FLAGELLUM STAINING 53 



must bo absolutely dean, and the growth be well diluted 

 before spreading, to render individuals sufficiently isolated. 

 A trace of an eighteen to twenty-four hour culture of the 

 organism is very gently mixed with a few drops of tap- 

 water in a watch-glass. A minute drop of water is placed 

 on a clean cover-glass which has been well heated over a 

 Bunsen flame and allowed to cool, and a very small loopful 

 of the bacterial emulsion is added. The cover-glass is 

 spread at once, and the material thereon should be suffi- 

 ciently small to cause the film to dry immediately after 

 spreading. The cover-glass is held in the fingers and 

 passed three times through the flame at such a rate that 

 the heat can be endured by the fingers. 



McCrorie's Night Blue Method. Solution A. Dissolve 

 0-5 gramme of McCrorie's night- blue in 20 c.c. of absolute 

 alcohol. 



Solution B. Dissolve 1 gramme of tannic acid in 20 

 c.c. of hot water. Add 1 gramme of alum dissolved in 

 20 c.c. of cold water. 



Add A to B slowly, shaking gently all the time. Filter. 



The stain is allowed to act for two minutes in a warm 

 place. 



M uir's Modified Pit field Method. 



A. The Mordant. 



Tannic acid (10 per cent. aq. sol., filtered) 10 c.c. 



Sat. aq. sol. mercuric chloride . . . . 5 



Sat. aq. sol. alum . . . . . . . . 5 ,, 



Ziehl's carbol-fuchsin . . . . . . 5 



A precipitate forms which may be allowed to deposit 

 or centrifuged. 



The clear solution is removed and will keep a week. 



B. The Stain. 



Sat. aq. sol. alum . . . . . . . . 10 c.c. 



Sat. alcoholic sol. gentian violet . . . . 2 



This keeps two days. 



The film is flooded with the mordant and steamed for 

 one minute. After well washing in water for two minutes, 

 it is very cautiously dried, flooded with the stain, steamed 

 for one minute and well washed in water. 



Capsule Staining. By considerably diminishing the 

 light, capsules may be observed in fresh preparations. 



