Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

Histological Staining Techniques

Giemsa for blood cells and parasites
Use a positive control when staining for parasites.

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 56^oC - 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: 

         
(Leishmaniasis)  (Cryptospridium)
Nuclei - blue/purple.
Acidophils - pink/red.
Basophils - blue.
Eosinophils - red/orange.
Mast cells - purple (granules).
Parasites - blue/dark blue.
 

Other Giemsa staining methods are as follows:

Thin films of blood or marrow

• Fix in methanol for 5 minutes.
• Place in a Coplin jar containing Giemsa stain diluted 1:10 with pH 6.8 buffer for 20 minutes.

(This time may need to be prolonged to adequately demonstrate parasites such as spirilla and trypanosomes).
• Rinse in distilled water.
• Drain and dry at room temperature.
• Mount in DPX or leave unmounted and use oil immersion.

Thick films for malaria parasites

• Do not fix the film.
• Place in a Coplin jar containing Giemsa stain diluted 1:50 with pH 7.2 buffer for 1 hour.
• Differentiate in distilled water for a few minutes.
• Drain and dry at room temperature.
• Mount in DPX or leave unmounted and use oil immersion.

Wax sections (overnight method)

• Dewax the sections, rinse in alcohol, rinse in water.
• Wash the sections in distilled water.
• Stain overnight, at room temperature, in a Coplin jar of Giemsa diluted 1:25 with pH 6.8 buffer.
• Rinse in distilled water.
• Differentiate in 1:1,500 acetic acid. (Control by viewing at intervals under a microscope. Sections should have an overall pink colour, with the nuclei blue and eosinophil granules red.)
• Rapidly rinse in distilled water.
• Dehydrate, clear and mount.

 

Giemsa stain
Mix 7.36g Giemsa powder in 500mls glycerol which is heated to 50^oC in a water bath. Leave for 30 minutes at 50^oC 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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