Trevan and Sharrock's
Methyl Green – Pyronin
Materials
Methyl green stock
| Material | Amount | |
|---|---|---|
| Methyl green | 2 | g |
| Distilled water | 100 | mL |
Purifying the methyl green
Before making the methyl green-pyronin stock solution the methyl green must be purified to remove any methyl violet which has formed.
- Place the 2% aqueous stock methyl green solution in a stoppered separating funnel.
- Add some chloroform and shake periodically for about a half hour.
- Crystal violet will discolor the chloroform as it is removed.
- Allow to separate, then drain off the methyl green and discard the chloroform.
- Repeat the extraction until the chloroform is colorless or almost so.
Pyronin Y stock
| Material | Amount | |
|---|---|---|
| Pyronin Y | 5 | g |
| Distilled water | 100 | mL |
Acetate buffer pH4.8
| Material | Amount | |
|---|---|---|
| 0.1M sodium acetate | 119 | mL |
| 0.1M acetic acid | 81 | mL |
Methyl green–pyronin stock
| Material | Amount | |
|---|---|---|
| Methyl green, 2% aqueous, purified | 10 | mL |
| Pyronin Y, 5% aqueous | 17.5 | mL |
| Distilled water | 250 | mL |
Staining solution
| Material | Amount | |
|---|---|---|
| Methyl green–pyronin stock | 1 | volume |
| Acetate buffer pH 4.8 | 1 | volume |
Make fresh.
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Avoid mercuric chloride.
Protocol
- Bring sections to water via xylene and ethanol.
- Rinse with distilled water, then gently blot dry.
- Place into the staining solution for 20–30 minutes,
- Rinse with distilled water, then gently blot dry.
- Dehydrate rapidly with acetone.
- Clear with xylene and mount with a resinous medium.
Expected Results
- DNA – blue–green
- RNA – Red
Notes
- Mercuric chloride fixation may cause the DNA to depolymerize during fixation and should be avoided. The differential staining depends on differences in the degree of polymerization of the two nucleic acids and depolymerization may cause the DNA to stain with pyronin.
- Acid decalcified tissues are often unsatisfactory and may stain excessively red with pyronin. EDTA decalcification is usually satisfactory.
- The extracted solutions of methyl green are reasonably stable, but changes in the expected nuclear coloration may require a freshly extracted solution of methyl green to be prepared.
- This method is reasonably selective for nucleic acids but should be controlled with ribonuclease and/or deoxyribonuclease enzyme extractions if specificity is required.
- Since this technique is quite simple, it is often recommended for plasma cells. Nissl substance also stains well.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Drury, R A, and Wallington, E A, (1967).
Carleton’s histological technique., Ed. 4, p. 164.
Oxford University Press, London, England.
