Wolbach's Giemsa
General Oversight stain

Solutions

Stock Giemsa
Working Giemsa
Stock Giemsa 2.5 mL
Methanol 3 mL
Distilled water 100 mL
Make the diluted solution just before using.

Stock Colophonium
Colophonium 10 g
Absolute ethanol 100 mL

Working Colophonium
Stock colophonium 5 mL
95% ethanol 40 mL

Tissue sample
Zenker or Helly fixed tissue is recommended, but many fixatives may be used, although staining results may vary. 3µ paraffin sections of neutral buffered formalin fixed tissue are usually suitable.

Method

  1. Bring sections to water with xylene and ethanol.
  2. Rinse well with distilled water.
  3. Place into working Giemsa for 1 hour.
  4. Place into fresh working Giemsa overnight.
  5. Rinse well with distilled water.
  6. Differentiate with working colophonium solution until colour contrast is satisfactory.
  7. Rinse well with 95% ethanol.
  8. Dehydrate with absolute ethanol.
  9. Clear with xylene and mount with a synthetic resinous medium.

Expected results

Notes

  1. 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.
  2. 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.
  3. Humason notes that the diluted Giemsa may be heated to 80-85°C then poured on the slide and applied for 10 minutes.

 

Reference
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

 


 

 

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