Laboratory Protocols
Histological Staining Techniques
Leishman's stain for blood cells and parasites
Leishman 1901.
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.
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.
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.