Monday, November 24, 2003

Description
Staining protocol

Procedure
For smears or cytospin preparations:

Fix prepared film in methanol - 3 to 5 minutes.
Stain in a mixture of 1ml of Giemsa stock plus 2mls of pH6.4 buffer and 47mls of distilled water - 40 to120 minutes.
Rinse rapidly in distilled water, blot dry and mount.
For paraffin wax sections:
Dewax sections, rinse in alcohol, rinse in water.
Stain in a coplin jar in a mixture of 1mls of Giemsa stock plus 45mls distilled water in a water bath at 56oC - 20 mins to 1 hour (maximum).
Rinse in distilled water.
Differentiate in 1/1,500 acetic acid for approx. 30 seconds total, rinse in distilled water. (Control by viewing at intervals under a microscope. Sections should have an overall pink colour, with the nuclei blue and eosinophil granules red.)
Rinse in distilled water.
Blot dry, rinse briefly in alcohol, clear and mount.
Results:
Nuclei - blue/purple.
Acidophils - pink/red.
Basophils - blue.
Eosinophils - red/orange.
Mast cells - purple (granules).
Parasites - blue/dark blue.


Recipes
Giemsa stain
Mix 7.36g Giemsa powder in 500mls glycerol which is heated to 50oC in a water bath. Leave for 30 minutes at 50oC with periodic mixing. Allow to cool and add 500mls methanol. Mix and filter.
Phosphate buffer (Sorensen)
Stock A: 0.2M sodium di-hydrogen orthophosphate (mw 156).
To prepare dissolve 3.12g in 100ml distilled water.

Stock B: 0.2M di-sodium hydrogen orthophosphate (mw 142).
To prepare dissolve 2.83g in 100ml ditilled water.

For pH 6.4 add 36.7ml of A to 13.3ml of B and make up to 100ml with distilled water.
For pH 6.8 add 25.5ml of A to 24.5ml of B and make up to 100ml with distilled water.
For pH 7.2 add 14.0ml of A to 36.0ml of B and make up to 100ml with distilled water.



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