MORDANTS AND DECOLORISING AGENTS 97 



For the general staining of films a saturated watery solution 

 of methylene-blue will be found to be the best stain to com- 

 mence with, the Gram method (v. infra) is then applied, and 

 subsequently any special stains which may appear advisable. 



The Use of Mordants and Decolorising Agents. In films of 

 blood and pus, and still more so in sections of tissues, if the above 

 methods are used, the tissue elements may be stained to such an 

 extent as to quite obscure the bacteria. Hence many methods 

 have been devised in which the general principle may be said to 

 be (a) the use of substances which, while increasing the staining 

 power, tend to fix the stain in the bacteria, and (b) the subse- 

 quent treatment by substances which decolorise the overstained 

 tissues to a greater or less extent, while they leave the bacteria 

 coloured. The staining capacity of a solution may be increased 



(a) By the addition of substances such as carbolic acid, 

 aniline oil, or metallic salts. 



(6) By the addition of alkalies, such as caustic potash or 

 ammonium carbonate, in weak solution. 



(c) By the employment of heat. 



(d) By long duration of the staining process. 



As decolorising agents we use chiefly mineral acids (hydro- 

 chloric, nitric, sulphuric), vegetable acids (especially acetic acid), 

 alcohol (either methylated spirit or absolute alcohol), or a com- 

 bination of spirit and acid, e.g. methylated spirit with a drop or 

 two of hydrochloric acid added, also various oils, e.g. aniline, 

 clove, etc. In most cases about thirty drops of acetic acid in a 

 bowl of water will be sufficient to remove the excess of stain 

 from over-stained films and sections. More of the acid may, of 

 course, be added if necessary. 



Hot water also decolorises to a certain extent ; over-stained 

 films can be readily decolorised by placing a drop of water on 

 the film and heating gently over a flame. 



When preparations have been sufficiently decolorised by an 

 acid, they should be well washed in tap water, or in distilled 

 water with a little lithium carbonate added. 



The methods embracing the use of a stain with a mordant, 

 and a decoloriser, are very numerous, and we can only enumerate 

 the best of them. 



Different organisms take up and retain the stains with 

 various degrees of intensity, and thus duration of staining and 

 decolorising must be modified accordingly. We sometimes have 

 to deal with bacteria which show a special tendency to be 

 decolorised. This tendency can be obviated by adding a little 



