Romanowsky – Giemsa
General Oversight stain

Solutions

Stock Giemsa or other Romanowsky stain e.g. Lieshman, Wright etc.
0.1% acetic acid in distilled water.
Working Solution
Stock Romanowsky stain 1 mL
Distilled water or pH 6.8 buffer 9 mL

Make the diluted solution just before using. Discard after a few hours.

Tissue sample
Most fixatives permit staining but the 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. Optionally, treat with pH 6.8 phosphate buffer for 30 minutes.
  3. Place into the staining solution for 1 hour.
  4. Rinse well with water.
  5. Differentiate with acetic acid until nuclear morphology is clear. Control microscopically.
  6. Rinse well with distilled water.
  7. Blot dry with filter paper, then flood with xylene.
  8. Repeat step 6 until section is transparent (usually 4-5 times).
  9. Mount with a synthetic resinous medium.

Expected results

Notes

  1. Giemsa is usually diluted 1 in 10. Lieshman, Wright and others are often diluted 1 in 3.
  2. Pretreatment with a pH 6.8 phosphate buffer is sometimes recommended immediately before placing in diluted stain. In that case, buffer with the same pH should be used to dilute the stock Romanowsky stain.
  3. Differentiation may also be carried out in the same buffer as used to dilute the stock stain, but may take some time.
  4. Drury and Wallington use pH 5.0 buffer mixed equal parts with methanol, and specify green Euparal as the mounting medium..
  5. Sections may be rapidly dehydrated with ethanol instead of blotting but this removes some blue staining and will likely destroy any metachromasia.
  6. Sections stained with Romanowsky stains do not usually show the same range of colours that are shown by the same stain on smears, and the choice of stock Romanowsky stain does not necessarily influence the final appearance.

 

Reference
Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
Cellular Pathology Technique, 4th ed.
Butterworths, London, UK.

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.

 


 

 

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