TUBERCLE STAINS 107 



(4) Wash well in xylol, and mount in xylol balsam. Film preparations 

 after being washed in xylol may be dried, and thereafter dilute carbol- 

 fuchsin may be used to stain bacteria which have been decolorised. 



This modification probably gives the most uniformly successful results. 



2. Nicolle's Modification. Carbol-gentian-violet is used as the stain. 

 Treatment with iodine is carried out as above, and decolorisation is 

 effected with a mixture of acetone (1 part) and alcohol (2 parts). 



3. Kiihnes Modification. (1) Stain for five minutes in a solution 

 made up of equal parts of saturated alcoholic solution of crystal-violet 

 (" Krystall- violet ") and 1 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 Kiihne : 



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. 



There is great variability in the avidity with which organisms stained 

 by Gram retain the dye when washed with alcohol, and sometimes 

 difficulty is experienced in saying whether an organism does or does not 

 stain by this method. 



Most bacteria are either frankly Gram-positive or frankly 

 Gram-negative, but cases occur when an organism, usually Gram- 

 positive or Gram-negative, tends when grown on certain media to 

 show an opposite tendency, and sometimes an organism is met 

 with in which the individuals in a film show slightly different 

 reactions to the Gram stains. 



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 at- 

 tempting to stain a film of a tubercle culture with such a 

 solution ; with the Gram method, however, a partial staining 

 is sometimes effected. Such bacteria require a powerful stain 

 containing a mordant, and must be exposed to the stain for a 

 long time, or its action may be aided by a short application of 

 heat. When once stained, however, they resist decolorising 

 even with very powerful acids; they are therefore called "acid- 

 fast." The smegma bacillus also resists decolorising with 

 strong acids (p. 280), and a considerable number of other acid- 

 fast bacilli are now known (p. 278). Any combination of 

 gentian-violet or fuchsin with aniline oil or carbolic acid or 



