GRAM'S STAIN 99 



to prevent access of light, (b) Make a saturated solution of gentian- 

 violet in alcohol. When the stain is to be used, 1 part of (b) is 

 added to 10 parts of (a), and the mixture filtered. The mixture should 

 be made not more than twenty-four hours before use. Stain sections 

 for a few minutes ; then decolorise with methylated spirit. Sometimes 

 it is advantageous to add to the methylated spirit a little hydrochloric 

 acid (2-3 minims to 100 c.c. ). This staining solution is not so much 

 used by itself, as in Gram's method, which is presently to be described. 



5. Carbol-Gcntian-Violet. 1 part of saturated alcoholic solution of 

 gentian-violet is mixed with 10 parts of 5 per cent solution of carbolic 

 acid. It is used as No. 4. 



6. Carbol-Fuchsin (see p. 101). This is a very powerful stain, and, 

 when used in the undiluted condition, |-1 minute's staining is usually 

 sufficient. It is better, however, to dilute with from five to ten times its 

 volume of water and stain for a few minutes. In this form it has a very 

 wide application. Methylated spirit with or without a few drops of 

 acetic acid is the most convenient decolorising agent. Then dehydrate 

 thoroughly, clear, and mount. 



Gram's Method and its Modifications. In the methods 

 already described the tissues, and more especially the nuclei, 

 retain some stain when decolorisation has reached the point to 

 which it can safely go without the bacteria themselves being 

 affected. In the method of Gram, now to be detailed, this does 

 not occur, for the stain can here be removed completely from 

 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 composition : 



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, and wash 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. 



