102 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 penetrating to the protoplasm. 

 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 methyl- 

 ated 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 1 per cent 

 is used the spores of many bacilli are readily decolorised. 



Hollers Method. The following method, recommended by Holier, 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) 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. 



(6) Richard Muirs Method (as recently modified). 



1. The film containing the bacteria must be very thin. It is dried 

 and stained in filtered carbol-iuchsin for half a minute, the preparation 

 being gently heated. 



