104 MICROSCOPIC METHODS. 



3. Kuhne's Modification. — (i) Stain for five minutes in a solution made up 

 of equal parts of saturated alcoliolic solution of crystal-violet (" Krystall-violet ") 

 and I per cent solution of ammonium carbonate. 



(2) Wash in water. 



(3) Place for two to three minutes in Gram's iodine solution, or in the 

 following modification by Kuhne : — 



Iodine 2 parts. 



Potassium iodide .... 4 „ 

 Distilled water 100 „ 



For use, dilute with water to make a sherry-coloured solution. 



(4) Wash in water. 



(5) Decolorise in a saturated alcoholic solution of fluorescein (a saturated 

 solution in methylated spirit does equally well). 



(6) Dehydrate, clear, and mount. 



Stain for Tubercle and Other Acid-fast Bacilli. — These bacilli 

 cannot be well stained with a simple watery solution of a basic 

 aniline dye. This fact can easily be tested by attempting to 

 stain a film of a tubercle culture with such a solution. They 

 require a powerful stain containing a mordant, and must be 

 exposed to the stain for a long time, or the action of the latter 

 may be aided by a short application of heat. When once 

 stained, however, they resist decolorising even with very power- 

 ful acids; they are therefore called "acid-fast." The smegma 

 bacillus also resists decolorising with strong acids (p. 256), and 

 a number of other acid-fast bacilli have recently been discovered 

 (p. 254). Any combination of gentian-violet or fuchsin with 

 aniline oil or carbolic acid or other mordant will stain the 

 bacilli named, but the following methods are most commonly 

 used : — 



Ziehl-Neelsen Carbol-fuchsin Stain. 



Basic fuchsin I part. 



Absolute alcohol . . .10 parts. 



Solution of carbolic acid (1-20) . 100 „ 



1 . Place the specimen in this fluid, and having heated it till steam rises, 

 allow it to remain there for five minutes, or allow it to remain in the cold 

 stain for from twelve to twenty-fo.ur hours. (Films and paraffin sections are 

 usually stained with hot stain, loose sections with cold ; in hot stain the latter 

 shrink.) 



2. Decolorise-with 20 per cent solution of strong sulphuric acid, nitric 

 acid, or hydrochloric acid, in water. In this the tissues become yellow. 



