THE STAINING OF FLAGELLA. 107 



1. Films containing the bacteria are dried and tlien fixed in the above fluid 

 for two minutes. 



2. Wash in water, then in spirit, and again in water. 



3. Stain with carbol-fuchsin for 2-3 minutes, heating gently. 



4. Wash in water, place the film in the mordant for 2-3 minutes, and wash 

 again in water. 



5. Stain for 2 minutes in a saturated watery solution of methylene- 

 blue. 



6. Differentiate in methylated spirit, dehydrate in alcohol, clear in xylol, 

 and mount in xylol-balsam. 



The bacteria are deep crimson and the capsules of a blue tint. Fig. 79 is 

 from a film stained by this method. 



The Staining of Flagella. — The staining of the ilagella 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 fla- 

 gella, young cultures on agar should be used, say a culture in- 

 cubated 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 overheat it. But as ordinarily 

 practised, there is far too much handling of the bacteria in the 

 preparing of films, whereby large numbers of the organisms are 

 more or less denuded of their flagella, in consequence giving 

 poor results. To avoid this, Kendall recommends the following 

 procedure : A tube containing 5 c.c. of sterile water is gently in- 

 oculated with enough of an 1 8-24-hour-old agar culture of a bac- 

 terium to produce a very faint turbidity in the upper half of the 

 water. The tube is then placed in the thermostat for one hpur, 

 so as to let any clumps sediment as much as possible and per- 

 mit of slight development. Without disturbing the fluid in any 

 manner, two or three loopfuls of this culture are placed upon a 

 clean cover-slip, without spreading, and dried in the thermostat, 

 when. they are to be fixed in the flame and stained by any of the 

 methods recommended. The cover-glasses used should always 



