GRAM'S STAIN. 103 



Iodine i part. 



Potassium iodide .... 2 parts. 



Distilled water ..... 300 „ 



The following is the method : — 



1. Stain in aniline oil gentian-violet or in carbol-gentian-violet (vide 

 pp. loi, 102) for about five minutes, and wasii in water. 



2. Treat the section or film with Gram's solution till its colour becomes a 

 purplish black — generally about half a minute or a minute is sufficient for the 

 action to take place. 



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 (i). As a contrast stain for other bacteria which 

 are decolorised by Gram's method carbol-fiichsin diluted with ten volumes of 

 water, or a saturated watery solution of Bismarck-brown, may be used before 

 stage (4). 



As applied to bacterial films special conditions must be 

 observed. The film must always be prepared from an agar 

 slant-culture between 12 and 24 hours old, and is then to be 

 treated as follows : — 



1 . Stain in aniline gentian-violet for i \ minutes. 



2. Wash in water. 



3. Stain in Gram's solution for \\ minutes. 



4. Decolorise in absolute alcohol for at least four minutes, or until all 

 stain is completely discharged. 



5. Mount in balsam. 



The following modifications of Gram's method may be 

 given : — 



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

 stages (i) 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 i). 



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

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

 may be used to stain bacteria which have been decolorised. 



This modification probably gives the most uniformly successful results. 



2. NicoUe'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 (i part) and alcohol (2 parts). 



