STAINING OF FLAGELLA 103 



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. 

 The preparation has a pale reddish appearance. 



6. Wash well in water. 



7. Counterstain with watery solution of ordinary methylene-blue for 

 half a minute. 



8. Dehydrate in alcohol, clear in xylol, and mount in balsam. 



The bacteria are a deep crimson and the capsules of a blue tint. The 

 capsules of bacteria in cultures may sometimes be demonstrated by this 

 method. 



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 two 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 ten 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 87. 



1. Pitfield's Method as modified by Richard Muir. 

 Prepare the following solutions : 

 A. The Mordant. 



Tannic acid, 10 per cent watery solution, filtered . 10 c.c. 



Corrosive sublimate, saturated watery solution 



Alum, saturated watery solution . . ; 5 ,, 



Carbol-fuchsin (vide p. 101) ..... 5 ,, 



Mix thoroughly. A precipitate forms, which must be allowed to 

 deposit, either by centrifugalising or simply by allowing to stand. 

 Remove the clear fluid with a pipette and transfer to a clean bottle. 

 The mordant keeps well for one or two weeks. 



B. The Stain. 



Alum, saturated watery solution . . . . 10 c.c. 

 Gentian- violet, saturated alcoholic solution . . 2 ,, 



