STAINING METHODS 29 



After step 3 : 



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

 blue, 90 per cent 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 per cent aqueous CuS04-5H20. 



4. Blot dry, and examine. 



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



Hisses 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. 



4. Wash off with 20 per cent aqueous CuS04-5H«0. 



5. Blot dry, and examine. 



Results: Capsules, faint blue; cells, dark purple. 



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



