STAINING, PRACTICAL AND THEORETICAL 



BENZIDINE METHOD 



(After Pickworth) 



For haemoglobin 



B. 



C. 



Technique: 



1. Fix pieces of tissue in 10% neutral formalin and embed in 

 paraffin wax or celloidin or Waterwax, or cut frozen sections. 



2. Take sections through to water. 



3. Wash in 70% alcohol. 



4. Wash in absolute methyl alcohol. 



5. Immerse in reagent A, in a Coplin jar, for ten minutes. 



6. Wash well with reagent B to remove excess benzidine. 



7. Wash in running water for fifteen minutes. 



8. Counterstain in neutral red for three minutes. 



9. Wash in distilled water. 



10. Dehydrate quickly through the usual graded alcohols. 



11. Clear in xylol. 



12. Mount in Cristalite or in D.P.X. or Emexel. 



Results: 



Haemoglobin and certain oxidase granules in leucocytes appear 

 dark blue. Nuclei : red. 



Note: 



Ivillie (1948) states that a further identification of the haemo- 

 globin lies in the fact that with acid formaldehyde fixation (pH 

 3 -0-3 -5), or with acid treatment of the sections for a sufficient 



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