Gram Weigert
for Fibrin and Gram Positive Bacteria
Materials
Eosin
| Material | Amount | |
|---|---|---|
| Eosin Y ws | 5 | g |
| Distilled water | 100 | mL |
Crystal violet
| Material | Amount | |
|---|---|---|
| Crystal violet | 1 | g |
| Distilled water | 100 | mL |
Gram’s iodine
| Material | Amount | |
|---|---|---|
| Iodine | 2 | g |
| Potassium iodide | 4 | g |
| Distilled water | 400 | mL |
Aniline-Xylene
| Material | Amount | |
|---|---|---|
| Aniline | 1 | volume |
| Xylene | 1 | volume |
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Place in eosin solution for 5 minutes.
- Rinse with tap water.
- Place in crystal violet 1 minute.
- Rinse with tap water.
- Flood with Gram’s iodine for 1 minute.
- Rinse with tap water.
- Gently blot the section, being careful not to damage it.
- Decolorise the section with aniline-xylene.
- Rinse with several changes of xylene to remove all aniline.
- 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
- Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London. - McManus, J.F.A. and Mowry, R.W., (1960),
Staining methods, histologic and histochemical,
Harper & Row, New York, NY, USA.
