STAINING THE FLAGELLA 63 



water suspension prepared at the last exercise and stain for 

 flagella. Use Johnston's and Mack's modified method, with 

 Loeffler's mordant and stain. The films should not be heated 

 either before or during the staining process, but if it does not 

 succeed, Loeffler's process may be tried. 



95. Johnston's and Mack's modified method, (a) Preparation 

 of films. Make a culture of the organism to be stained on slant 

 agar and incubate for from 18 to 24 hours. Prepare a tube with 

 6 to 8 cc. of sterile water, and keep it in the incubator until 

 it is of the same temperature. With a sterile platinum loop 

 scrape away some of the growth from the agar surface, using 

 care not to remove any of the agar, and rinse it off carefully 

 in the tube of water previously prepared for this purpose. 

 There should be enough to impart to the water a faint cloudi- 

 ness. This should be done in a warm room and with con- 

 siderable care. Replace this tube in the incubator and the 

 bacilli will distribute themselves quite evenly through the water, 

 but any clumps or masses settle to the bottom. The tube is 

 left in the incubator for from 2 to 3 days before preparing 

 the films. Place a tray of properly prepared cover glasses in 

 the incubator to warm ; then, still in the warm room, with a 

 platinum loop put a drop or two from the tube on each cover 

 ( 4). Replace the tray in the incubator until the water has 

 evaporated, when the films are ready to stain. The films 

 require no fixing other than by the mordant. The small 

 amount of organic matter in films prepared in this way gives 

 but little background when stained. 



(b) Mordants and stains. Different mordants and staining 

 solutions may be used. Those of Loeffler or Pitfield give the 

 most uniform and satisfactory results. 



LOEFFLER'S MORDANT 



Twenty per cent aqueous solution tannic acid . . 10 cc. 

 Saturated aqueous solution iron sulphate . . . 5 cc. 

 Saturated alcoholic solution basic fuchsin . . . I cc. 



Mix, let stand two or three hours, and filter. 



