GRAM'S STAIN. 1 1 1 



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 . . i part 



Potassium iodide . . 2 parts 



Distilled water . 300 parts 



The following is the method : 



1. Stain in aniline oil gentian- violet (vide p. 109) 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. 



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. 



Before (4) a contrast stain is often used (vide infra}. 



Gram's method, when carefully used, generally gives 

 quite satisfactory results, but sometimes a precipitate of 

 the gentian-violet is left in the tissues, and sometimes the 

 specimen decolorises very slowly and the bacteria lose the 

 stain in the process. We find that the result is more 

 certain if in (i) the alcoholic gentian- violet be mixed 

 with carbolic acid solution (i : 20) instead of aniline oil 

 water, and if in (3) clove oil be used in decolorising 

 after the specimen has been sufficiently dehydrated with 

 alcohol or methylated spirit. The clove oil decolorises 

 rapidly, and must then be washed thoroughly out with 

 xylol, or, if a contrast stain is to be used, with alcohol. 

 Nicolle has modified Gram's method by staining with 

 carbol-gentian-violet as described, and, in (3) decolorising 

 with a mixture of acetone one part and alcohol two parts. 

 This also gives very good results. In Weigert's modifica- 

 tion, aniline oil is used in (3) as the decolorising agent 



