ioo MICROSCOPIC METHODS. 



The following is the method : 



1. Stain in aniline oil gentian-violet (v. supra No. 4) 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, and the tissue appears only a 

 very light violet. 



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 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 modification, aniline oil is used in (3) as the 

 decolorising agent instead of alcohol. Other modifications 

 have been introduced, of which only Kiihne's need be 

 mentioned. 



Kiihne's Modification of Grants Method. 



1. Stain for five minutes in a solution made up of equal parts of 

 saturated alcoholic solution of crystal-violet (" krystall- violet ") and i 

 per cent solution of ammonium carbonate. 



2. Wash in water. 



3. Place for two to three minutes in Gram's iodine solution, or in 

 the following modification by Kiihne : 



