16 MATERIALS AND PROCESSES OF SLIDE-MAKING 



best techniques are those of Dobell and Regaud, though several dozen 

 variants of these have been proposed from time to time. 



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 solu- 

 tion uses hematein, which is an oxidation product, instead of hematoxylin, so 

 that no ripening is required. This method can be used only on very thin sec- 

 tions or on protozoan smears because it gives such an intense coloration that 

 thick sections cannot be differentiated in practice. 



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 minutes. 



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



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 quite 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. 



Regaud's Mordant Hematoxylin Stain: 

 Mordanting solution 

 5% Iron alum 



Staining solution 



Water 80 ml. 



Hematoxylin 1 Gm. 



Glycerin 10 ml. 



Alcohol 10 ml. 



Differentiating solution 

 Alcohol 65 ml. 



Water 35 ml. 



Picric acid 0.5 Gm. 



