MICROSCOPIC EXAMINATION 133 



purpose. There should be enough to impart to the water a 

 faint cloudiness. This should be done in a warm room and 

 with considerable 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 pre- 

 paring the films. Place a tray of properly prepared cover- 

 glasses in the incubator to warm them ; then, still in the warm 

 room, with a platinum loop put a drop or two from the tube 

 on each cover. 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. 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 1 cc. 



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



Tannic acid solution should be freshly prepared, but the 

 iron sulphate solution is better if it stands until it begins to 

 turn brownish by oxidation, but it should not be too old. If 

 when this mordant is used it gives a precipitate, filter again. 

 When properly prepared it should have much the same color 

 as a solution of hematoxylin. 



LOEFFLER 'S STAIN (zlEHL's CARBOL FUCHSIN) 



Saturated alcoholic solution basic fuchsin 31 cc. 



Five per cent carbolic acid 20 cc. 



If not clear, add fuchsin solution drop by drop until it 

 clears, then filter. 



Loeffler 's Method. 1 Place 2 loopfuls of sterilized dis- 

 tilled water or normal salt solution on the center of the cover- 



1 Loeffler. Centralbl. f. Bakteriologie, Bd. VI (1889) p. 209. 



