106 MICROSCOPIC METHODS. 



The Staining of Spores. If bacilli containing spores are 

 stained with a watery solution of a basic aniline dye, the spores 

 remain unstained. The spores either take up the stain less 

 readily than the protoplasm of the bacilli or they have a resist- 

 ing envelope which prevents the stain penetrating to the proto- 

 plasm. Like the tubercle bacilli, when once stained they retain 

 the colour with considerable tenacity. The following is the 

 simplest method for staining spores : - 



i. Stain cover-glass films as for tubercle bacilli. 



2'. Decolorise with i per cent sulphuric acid in water or with methylated 



spirit. This removes the stain from the bacilli. 



3. Wash in water. 



4. Stain with saturated watery methylene-blue for half a minute. 



5. Wash in water, dry, and mount in balsam. 



The result is that the spores are stained red, the protoplasm of the 

 bacilli blue. 



The spores of some organisms lose the stain more readily than those of 

 others, and for some, methylated spirit is a sufficiently strong decolorising 

 agent for use. If sulphuric acid stronger than i per cent is used, the spores of 

 many bacilli are readily decolorised. 



Moller*s Method. The following method, recommended by Mb'ller, is 

 much more satisfactory than the previous. Before being stained, the films are 

 placed in chloroform for 2 minutes, and then in a 5 per cent solution of chromic 

 acid for $-2 minutes, the preparation being well washed after each reagent. 

 Thereafter they are stained and decolorised as above. 



The Staining of Capsules. The two following methods may 

 be recommended in the case of capsulated bacteria : 



(a} Welches Method. This depends on the fact that in many cases trie 

 capsules can be fixed with glacial acetic acid. 



Films when fixed are placed in this acid for a few seconds. 



The superfluous acid is removed with filter paper and the preparation is 

 treated with gentian-violet in aniline-oil water repeatedly till all the acetic acid 

 is removed. 



Then wash with 0.85-2.0 per cent solution of sodium chloride and examine 

 in the same solution. 



Occasionally such preparations can be kept permanently in a balsam 

 mounting. 



The capsule appears as a pale violet halo around the deeply stained 

 bacterium. 



(b) Richard Muir^s Method. The following fixative and mordant is 

 made up. 



Saturated watery solution of corrosive sublimate . 2 

 Tannic acid solution 20 per cent ... . .2 

 Saturated solution of potash alum 5 



