METHODS OF CULTURE AND OF EXAMINATION. 109 



to the action of five per cent, chromic acid, is again rinsed 

 in water, and is stained for two minutes in steaming con- 

 centrated carbol-fuchsin solution. The preparation is then 

 treated for a short time with five per cent, sulphuric acid 

 (being passed through once or at most twice), is thoroughly 

 rinsed in water, is counterstained quite deeply with 

 methylene-blue or malachite-green, is again rinsed, and is 

 dried and mounted in Canada balsam. The spores appear 

 deep red, the bodies of the bacteria blue or green. 



Staining of Flagella (Loffler). In staining flagella a 

 mordant is first used : Twenty grams of tannic acid are 

 dissolved in 80 cu. cm. of hot water, and 50 cu. cm. of a 

 saturated watery solution of ferrous sulphate that has stood 

 in the cold for twenty-four hours with an excess of ferric 

 sulphate are added, and finally 10 cu. cm. of a concentrated 

 alcoholic solution of fuchsin. This fuchsin-solution may 

 be advantageously permitted to stand exposed to air for 

 several weeks ; it stains the better the older it is. 



The specimens themselves must be prepared with cover- 

 slips most carefully cleansed, and in such a way that an 

 extremely thin layer is spread upon the cover-slip with 

 little rubbing so much of the material as will adhere to 

 the tip of a platinum needle must yet be largely diluted. 

 Quite young agar-cultures, from fourteen to eighteen hours 

 old, and at the most twenty-four hours, are employed, as 

 the flagella can not be stained in older cultures. After the 

 preparations have been thoroughly dried in the air, the mor- 

 dant is dropped upon them, and its action is permitted to 

 continue for a minute. It is then entirely washed off, the 

 cover-glass is dried, and upon it is filtered aniline-water 

 gentian-violet or methyl-violet or fuchsin-solution. Heat 

 is now applied carefully by means of a small flame, until 

 vapors of steam arise. Then, after a minute, the prepara- 

 tion is washed in water, is dried, and is mounted. This 

 modification by Gunther of Loffler's method of staining 

 flagella yields quite satisfactory results. By another method 

 the preparations, dried in air, are exposed for from six to 

 twelve hours to the action of a two per cent, solution of tannic 

 acid and one-half per cent, hydrochloric acid, are rinsed in 

 water, immersed for an hour in a watery solution of iodin, 

 again washed in water, and then stained for half an hour in 

 aniline-water gentian-violet. The specimen should then be 

 mounted, not in Canada balsam, but in solution of iodin. 



