110 MICROSCOPIC METHODS 



Leprosy bacilli are stained in the same way, but are rather 

 more easily decolorised than tubercle bacilli, and it is better 

 to use only 5 per cent, sulphuric acid in decolorising. 



In the case of specimens stained either by the original Ziehl- 

 Neelsen method, or by Fraenkel's modification, the tubercle or 

 leprosy bacilli ought to be bright red, and the tissue blue or 

 brown, according to the contrast stain used. Other bacteria 

 which may be present are also coloured with the contrast stain. 



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 resisting 

 envelope which prevents the stain from 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 : 



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



2. Decolorise with 1 per cent, sulphuric acid in water or with methy- 

 lated 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 staining some, methylated spirit is a sufficiently 

 strong decolorising agent to use. If sulphuric acid stronger than 1 per 

 cent, is used, the spores of many bacilli are readily decolorised. 



Hollers Method. The following method, recommended by Moller, is 

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

 films are placed in chloroform for two minutes, and then in a 5 per cent, 

 solution of chromic acid for \-1 minutes, the preparation being well 

 washed after each reagent. Thereafter they are stained and decolorised 

 as above. 



The Staining of Capsules. The following methods may be 

 recommended in the case of capsulated bacteria : 



(a} Welch's Method. This depends on the fact that in many cases 

 the capsules can be fixed with glacial acetic acid. 



Films when still wet 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 1-2 per cent, solution of sodium chloride, and examine 

 in the same solution. 



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

 bacterium. 



(b) Hiss's Method. The staining solution consists of 1 part of a 

 saturated alcoholic solution of fuchsin or gentian-violet and 19 parts of 



