STAINING, PRACTICAL AND THEORETICAL 



C. Solution B . . . . • • 4 nil. 



Hydrochloric acid, 

 Alcohol, 95% 



D. Solution A 

 Solution C 



cone. 



I ml. 

 95 ml. 



I volume 

 I volume 



Note: Solution D should be prepared only as and 

 when required for immediate use. 



Technique: 



1. Fix material in Bouin, Helly, Zenker, Susa-Heidenhain, 

 Susa with picric acid, or Flemming, and embed in paraffin wax in 

 the usual manner. 



2. Cut sections at 4 to 5/x (thicker sections are of little value 

 for phase contrast microscopy), and fix them to slides. 



3. Dewax with xylol, and wash with absolute alcohol followed 

 by 90% and 70% alcohols. 



4. If a mercury-containing fixative has been used, treat for the 

 removal of mercurial precipitate in the usual way, then return to 

 70% alcohol, otherwise this step should be omitted. 



5. Immerse for one and a half hours in solution D. 



6. Rinse in 70% alcohol. 



7. Dehydrate through the alcohols; clear in xylol and mount 

 in a neutral synthetic resinous medium such as D.P.X. (Lendrum 

 & Kirkpatrick), or Cristalite, Clearmount, or Emexel, etc. 



Results: 



Observed with dark-contrast medium objectives, cellular and 

 nuclear membranes, chromatin and cytoplasmic structures appear 

 black against a green background. 



Notes: 



{a) The author (J. A. Green) states that although the phase 

 contrast microscope was designed primarily for the study of 

 living material, it has proved to be a useful tool for the study of 

 unstained sectioned material, and that it is, in addition, an 

 excellent substitute for counterstaining to show cellular detail 

 after periodic acid-Schiff, elastin stains, Feulgen stain and a very 

 large number of other highly selective staining procedures. 

 However, when the phase contrast microscope is employed with 



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