INTRODTJCTION. 43 



material to be examined is thick, it should be emulsified with 

 physiological salt solution. In the case of blood, fluid from 

 blood or serum culture media, tissues, faeces, in fact any 

 hquid containing albuminous matter, the film will stick to 

 the sUde or the cover-shp during the processes of fixation and 

 staining. The usual fixatives and stains are described here- 

 after. 



Staining the Organisms fixed on a Cover-slip. — ^Make a number 

 of smears of the fluid containing the organisms on cover-sHps, 

 and when the fluid, has partially dried, invert the cover-shps 

 and let them float on the surface of the flxative contained in 

 a dish ; or put a small drop of fluid containing the organism 

 on a cover-shp and add with a pipette twice the quantity 

 of hot fixative. When the organisms have been fixed on the 

 cover-shps, pass successively through the washing fluid, 

 alcohols, stain, clearing-fluid, etc., all these reagents being 

 contained in shallow dishes. In all these subsequent stages 

 put the cover-shps at the bottom of the fluid in the dish, 

 with the face bearing the organisms upwards. Finally, 

 remove the cover-shps and put them, face downwards, on 

 shdes on each of which a drop of Canada balsam has been 

 put. In this way quite a large number of smears or preparations 

 can be fixed and stained in practically the same time as would 

 be taken to make one preparation. 



Fixatives. — The foUowing are commonly employed for fixing 

 the Protozoa : — 



(1) Concentrated solution of mercuric chloride, hot or 

 cold : (2) Schaudinn's subHmate alcohol (2 parts saturated 

 aqueous solution of mercuric chloride, 1 part absolute alcohol ; 

 immediately before use, add acetic acid to the quantity to 

 be used to the strength of 5 per cent.) : (3) Zenker's fiuid 

 (mercuric chloride 5 grm., potassium bichromate 2-5 grm., 

 sodium sulphate 1 grm. in 100 c.c. of distifled water, with 

 2-5 to 5 per cent, of glacial acetic acid added before use) : 

 (4) Bouin's fluid (saturated aqueous solution of picric acid 

 75 parts, formol 25 parts, and acetic acid 5 parts) : (5) Bouin's 

 alcohoHc fixative (picric acid 1 grm., 80 per cent, alcohol 

 150 c.c, formol 60 c.c, acetic acid 15 c.c) : (6) formahn : 

 and (7) vapour of 4 per cent, solution of osmic acid. Fixation 

 is usuaUy complete in 15 to 30 minutes. 



Bouin's fixatives are the simplest to use, as the picric acid 

 is more easfly washed out after fixation than the mercuric 

 chloride contained in the others. After Bouin's fixative, 

 wash the films in several changes of distilled water till all the 

 picric acid has been removed. In the case of alcohohc Bouin, 

 if an aqueous stain is to be used, commence washing in 

 70 per cent, alcohol and, passing the film through graded 

 alcohols, bring down to distilled water. After Schaudinn's 



