78 BASIC METHODS FOR EXPERIMENTS 



are: Heidenhain's Iron-alum haematoxylin and safranin (or 

 gentian violet). 



(a) Heidenhain's Iron Haematoxylin. — Heidenhain's iron 

 haematoxylin is a 0.5 per cent aqueous solution of the dye. I 

 find that the solution may be made with tap water or distilled 

 water. Although many workers prefer to use the solution only 

 after it has been standing for some days, I get best results with a 

 freshly prepared solution. 



Before coming into the stain, the sections, previously washed 

 in water, are kept in 4 per cent aqueous solution of iron alum for 

 15 to 60 minutes, depending upon their thickness. The sections 

 are now carefully rinsed in water to remove the superfluous iron- 

 alum. They are then brought into tubes of the staining solution 

 for I to 16 hours, depending upon the depth of color one desires 

 to obtain; shorter baths in the iron alum and in the dye stain 

 the chromosomes blue, longer ones, black. As will be noted 

 beyond, the shorter baths stain plasma and cytoplasmic inclu- 

 sions after fixatives containing osmic acid and chromic acid as 

 that of Meves. 



From the staining solution the sections are brought Into 

 water and thoroughly washed. This is best accomplished in a 

 large volume of water where the slide is kept until no more color 

 is discharged. The slide is now brought into a 2 per cent 

 solution of ferric alum and then placed under the low power of 

 the microscope illuminated with daylight. The ferric alum 

 brings about discharge of color. This should be carefully con- 

 trolled by dipping the slide into water which arrests destaining. 

 (Caution: Filter both ferric alum solutions before using them!) 



At the point of the desired difi^erentiation, the slide is removed 

 to a tube which is placed under a gentle stream of tap water for 

 one hour. After this, the slide is run up through successive 

 grades of alcohol: 35, 50, 70, 80, 90, 95, 95, 100, 100, percent, 

 each for 5 to 10 minutes. The sections are cleared by placing 

 them in two changes of xylene or toluene for 5 minutes each. 

 They are then mounted in a thin solution of neutral xylene 

 balsam. 



I. Author'' s modification of Heidenhain's iron haematoxylin. — - 

 I frequently take, instead of a 0.5 aqueous solution, as used in 

 the Heidenhain method, a 0.25 or a 0.125 aqueous solution. 



