106 MICROSCOPIC METHODS 



the ordinary tissues, and left only in the bacteria. All kinds 

 of bacteria, however, do not retain the stain in this method, 

 and therefore in the systematic description of any species it is 

 customary to state whether it is, or is not, stained by Gram's 

 method by this is meant, as will be understood from what 

 has been said, whether the particular organism retains the 

 colour after the latter has been completely removed from the 

 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 net for one 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. Tlie period of time tor 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 decolorisation 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. Weigert's 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). 



