GRAM'S METHOD AND ITS MODIFICATIONS 107 



tissues. It must, however, be remarked that some tissue 

 elements may retain the stain as firmly as any bacteria, e.g., 

 keratinised epithelium, calcified particles, the granules of mast 

 cells, and sometimes altered red blood corpuscles, etc. 



In Gram's method the essential feature is the treating of the 

 tissue, after staining, with a solution of iodine. This solution 

 is spoken of as Gram's solution, and has the following com- 

 position : 



Iodine ..... 1 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 

 supra) for about five minutes. 



2. Without washing in water, now treat the section or film with 

 repeated doses of Gram's solution till its colour becomes a purplish 

 black, and allow the solution to act for 1 minute. 



3. Again without washing with water, decolorise with absolute alcohol 

 or methylated spirit till the colour has almost entirely disappeared, the 

 tissues having only a faint violet tint. The period of time for which the 

 alcohol is allowed to act varies in different laboratories. The best period 

 is probably about three minutes. 



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 decolorisatioii is more satisfactorily per- 

 formed 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 twenty volumes of water or a saturated watery solution of Bismarck- 

 brown may be used before stage (4) ; the former should not be applied 

 for longer than a few seconds. 



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



1. Weiyerfs 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. Kuhne's Modification. (\) Stain for five minutes in a solution 



