114 MICROSCOPIC METHODS 



2. Leishman's Stain. The following solutions are prepared : (a) to 

 a 1 per cent, solution of medicinal methylene-blue is added "5 per cent, 

 sodium carbonate ; the mixture is kept at 65 C. for twelve hours, and 

 then for ten days at room temperature ('25 per cent, formalin may be 

 added as a preservative) ; (&) 1-1000 solution of eosin, extra B.A., in 

 distilled water. Equal volumes of the two solutions are mixed and 

 allowed to stand for six to twelve hours with occasional stirring, the 

 precipitate is collected, filtered, washed with distilled water, and dried. 

 For use, '15 per cent, is dissolved in Merck's methyl alcohol ("for 

 analysis, acetone free") as follows: The powder is placed in a clean 

 mortar, a little of the alcohol is added and well rubbed up with a 

 pestle ; the undissolved powder is allowed to settle and the fluid 

 decanted into a dry bottle ; the process is repeated with fresh fractions 

 of the solvent till practically all the stain is dissolved, and the bottle 

 is well stoppered. The stain will keep for a long period. For the 

 staining of films a few drops of the stain are placed on the unfixed 

 preparation for fifteen to thirty seconds so as to cover it with a 

 shallow layer (the stain may be conveniently spread over the film 

 with a glass rod), and the film is tilted to and fro so as to prevent 

 drying. This treatment efficiently fixes the film by the action of the 

 methyl alcohol. About double the quantity of distilled water is now 

 dropped on the film, and the stain and diluent are quickly mixed with 

 the rod. Five minutes are now allowed for staining, and the stain is 

 then gently washed off with distilled water. A little of the water is 

 kept on the film for half a minute to intensify the colour contrasts in 

 the various cells. For certain special structures, such as Schuffner's dots 

 or Maurer's dots in the malarial parasite, a longer staining (up to one 

 hour) may be necessary, and in any case it is well to practise being able 

 to control the depth of the staining effect by observation with a low- 

 power objective. If a preparation is to be stained for a long time it 

 must be kept covered, and if in such cases a granular deposit is formed 

 this may be got rid of by a quick wash with absolute alcohol. If in blood 

 films the red corpuscles appear bluish instead of pink, the colour may 

 be restored by washing the film with acetic acid, 1-1500. The film is 

 dried between filter-paper and mounted. 



For staining sections a little modification is necessary. A paraffin 

 section is taken into distilled water as usual, the excess of water is drained 

 off, and a mixture of one part of stain and two parts of distilled water 

 is placed on it. The stain is allowed to act for five to ten minutes till the 

 tissue appears a deep Oxford blue ; it is then decolorised with 1-1500 

 acetic acid the effect being watched under a low-power lens. The blue 

 begins to come out, and the process is allowed to go on till only the 

 nuclei remain blue. The section is then washed with distilled water, 

 rapidly dehydrated with alcohol, cleared, and mounted. If, as some- 

 times happens, the eosin tint be too well marked, it can be lightened 

 by the action of 1-7000 solution of caustic soda, this being washed off 

 whenever the desired colour has been attained. 



In certain cases, e.g. for the staining of old films or of trypanosomes 

 or Leishmanife in sections, Leishrnan recommends an initial treat- 

 ment of the preparation with serum. This modification is described in 

 Appendix E. 



3. J. II. Wright's Stain. In this modification 1 per cent, methylene- 

 blue (BX or Ehrlich's rectified) and ^ per cent, sodium carbonate (both 

 in water) are mixed and placed in a Koch's steriliser for an hour. When 



