MOKDANTS AND DECOLOKISING AGENTS 101 



staining, a drop of water is placed at one side of the cover-glass 

 and a little piece of filter paper at the other side. The result is 

 that the stain is sucked out by the filter-paper. By adding 

 fresh drops of water and using fresh pieces of filter-paper, the 

 specimen is washed without any violent application of water, 

 and the bacteria are not displaced. 



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 (vide infra) is also used, 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 sub- 

 sequent 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. 



(b) 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 often 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. 



Different organisms take up and retain the stains with various 

 degrees of intensity, and thus duration of staining and decoloris- 

 ing must be modified accordingly. We sometimes have to deal 



