66 Neurological Technique 



7. To stain. — Remove the mordant and wash in several 

 changes of distilled water for 20-30 minutes. 



Then replace the water with 40-50 cc. of the stain (C) for 

 4-24 hours. 



8. To decolorize. — Remove the stain and wash well in 2 or 3 

 changes of water. The sections are now a deep blue-black. 



Next dilute 20-30 cc. of the mordant (B) with an equal vol- 

 ume of distilled water (2 per cent, iron alum) and place the 

 sections in it. Use a shallow dish and work over a white surface 

 in order to watch the decolorization better. When the gray 

 substance of the nervous tissue becomes clearly differentiated 

 and the white substance is a steel blue, the section is decolorized 

 sufficiently. This usually occurs in less than 5 minutes, but may 

 take 15 minutes. 



As soon as a section becomes sufficiently decolorized, remove 

 it to distilled water. To stop the decolorization it is necessary 

 that all the decolorizer be washed out. Use plenty of water, 

 and change it whenever it becomes cloudy. Wash for at least 

 20 minutes. 



9. If desired, the sections may now be counterstained by 

 transferring them to 10-20 cc. of the Orange G. solution (D) 

 for 5-10 minutes. Counterstaining is not always desirable. It 

 gives more prominence to the nerve-cells and brings out the con- 

 nective tissue investments and trabeculae which are left unstained 

 by the haematoxylin, but for the study of the medullated fibers 

 it detracts from the clearness and brilliancy of the section. 



10. To dehydrate, clear, and mount. — {a) After counterstaining 

 or omitting the same, wash in distilled water for 2 or 3 minutes. 

 Then replace the water with 50 per cent, alcohol for 3-5 minutes. 

 Replace 50 per cent, with 70 per cent, alcohol, 5 minutes; change 

 to 95 per cent, alcohol for 10-15 minutes. 



[b) With a section lifter carefully transfer the sections, one 

 at a time, from the 95 per cent, alcohol to a shallow dish of the 

 clearing fluid (IX, F, p. 56). The section, as it is placed in the 

 fluid, will usually straighten out and become cleared thus. The 

 sections will become cleared in 5-10 minutes. 



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