IN- VITRO STAINING 25 



jelly film which contains the stain in solution. To 

 5 cc. of 2-per-cent. agar jelly (with or without the 

 addition of salts) stain and alkali to the required 

 amount are added, and the total bulk is made up 

 to 10 cc. with distilled water. This is melted and 

 poured on a microscope slide, where it sets when 

 cold. A platinum loopful of amoebae is smeared on 

 a cover-glass, which is then inverted and allowed 

 to fall on the jelly film, when the stain diffuses 

 into the cell while it is still alive. The coefficient 

 of diffusion of this amoeba is very low, viz. with 

 0'2 cc. of 5-per-cent. sodium bicarbonate (alkali) 

 and O'l cc. of Unna's polychrome methylene blue 

 (stain) the nucleus stains a pale purple colour in 

 ten minutes at 20 C. The coefficient of diffusion 

 (cf) is therefore: 



Stain Alkali Heat Time 



Cf = (Is + 4a + 3h + 1 t) = 9 



Stages of nuclear division are shown by the jelly 

 method with ease (PL IV.), and the degree of 

 staining can be regulated by means of the alkali 

 while the specimen is under observation. Staining 

 of the granules does not cause death of the amceba, 

 and it may even move for some minutes after the 

 nucleus stains, but it then rapidly dies and becomes 

 spherical (PI. V.). Amoebae may be fixed before 

 or after staining by means of formalin, absolute 

 alcohol, or Schaudinn's fluid. In the preparation 

 of fixed films from test-tube cultures, it is un- 

 satisfactory simply to make cover-slip smears which 

 are then immediately fixed and stained, because 

 the amoebae have shrunk from the shock of the 

 removal and their contents are massed together. 



