STAINING OF CAPSULES 107 



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. 

 ' Mailer's Method. — The following method, recommended by Mbller, 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 J-2 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 aeid 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) Hiss's Method. — The staining solution consists of 1 part of a 

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

 distilled water. A few drops of the stain are placed on a film, previously 

 dried and fixed by heat, and the preparation is steamed for a few seconds over 

 a flame. The staining solution is washed off with a 20 per cent, solution 

 of copper sulphate, the preparation (without being washed in water) is dried 

 between filter-papers, and when thoroughly dry is mounted in balsam. The 

 capsules of pneumococci in exudates or growing in a fluid serum medium 

 can be readily demonstrated by this method ; in the case of solid cultures, 

 films should be made without any diluent, or a drop of fluid serum should 

 be used. The method is easily applied, and gives excellent results. 



(c) Richard Muir's Method (modified). 



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

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

 being gently heated. 



2. Wash slightly with spirit and then well in water. 



3. Place in following mordant for a few seconds : — 



Saturated solution of corrosive sublimate . . 2 parts. 



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



Saturated solution of potash alum . . . 5 



4. Wash well in water. 



5. Treat with methylated spirit for about a minute. 



