STRUCTURE OF THE PARASITE 773 



the bottom by means of a little piece of glass rod or a protuberance of the 

 vessel ; otherwise a precipitate is generally deposited on it. 



If the film has been made on a cover-glass this may be floated, film down- 

 wards, on the surface of the stain in a watch-glass. 



After staining for the necessary length of time (5-10 minutes in the case 

 of recently prepared films of malarial blood, and somewhat longer for older 

 films, and several hours in the case of the Hsematozoa of birds) the films are 

 washed in a liberal quantity of water, treated with a 5 per cent, watery solu- 

 tion of tannin for about 2 minutes and finally washed again in distilled water 

 and dried. 



If the staining be too intense or if a copious precipitate be formed the 

 preparations mast be washed in absolute alcohol or clove oil and then in 

 xylol. 



If the preparation is to be kept it is better not to mount it in balsam or 

 cedar wood oil, which will soon dissolve the stain. 



The red cells are stained pink and the nuclei of the leucocytes deep violet : 

 the protoplasm of the parasite assumes a pale blue colour and the chromatin 

 of its nucleus becomes violet or purplish red : pigment granules (Schiiffner's 

 dots) appear in the bodies of the red cells containing parasites. 



Romanowsky's method (p. 210) is applicable to the staining of Hsematozoa. 

 The nuclei of the parasites are stained purplish-red and their protoplasm blue. 



This method is merely of historical interest and is now no longer used in practice : the 

 results are often disappointing. 



The success of the method depends upon the formation, under certain conditions, of 

 violet and azur from methylene blue ; these colour-changes are produced either by treating 

 methylene blue with a dilute alkali (polychrome blue) or by obtaining azur from methylene 

 blue by more or less complicated processes. Most of the Romanowsky methods have 

 been described in connexion with the spirochsete of Syphilis (p. 727) : in this chapter 

 only those which are especially applicable to the study of the Hsematozoa will be con- 

 sidered. 



Leishman's stain. Leishman's method is a modification of that described 

 by Nocht, which depends upon the action of sodium carbonate on methylene 

 blue. 



Prepare a 1 per cent, aqueous solution of medicinal methylene blue (Grubler) and 

 add 0*5 per cent, sodium carbonate : heat for 12 hours at 65 C. and keep for 10 

 days at the temperature of the laboratory. Prepare also a O'l per cent, aqueous 

 solution of eosin (eosin BA Grubler). Mix equal volumes of the two solutions and 

 leave for 10 hours or so, shaking the mixture at .frequent intervals. Filter, wash 

 the precipitate with distilled water until the washings are a very pale blue, collect 

 the precipitate on the filter, dry and powder. (Leishman's stain in powder can now 

 be obtained from Grubler). 



The staining solution is prepared by dissolving the powder in absolute methyl 

 alcohol (0*15 per cent.). For use, 3 or 4 drops of the stain are poured on the film, 

 and after 30 seconds 6-8 drops of water are added and the diluted stain allowed 

 to act for a further 5-10 minutes. Wash in water and leave the water on the film 

 for a minute. Dry and mount in balsam. When stained by this method the red 

 cells are pale pink or greenish pink in colour, the nuclei of the leucocytes red, the 

 Hsematozoa blue and their chromatin ruby-red. 



Tenner's stain. This method, like that of Wright, is based upon the use 

 of the product obtained by the interaction of solutions of methylene blue 

 and eosin. The stain is sold in the dry form by Grubler. 



The film, which does not require fixing, is stained for 3-5 minutes in the following 

 solution. 



Jenner's stain in powder (Grubler), .... l gram. 



Pure absolute methyl alcohol, - - 100 c.c. 



The preparation should be covered during the staining process to prevent evaporation 

 of the alcohol. 



