106 MICROSCOPIC METHODS 



new body the reddish hue is due, but it may be to methyl-violet 

 or to methyl-azure, both of which result from the action of alkali 

 on methylene-blue. The stains are much used in staining blood- 

 films (in which the characters of both nucleus and cytoplasm are 

 beautifully brought out), in staining bacteria in tissues or 

 exudates, the malaria parasite, trypanosomes, the pathogenic 

 spirochsetes (such as the spirochaete pallida), and protozoa 

 generally. 



The following are the chief formulae in use : 



1. Jenners Stain. This is an excellent blood stain, but is not so good 

 for the study of parasites as the others to be mentioned. In its 

 preparation no alkali is used. It is made by mixing equal parts of (a) 

 a 1 '2 to 1 '25 per cent solution of Griibler's water soluble eosin (yellow 

 shade) in distilled water and (b) 1 per cent Griibler's medicinal methy- 

 lene-blue (also a watery solution). The mixture is allowed to stand 

 twenty-four hours, is filtered, and the residue is dried at 55 C. ; the 

 powder is shaken up in distilled water, filtered, washed with distilled 

 water and dried. Of the powder, 5 grms. are dissolved in 100 c.c. Merck's 

 methyl alcohol. For use a few drops are placed on the dried unfixed 

 film for one to three minutes, the dye is poured off, and the preparation 

 washed with distilled water till it presents a pink colour ; it is then 

 dried between filter-paper and mounted in xylol balsam. 



2. Leishmans 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) ; (b) 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 Schiiffner'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 



