Leishmans stain for blood cells and parasites

Monday, November 24, 2003

Description
Staining protocol

Procedure
Prepare cytospin slide or smear of sample.
Either spray the wet preparation with Smear Fix? leave for 1-2 mins, wash off the fixative with distilled water and drain....
Or lower the slide gently into a coplin jar of acetic alcohol (3% acetic acid in 95% methanol), fix for 1 minute, wash off the fixative with distilled water and drain.
Put slides on a rack and cover with 1ml of Leishman stock - 20 seconds.
Add 2ml of pH6.8 buffer and tip the rack up and down to mix the solutions, stain for 7 minutes.
Rinse quickly in distilled water then treat with pH6.8 buffer - 2 minutes.
Rinse quickly in distilled water, shake off the excess and dry on a warm (50oC) hotplate, or carefully blot dry with fibre-free blotting paper.
Clear and mount.
Results: .
Red blood cells - red to yellowish red
Neutrophils - dark purple nuclei, pale pink cytoplasm, reddish-lilac small granules
Eosinophils - blue nuclei, pale pink cytoplasm, red to orange-red large granules
Basophils - purple to dark blue nucleus, dark purple, almost black large granules
Lymphocytes - dark purple to deep bluish purple nuclei, sky blue cytoplasm
Platelets - violet to purple granules
Parasites (Leishmania, malaria, etc.) - dark blue-black.

Recipes
Leishman stain
0.15% Leishman powder in 100% methanol. Use after 24hrs.

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 6.8 add 25.5ml of A to 24.5ml of B and make up to 100ml with distilled water.

Supplies

Tips

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Leishmans stain for blood cells and parasites