STAINING OF FLA GEL LA. 115 



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 de- 

 colorising agent for use. If sulphuric acid stronger than I per cent is 

 used the spores of many bacilli are readily decolorised. 



Moller recommends that before being stained, the films should be 

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

 of chromic acid for ^-2 minutes. This procedure has an advantage in 

 some cases, though in many it is unnecessary. 



The Staining of Capsules. In many capsulated bacteria the 

 capsule can be fixed by means of glacial acetic acid. Films are made 

 and while wet are placed in this acid for a few seconds. The super- 

 fluity is removed with filter-paper, and the preparation treated with 

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

 removed. It is then washed with a 1-2 per cent solution of sodium 

 chloride and examined in the same solution. 



The Staining of Flagella, The staining of the flagella 

 of bacteria is the most difficult of all bacteriological 

 procedures, and it requires considerable practice to ensure 

 that good results shall be obtained. Many methods have 

 been introduced, of which the three following are the most 

 satisfactory. 



Preparation of Films. In all the methods of staining 

 flagella, young cultures on agar should be used, say a 

 culture incubated for from twelve to eighteen hours at 37 C. 

 A very small portion of the growth is taken on the point 

 of a platinum needle and carefully mixed in a little water 

 in a watch glass ; the amount should be such as to produce 

 scarcely any turbidity in the water. A film is then made 

 by placing a drop on a clean cover-glass and carefully 

 spreading it out with the needle. It is allowed to dry 

 in the air and is then passed twice or thrice through a 

 flame, care being taken not to over-heat it. The cover- 

 glasses used should always be cleaned in the mixture of 

 sulphuric acid and potassium bichromate described on 

 page 95- 



