82 The Preparation of Microscope Slides 



duce this blue color. If it is not, a pinch of sodium bicarbonate should 

 be added to the coplin jar containing the tap water. 



Dobell's Mordant Hematoxylin Stain: 



Mordanting solution 



1% iron alum in 70% alcohol 

 Staining solution 



1% hematein in 70% alcohol 

 Differentiating solution 



0.1% hydrochloric acid in 70% alcohol 



All these solutions are stable, and it will be noticed that the staining 

 solution uses hematein, so that no ripening is required. This method can 

 be used only on very thin sections or on protozoan smears because it 

 gives such an intense coloration that thick sections can be differentiated 

 only with difficulty. 



Procedure Is as Follows: 



1. Accumulate thin sections or protozoan smears in 70 per cent alcohol. 



2. Transfer them to the mordanting solution for about 10 min. 



3. Without rinsing, transfer the sections directly to the stain for 10 min. 



4. Rinse them quickly in 70 per cent alcohol. 



5. Differentiate sections in the last solution until only the chromosomes 

 or nuclei remain stained. Differentiation is fairly rapid. Remove sections 

 from the differentiating solution at intervals, wash thoroughly in 70 per 

 cent alcohol, and then examine under the microscope. If a number of 

 slides are being stained at the same time, it is desirable to differentiate 

 one carefully, noting the time needed for the operation, and to use this 

 same amount of time to bring the remaining sections through in one batch. 



6. Wash them thoroughly in 70 per cent alcohol. 



7. Counterstain the sections, if desired, by one of the methods to be de- 

 scribed later or mount them in balsam after dehydration and clearing. 



Direct Hematoxylin Staining. The solutions used for these techniques 

 contain both dye and mordant in one solution. The commonest mordants 

 are either potassium or ammonium alum and most formulas contain 

 glycerin as a stabilizing agent. Literally hundreds of mixtures have been 

 proposed, but they may be broadly divided into acidified and not-acidified 

 mixtures. The former are generally used in histological and pathological 

 routines to stain nuclei before eosin counterstaining; the latter are used, 

 although less frequently, for the same purpose and for staining xylem in 

 plant sections. 



Both types of stain must be "blued" after differentiation. The acid 

 solution used for differentiation leaves the nuclei colored red, in which 



