STAINING 2 1 



MORDANT FOR CAPSULE STAINING (MUIR'S METHOD) 



Saturated aqueous solution mercuric chloride, 2 cc. 



25 per cent, aqueous solution tannic acid, 2 cc. * 



Saturated aqueous solution potash alum, 5 cc. 



Spores do not stain by the methods generally employed for bacteria. 



When a bacillus containing an endospore is stained with eosin, fuchsin or 

 methylene blue, the bacillus red or blue, the spore colorless, by contrast it is 

 apparent. 



To stain the spores and maintain the contrast, use the following method: 



METHOD FOR STAINING SPORES 



1. Stain with carbol fuchsin for 15 minutes. 



2. Wash with i per cent, aqueous solution of sulphuric acid until bleaching 

 is noticed. 



3. Wash in water. 



4. Apply methylene blue for 30 seconds. 



5. Wash in water, dry and examine. 

 Spores will be red and bacteria blue. 



BLOOD-CELL STAINING 



It is occasionally necessary to stain blood, serous fluids or exudates con- 

 taining bacteria by a method that will show and differentiate the white blood 

 cells. For this purpose the preparations are made and stained by the methods 

 commonly used in the examination of blood cells : 



A drop of the blood or serous fluid is placed on the center of a square cover 

 glass and a second cover glass dropped diagonally on it. As soon as the fluid 

 ceases to spread the glasses are slid apart; or, a drop of the fluid is placed on a 

 slide, near one end, and is drawn along the slide with the edge of a second slide 

 held at an angle of about 45. By whichever method the spread is made, it 

 must be done rapidly and the film must be thin and even. It is gently heated 

 until dry and then stained with Romanowsky's, Giemsa's, or any of the modifica- 

 tions of these. The preparation is covered with the stain, from i to 2 minutes 

 later distilled water is added to the stain until a metallic scum appears, usually 

 it requires as much water as stain to effect this: 5 to 10 minutes later the slide 

 or cover glass is washed with water, dried and examined. 



Saturated alcoholic solutions of fuchsin, methylene blue and gentian violet 

 keep until used. It is a great convenience to have such solutions in stock. 

 They should be stored in dark glass-stoppered bottles. Absolute alcohol is 

 used in their preparation and the addition of 10 Gm. of powdered stain to each 

 100 cc. of alcohol is more than sufficient to saturate it. When made saturated, 

 solutions are thoroughly shaken and then allowed to sediment for at least 

 24 hours before use. Care must afterwards be taken when pouring that no 

 undissolved particles get into staining fluids. These latter must all be per- 

 fectly clear and free of undissolved particles. When procuring stains it is 

 best to specify "Grubler's." 



