STAINING OF FLAGELLA HI 



Klein's Method. See chapter on the Anthrax Bacillus. 



Staining of Flagella. Flagella, like spores, cannot be dyed by the 

 ordinary methods, and it is difficult to get a good flagellar stain. It 

 is necessary to prepare the cover-glasses in a special manner. They 

 must first be carefully washed successively in strong mineral acid, 

 water, alcohol, and ether, so that they are absolutely free from dirt, 

 grease, etc. The steps in the preparation of the clean cover-glasses 

 are then as follows: 



1. Place six clean cover-glasses in a row, and with the platinum 

 loop place a small drop of water on each. 



2. Inoculate the drop on cover-glass No. 1 three times from the 

 margin of the growth of a young agar culture, about eighteen hours 

 old, and not over twenty-four hours old. Mix up the growth well 

 with the water to make a uniform emulsion. 



3. Now inoculate drop on cover-glass No. 2 three times from 

 emulsion No. 1 (on cover-glass No. 1), and having made a uniform 

 emulsion on No. 2. 



4. Inoculate No. 3 three times from No. 2, and so on until all of 

 the six drops have been inoculated. 



5. Allow the six cover-glasses to become air dry, then fix in the 

 following manner: Do not pick up cover-glasses with forceps but 

 hold in the right hand between thumb and index finger, and while 

 in this position fix by passing three times through a flame. In this 

 manner the temperature sense of the worker will prevent an over- 

 heating of the cover-glasses and a burning of the delicate flagella. 



Treat and stain all six cover-glasses by one of the following methods, 

 and if successful, several good preparations are generally obtained. 



Loeffler's Method. 1. Apply to fixed cover-glass the following 

 mordant :* 



20 per cent, watery solution of tannic acid, prepared by heating. . 100 c.c. 



Watery solution of sulphate of iron, saturated in cold 50 c.c. 



Saturated alcoholic solution of fuchsin 10 c.c. 



Use the mordant, moderately heated, for one-half to one minute. 



2. Remove the mordant and wash well in water. 



3. Wash in alcohol. 



4. Stain with a warm anilin-water gentian-violet solution to which 

 has been added a trace of a very dilute caustic soda solution. 



5. Wash in water, dry between filter paper, and mount in Canada 

 balsam. 



Loeffler's method for staining flagella was the first one used and 

 published. It is a difficult one on account of the varying amounts 

 of alkali which must be added to the gentian stain. The following 

 method furnishes better and more uniform results : 



1 The word mordant means a preparation which will so act upon a substance that the latter 

 will take a stain more easily and hold it more firmly for instance, tannic acid or sulphate of 

 iron are used as mordants in the dyeing of wool in technical establishments. 



