PROTOZOA 581 



seconds. Then remove slide, wiiich should be now dry, and 

 place it in absolute alcohol for ten minutes. It may then be 

 allowed to dry or be placed at once in Giemsa's stain (see below). 

 Osmic acid solution may also be used as a fixative (see § 40). 



1145. Iodine is well known to be an excellent fixative (§ 88). 

 E. S. Goodrich {Quart. Journ. Micr. Sci., Ixiv, 1019, p. 38) 

 modifies Kent's method by following the dilute iodine in potassium 

 iodide solution with a definitive fixative, such as Bouin's, and 

 obtains excellent results with leucocytes of invertebrates. Good 

 results have also been obtained with free-living amoebae, ciliates 

 and flagellates by this method, especially when fixing them en 

 masse by the centrifuge method, § 1127. 



The two fixatives may be mixed before use, e.g. 1 drop of 

 Lugol's iodine solution to about 10 of Bouin's fluid makes a very 

 satisfactory mixture in which to fix films. 



CHIEF STAINS USED FOR PROTOZOA 



1146. A. Hwmatoxylin. For most protozoa iron ha^matoxylin 

 staining is unsurpassed. 



(a) Heidexhain's Iron Haematoxylin. Use, as a mordant, a 

 dilute solution of iron alum in distilled water (about 3-5 per cent.). 

 As stain, 0-5 per cent, ripened solution of haematoxylin in distilled 

 water. 



To ripen the solution, allow to stand for some weeks until the reddish 

 solution becomes deep brown owing to some haematoxylin being oxidised 

 to haematein. Dobell and O'Connor recommend putting it " in a flask 

 plugged with cotton-wool, in a warm place — if possible, in sunlight — 

 and shaking from time to time." See also p. 162. 



Mordant films or sections taken from distilled water for six 

 hours or more — overnight is a suitable time. Rinse in distilled 

 water and place in the stain for a similar time. Then again 

 rinse the preparation — now black — in distilled water, and 

 differentiate by washing out the stain in the iron-alum solution, 

 diluted if necessary. The result should be checked and controlled 

 by examining under the microscope from time to time. 



The preparation should be greyish, due to the chromatin being 

 black. Wash it in distilled water and then in running tap-water, 

 for at least half an hour. 



The mordanting and staining may be shortened to two or 

 three hours each, but then the chromatin will only stain blue, 

 instead of black as by the longer method, or alcoholic solutions 

 may be used and the time shortened still more. 



(6) Mallory's Ferric Chloride Haematoxylin {Journ. Exper. 

 Med., V, 1900, p. 18). Use as mordant a 10 per cent, aqueous 

 solution of ferric chloride, and as stain a freshly prepared 1 per 

 cent, solution of ha?matoxvlin. Mordant films or sections for 



