100 MICROSCOPIC METHODS 



3. Decolorise with absolute alcohol or methylated spirit till the 

 colour has almost entirely disappeared, the tissues having only a faint 

 violet tint. 



4. Dehydrate completely, clear with xylol and mount. In the case 

 of film preparations, the specimen is simply washed in water, dried and 

 mounted. 



In stage (3) the process of decolorisation is more satisfactorily 

 performed by using clove oil after sufficient dehydration with alcohol, 

 the clove oil being afterwards removed by xylol. 



As a contrast stain for the tissues carmalum or lithia carmine is used 

 before staining with gentian-violet (1). As a contrast stain for other 

 bacteria which are decolorised by Gram's method carbol-fuchsin diluted 

 with ten volumes of water or a saturated watery solution of Bismarck - 

 brown may be used before stage (4). 



The following modifications of Gram's method may be given : 



1. Weigerfs Modification. The contrast staining of the tissues and 

 stages (1) and (2) are performed as above. 



(3) After using the iodine solution the preparation is dried by 

 blotting and then decolorised by aniline-xylol (aniline-oil 2, xylol 1). 



(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. Nicolles 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. Kuhnes 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. 



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 its action may be aided 



