Method III 29 



and again wipe the under side and edges dry. Then again lay 

 the slide level and flood as above with a layer of the toluidin 

 blue solution (F), and again place in the thermostat (same tem- 

 perature) for 10-12 minutes. 



{e) To decolorize and differentiate: Drain off the warm 

 stain, quickly rinse off _the surplus by merely dipping through 

 distilled water, and then transfer the slide to a vessel of 96 per 

 cent, alcohol till the color is no longer given off. This will take 

 anywhere from 2 to 8 minutes. 



The sections should now appear a light reddish violet or 

 lilac. If they appear a deep or murky blue, further attempts to 

 differentiate will avail little toward improving the preparation. 

 On the other hand, if, at the end of 2 minutes, the sections are 

 pale blue and do not show the violet color, shortening the time 

 of differentiation avails little. 



16. To dehydrate, transfer the slide froin the differentiating 

 96 per cent, alcohol to fresh absolute alcohol for 10 minutes. 



17. To clear, transfer from absolute alcohol to a covered 

 vessel of pure fresh xylol for 15-20 minutes. 



It is essential that the xylol be free from water or alcohol. 

 Clouds should not be produced as the slide is placed in the 

 xylol. It is better to have special jars of absolute alcohol and 

 xylol for this part of the process. 



18. Mount in xylol balsam (I, G) or colophonium (II, E, 

 p. 21). 



The neurofibrils can be observed in detail only with an oil 

 immersion lens and with good light. 



Even if the previous steps have been carefully and correctly 

 followed, failures may often result from unfortunate manipula- 

 tion in the staining and differentiation in step 15. The most 

 favorable duration of the action of the water in the thermostat 

 and the optimum strength and duration of the stain must neces- 

 sarily be determined by some experimentation. The two seem 

 to be interdependent to some extent. The action of the water, 

 which Bethe calls "differentiation," is thought to extract a cer- 

 tain amount of the molybdate remaining in the tissue either free 



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