Wolbach's Giemsa
General Oversight Stain
Materials
- Stock Giemsa
- Working Giemsa
Material Amount Stock Giemsa 2.5 mL Methanol 3 mL Distilled water 100 mL Make the working solution just before using.
- Stock Colophonium
Material Amount Colophonium 10 g Absolute ethanol 100 mL
Tissue Sample
Zenker or Helly fixed tissue is recommended, but many fixatives may be used, although staining results may vary. 3 µ paraffin sections of neutral buffered formalin fixed tissue are usually suitable.
Protocol
- Bring sections to water with xylene and ethanol.
- Rinse well with distilled water.
- Place into working Giemsa for 1 hour.
- Place into fresh working Giemsa overnight.
- Rinse well with distilled water.
- Differentiate with working colophonium solution until color contrast is satisfactory.
- Rinse well with 95% ethanol.
- Dehydrate with absolute ethanol.
- Clear with xylene and mount with a synthetic resinous medium.
Expected Results
- Nuclei – blue-purple
- Cytoplasm – pink
- Inclusions – according to type – pink or blue
Notes
- Humason says to treat sections with 1% acid alcohol for 5 minutes immediately after bringing to water. They are then thoroughly washed with running tap water for 5 minutes. This would have the effect of improving staining by the eosin component of the Giemsa.
- Putt says to add 3-4 drops of 0.5% aqueous sodium bicarbonate to the water used to dilute the Giemsa. This would have the effect of improving staining by the basic dye component of the Giemsa.
- Humason notes that the diluted Giemsa may be heated to 80-85°C then poured on the slide and applied for 10 minutes.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Humason, G.L., (1967)
Animal Tissue Techniques
W.H. Freeman & Co., San Francisco, CA, USA - Putt, F.A., (1972)
Manual of histopathological staining methods
John Wiley & Sons, New York, NY, USA