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Gram Weigert for Fibrin and Gram Positive Bacteria

Protocol

Gram Weigert

for Fibrin and Gram Positive Bacteria

11
steps
9
materials
print

Materials

Eosin

MaterialAmount
Eosin Y ws5g
Distilled water100mL

Crystal violet

MaterialAmount
Crystal violet1g
Distilled water100mL

Gram’s iodine

MaterialAmount
Iodine2g
Potassium iodide4g
Distilled water400mL

Aniline-Xylene

MaterialAmount
Aniline1volume
Xylene1volume

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in eosin solution for 5 minutes.
  3. Rinse with tap water.
  4. Place in crystal violet 1 minute.
  5. Rinse with tap water.
  6. Flood with Gram’s iodine for 1 minute.
  7. Rinse with tap water.
  8. Gently blot the section, being careful not to damage it.
  9. Decolorise the section with aniline-xylene.
  10. Rinse with several changes of xylene to remove all aniline.
  11. Mount with a resinous medium.

Expected Results

  • Gram positive bacteria  –  blue
  • Fibrin  –  blue
  • Background  –  pink

Notes

  • Control the differentiation with aniline-xylene microscopically. To examine, place the section in xylene to stop dye removal. Return to aniline-xylene if more differentiation is needed. Stop differentiation when the target element has good contrast.
  • Increasing the aniline content of the aniline-xylene will increase the speed of dye removal. Decreasing it will slow dye removal.
  • If the background is not pink enough, increase the time in eosin, stain in eosin at elevated temperature, or increase the eosin concentration.
  • The eosin counterstain may be omitted entirely if wished.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling, C.F.A., (1963)
    Handbook of histopathological techniques, 2nd ed.
    Butterworths, London.
  2. McManus, J.F.A. and Mowry, R.W., (1960),
    Staining methods, histologic and histochemical,
    Harper & Row, New York, NY, USA.