STAINS AND STAINING 17 



This solution is the best of the general purpose "iron hematoxylins" and 

 should be widely used to stain the nuclei before complex afterstaining. The 

 staining solution contains hematoxylin, and, therefore, must be ripened for 

 some time before use. The most convenient method of doing this is to pre- 

 pare a 10 per cent solution of hematoxylin in alcohol and then to dilute this 

 with the glycerin in water immediately before use. The alcohol solution of 

 hematoxylin should be at least a month old. This stain is very slow if it is 

 used cold, so that it is customary to heat both the mordanting and staining 

 solutions to about 50° C. before use. 



SECTIONS ARE STAINED AS FOLLOWS: 



1. Accumulate the sections in distilled water. 



2. Transfer the slides to the mordanting solution for 30 minutes at 50° C. or 

 overnight at room temperature. 



3. Rinse each slide in distilled water to avoid carrying over too much of the 

 mordant into the staining solution. 



4. Transfer the slides to the staining solution for 30 minutes at 50° C. or 

 overnight at room temperature. 



5. Transfer the slides to distilled water and wash until no more stain 

 comes away. 



6. Dip each slide up and down in the differentiating solution until it ap- 

 pears to be partly differentiated and transfer to tap water until no further 

 color comes away. Then examine the slide under the microscope. If fur- 

 ther differentiation is required, repeat the process. 



7. Transfer all the slides to tap water until they have turned blue. If the tap 

 water becomes yellow from traces of picric acid, it should be changed or 

 differentiation will continue. Hematoxylin is very sensitive to acids and, 

 as it comes from the differentiating solution, has a reddish brown color, 

 as distinct from the clear blue color required on the finished slide. In 

 many parts of the world, natural tap water is sufficiently alkaline to pro- 

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

 be added to the coplin jar containing the tap water. 



Direct Nuclear Hematoxylin Staining 



These stains are not, in general, very satisfactory, for they give neither as 

 sharp staining as the mordant methods nor as good a diffuse stain as the in- 

 direct methods. 



The method of Ehrlich, however, is so well known that it is given here: 



