104 MICROSCOPIC METHODS 



solution of eosin (which is an acid dye), and then to use a blue 

 which will stain the bacteria and the nuclei of the cells. The 

 Romanowsky stains (vide p. 114) are here most useful, as by these 

 the preparations are fixed as well as stained. Fixation by heat, 

 which is apt to injure delicate cellular structures, is thus avoided. 

 In the case of films made from urine, where there is little or 

 no albuminous matter present, the bacteria may be imperfectly 

 fixed on the slide, and are thus apt to be washed off. In such 

 a case it is well to modify the staining method. A drop of 

 stain is placed on a slide, and the cover-glass, film-side down, 

 lowered upon it. After the lapse of the time necessary for 

 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 

 (6) the subsequent 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 



