STAINING AND EXAMINATION OF MICRO-ORGANISMS 47 



mixed, as in Ziehl's solution, with 100 c.c. of a 5 per cent, 

 aqueous solution of carbolic acid. 



The decoloration of preparations. In making mi- 

 croscopic preparations it is often necessary to obtain 

 greater definition by staining in two colours. For this 

 purpose methods have been devised by the use of which, 

 whilst one part of the specimen remains coloured, the 

 other portion is made to give up the stain, after which 

 it is treated with some other colour the application of 

 which does not affect in any way the already stained 

 portion of the preparation. This decoloration is essen- 

 tially the principle of Gram's well-known method. 

 The strongest decolorising agents are acids. Even a 

 weak solution of acetic acid already exerts a strong 

 decolorising power, whilst weak solutions of hydro- 

 chloric, nitric, and sulphuric acid act still more power- 

 fully in this manner. The strongest agents of all for 

 this purpose are acids combined with alcohol. The 

 following are the principal decolorising agents in 

 use : 



(1) 5 per cent, aqueous solution of acetic acid. 



(2) 20 per cent, aqueous solution of nitric acid. 



(3) 3 per cent, alcoholic solution of hydrochloric acid (100 parts abso- 

 lute alcohol and 3 parts hydrochloric acid). 



Grams method. The particular feature of this 

 method, which is of primary importance in the stain- 

 ing of tissues, is that in a section of animal tissue it 

 leaves the micro-organisms stained whilst removing 

 the stain from the animal nuclei. It consists in staining 

 the cover-glass preparation or section in an aniline- 

 water solution of gentian violet for about five minutes, 

 after which it is placed in a solution of iodine and po- 

 tassium iodide (1 iodine, 2 potassium iodide, 300 parts 

 water) for two minutes, and then washed with alcohol 

 until no more colour is removed ; it is then placed in 



