STAINING METHODS iv«-19 



methods which follow give satisfactory results. The student is 

 specially urged, however, not to pronounce any organism capsulated, 

 as a result of any of these staining procedures, until he has carefully 

 compared it with other organisms generally recognized as having 

 capsules. 



LEIFSON METHOD 



Leifson (1930) 

 This method is described in detail above (p. 17) and does not need 

 to be repeated here. The special methods of handling slides and 

 cultures, outlined for flagella staining, do not need to be observed, 

 but the following is essential: 



After step 3; 



4. Stain 5-10 min., without heating, in borax methylene blue 



(methylene blue, 90% dye content, 0.1 g.; borax 1 g. ; distilled 

 water 100 ml.). 



5. Wash in tap water. 



6. Dry and examine. 

 Results: capsules red; cells, blue, 



Anthony's method 

 WITH Tyler's modification 

 Anthony (1931) 

 Original formula Tyler's modification^ 



Crystal violet (85% dye content) 1 g. Crystal violet (85% dye con- 

 Distilled water 100 ml. tent) 0.1 g. 



Glacial acetic acid 0.25 ml. 



Distilled water 100 ml. 



Staining schedule: 



1. Prepare smears and dry them in the air. 



2. Stain 2 min. in the above aqueous crystal violet; or according 



to Tyler 4-7 min. in the above acetic crystal violet. 



3. Wash with 20% aqueous CuS04-5H20. 



4. Blot dry, and examine. 



Results: capsules, blue violet; cells, dark blue. 



hiss's method 

 Hiss (1905) 



ORIGINAL STATEMENT OF FORMULA EMENDED FORMULA 



Sat. ale. basic fuchsin or gentian Basic fuchsin (90% dye con- 

 violet 5-10 ml. tent) 0.15-0.3 g. 



Water to make 100 ml. Distilled water 100 ml. 



or 

 Crystal violet (85% dye con- 

 tent) 0.05-0.1 g. 



Distilled water 100 ml. 



Staining schedule: 



1. Grow organisms in ascitic fluid or serum medium, or mix with 



drop of serum and prepare smears from this mixture. 



2. Dry smears in the air and fix with heat. 



3. Stain with one of the above solutions a few seconds by gently 



heating" until steam rises. 



'See Park and Williams (1933), p. 84. 



