SECTION TWO 



AZAN STAIN (HEIDENHAIN) 

 A general purpose stain 



Solutions required: 



Azocarmine B 

 Distilled water 

 Glacial acetic acid . . 



Dissolve by warming ; cool and filter. 



100 ml 



0-5 gm. 

 lOO ml. 

 I ml. 



B. 96% alcohol 

 Aniline oil 



C. 95% alcohol 

 Glacial acetic acid . . 



D. Phosphotungstic acid 

 Distilled water 

 Methyl alcohol 



E. AniHne Blue, water soluble 

 Orange G 



Glacial acetic acid . . 

 Distilled water 



o-i ml. 



99 ml. 

 I ml. 



75 ml. 

 25 ml. 



0-5 gm. 

 2gm. 

 8 ml. 



100 ml. 



Dissolve by warming ; cool and filter. 



For staining, dilute 1 volume of this solu- 

 tion with 3 volumes of distilled water. 



Technique: 



1 . Zenker-, Bouin- or Carnoy-fixed tissues are stained from forty- 

 five to sixty minutes at 55° C. in Solution A; then at room tem- 

 perature for five to ten minutes. 



2. Wash in distilled water; then diflFerentiate in Solution B 

 until cytoplasm is pale pink, and nuclei are red and clear. 



3. Rinse for one half to one minute in Solution C. 



4. Transfer to Solution D for about one to three hours or 

 until the connective tissue is completely decolorized ; then wash 

 quickly in distilled water. 



5. Stain for one to two hours in the diluted Solution E, exam- 

 ining at ten- or fifteen-minute intervals to prevent over-staining. 



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