772 THE ILEMATOZOON OF MALARIA 



blue in the manner just described. The preparation is thus double stained, 

 the red cells being pink, the nuclei of the leucocytes and the parasites blue. 

 This method is recommended by Laveran. 



(c) The films may be stained with the eosin-methylene-blue mixture of 

 Chenzinsky (p. 210). 



2. Violet stains. The films may be stained, for a few seconds only, in a 

 saturated aqueous solution of gentian-violet or violet-dahlia or in Boehmer's 

 hsematoxylin. If carbol-thionin be used the staining should be prolonged 

 to 5 minutes. After staining the films are washed, dried and mounted in 

 balsam. The red cells are unstained while the nuclei of the white cells and 

 the parasites are stained violet ; the pigment granules are barely visible. 



Ross's method for the detection of the parasites. The parasites are present 

 in the blood in small numbers only as a rule, and their detection in thin films 

 on cover-glasses is often a tedious undertaking. Ross therefore takes about 

 20 cm. of blood and spreads it in a thick layer on a slide which, after drying 

 over a flame but without fixing the film, is washed in water ; in this way the 

 haemoglobin is dissolved and removed. The film is now stained for 1 minute 

 in a 1 per cent, aqueous solution of eosin followed by an alkaline solution of 

 methylene blue for 15-30 seconds (1 per cent, aqueous solution of methylene 

 blue to which 0'5 per cent, of carbonate of soda has been added and heated 

 until it acquires a purple tint) : it is finally washed in water, dried and mounted 

 in balsam. This method yields a transparent preparation in which only the 

 leucocytes and parasites are stained and so allows of the latter being readily 

 found. Ruge advises the use of Ross's method, taking care to fix the prepara- 

 tion (so as to obviate the chance of the blood being detached during washing) 

 in a 2 per cent, solution of formalin containing 1 per cent, acetic acid. This 

 method of fixing in no way interferes with the solution of the haemoglobin. 



Le Dantec's method. In cases in which the parasites are present in very 

 small numbers, Le Dantec advises hsemolyzing 1 c.c. of blood by collecting 

 it in 20 c.c. of water, centrifuging the mixture and examining the deposit 

 for parasites. 



Structure of the parasite. 



The staining of the nuclei of Heematozoa is difficult and requires special 

 methods. 



Laveran's method. Recommended. This method is based on the use of 

 Borrel's blue. 



Preparation of Borrel's blue. Dissolve some crystals of silver nitrate in 50 c.c. of 

 distilled water in a flask of about 150 c.c. capacity. Fill the flask with a 10 per cent, 

 solution of soda and shake. The black precipitate of silver oxide which is thus obtained 

 is then carefully washed several times in distilled water and after decanting the last 

 washing a saturated aqueous solution of medicinal methylene blue (Hochst) is added. 

 The solution is shaken several times and allowed to stand for 10 to 15 days ; the super- 

 natant fluid which is then decanted constitutes Barrel's blue. 



The dried blood films after fixing for 10 minutes in absolute alcohol are 

 stained with the following solution, which must be made up just before use : 

 Borrel's blue, 1 - 1 c.c. 



O'l per cent, aqueous solution of eosin, 2 5 ,, 



Distilled water, - - - 4 ,, 



Mix carefully. The eosin and methylene blue solutions must be filtered just before mixing 

 but the mixture itself should not be filtered. 



The stain is poured into a flat vessel (Petri dish or special rectangular dish) 

 and the slide immersed film side downwards, being prevented from touching 



1 The solution of Borrel's blue ought to be prepared afresh should it rapidly give a 

 heavy precipitate after mixing with the eosin solution. 



2 Water-soluble eosin (Hochst). 



