. For the Typhoid bacillus i cc of a i per cent, solution of sodium hy- 

 drate must be added to th^ 16 cc. of mordant. By first determining whether the 

 germ in question is an alkali or acid producing organism, the necessary quantity 

 of the acid or alkaline solution to be added to the mordant can easily be deter- 

 mined by actual experiment. 



(2) The Staining Fluid. — The staining fluid consists of a saturated solution 

 of crystal fuchsinin the ordinary aniline water. As the aniline water is very 

 nearly neutral a saturated solution of fuchsin in it is sufficient. Better results 

 may possib^' be obtained by adding to this as much of a i to 1000 solution of 

 sodium hydrate as it is necessary to bring it almost to a point of precipitation. 



Cover-glass preparations should be made of the bacteria to be studied in such 

 a manner as to avoid all albuminous material. This is best accomplished by 

 transferring a very small quantity of the growth from an agar or gelatine cul- 

 ture to a drop of sterile water on a cover-glass and thoroughly mixing; a small 

 quantity of this is conveyed to a second cover-glass 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 bacteria are isolated in an 

 aqueous medium. The preparation is allowed to drj' in the air. Sterilized 

 hydrant water is preferred to distilled water for diluting the culture. It is of the 

 utmost importance that the cover-glass should be free from all impurities. The 

 film on the cover-glass is fixed by heat, but care must be taken not to overheat 

 the preparation. The desired temperature 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 overheating is avoided. After 

 heating, the film on the cover-glass is covered with the mordant and held over 

 a flame until steam is given otf. It is then removed, and after h to i minute the 

 cover is rinsed in water, then in absolute alcohol, and again in water, until the 

 mordant is completely removed. Care must be taken to remove all traces of the 

 mordant from the cover-glass, as it would form, if present, a very troublesome 

 precipitate with the staining fluid. The film is then covered with a few drops of 

 the staining solution 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 one minute, the cover is washed in a stream of water. The prepa- 

 ration can be examined immediately in water or allowed to dry and be mounted 

 in balsam. The bacteria, with theiV flagella, should be deeply stained, resting 

 upon a colorless background if they are distributed in a purely aqueous sub- 

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

 stained medium, the intensitv of which depends upon the quantity of albumen 

 present. 



By the use of this method the flagella have been stained on not only 

 a large number of saprophytic, but also on all of the known motile 

 pathogenic bacteria. Unfortunately, the results usually obtained by 

 this process are not satisfactory for the diftereutial purposes suggested in 

 a previous paragiaph. The difficulty is not in simply demonstrating 

 their presence, but in the inability to determine the number and arrange- 



