Overview
Nucleoproteins combine basic proteins with nucleic acids — DNA in the nucleus and RNA mainly in cytoplasmic ribosomes. Because both molecules react with specific dyes or reagents through their sugar and phosphate groups, dedicated histochemical methods can selectively demonstrate DNA, RNA, and cytoplasmic organelles such as mitochondria.
This lesson covers the Feulgen technique for DNA, the methyl green-pyronin method for RNA, and Altman's technique for mitochondria, along with the fixation requirements and expected colour results for each method.
Learning Objectives
After this lesson you will be able to…- Differentiate the locations and chemical composition of DNA and RNA
- Describe the principle and procedure of the Feulgen technique for DNA
- Perform the methyl green-pyronin method to demonstrate RNA
- Explain why fixation choice is critical for nucleic acid demonstration
- Describe Altman's technique for demonstrating mitochondria
Clinical Story
Why This MattersA pathology resident examining a plasma cell-rich lesion wants to confirm the abundant RNA content typical of active protein-synthesising plasma cells. A methyl green-pyronin stain is requested, turning the RNA-rich cytoplasm a striking magenta-red while the DNA-containing nuclei remain green-blue — a classic histochemical confirmation of plasma cell identity.
Core Concepts
Mild acid hydrolysis with 1M hydrochloric acid at 60°C breaks the purine-deoxyribose bond in DNA, exposing aldehyde groups. These aldehydes then react with Schiff's reagent to produce a red-purple colour, specifically staining DNA in the nucleus while the cytoplasm is counterstained green with light green.
Methyl green and pyronin are both cationic dyes. When chloroform-washed (pure) methyl green is combined with pyronin Y in an acetate buffer at pH 4.8, methyl green binds preferentially to DNA (green-blue) while pyronin binds to RNA (red), allowing simultaneous differential staining of both nucleic acids.
Mitochondria, the cell's energy-producing organelles, can be demonstrated histologically using Altman's technique, which uses aniline-acid fuchsin after fixation in Champy's or Helly's fluid, followed by controlled differentiation in picric acid solutions until only mitochondria and red blood cells retain the red stain.
Laboratory Principle
Nucleic acid demonstration exploits either the reaction of dyes with phosphate groups (methyl green-pyronin) or the generation of aldehydes from sugar residues after acid hydrolysis (Feulgen reaction), which then react with Schiff's reagent. Mitochondrial staining relies on selective retention of acid fuchsin dye within mitochondrial membranes after careful differentiation, a phenomenon dependent on the mitochondria's lipid-rich membrane composition.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| 1M hydrochloric acid | 8.5 ml conc. HCl in 91.5 ml distilled water | Acid hydrolysis for Feulgen reaction | Room temperature |
| Methyl green-pyronin Y solution | 2% methyl green (chloroform washed) 9ml + 2% pyronin Y 4ml + acetate buffer + glycerol | RNA/DNA differential staining | Prepare fresh, mix well |
| Aniline-acid fuchsin | Saturated acid fuchsin in 5% aniline in distilled water | Mitochondria demonstration (Altman's technique) | Room temperature |
Step-by-Step Procedure
Fix tissue avoiding Bouin's fixative for DNA studies (Feulgen); use Carnoy's fixative (preferred) or formalin for RNA studies (methyl green-pyronin).
Place sections in 1M HCl at 60°C to expose aldehyde groups from the DNA sugar-phosphate backbone.
Transfer hydrolysed sections to Schiff's reagent for 45 minutes (Feulgen), or place directly in methyl green-pyronin Y solution for 25 minutes (RNA method).
Rinse in bisulfite solution (Feulgen) or acetate buffer (methyl green-pyronin) to remove excess unbound dye.
Counterstain if required, dehydrate rapidly through ascending alcohols, clear in xylene, and mount for permanent examination.
Flow Diagram
Quality Control
Include a tissue known to have abundant nucleic acid content (e.g. small intestine crypts for DNA, plasma-cell-rich tissue for RNA) as a positive control to confirm correct staining intensity and colour.
Where molecular confirmation methods (in-situ hybridisation) are available, periodically cross-validate histochemical DNA/RNA findings against these more definitive techniques.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Strong red-purple nuclear staining (Feulgen) | Confirms DNA content, useful in ploidy/cytometry studies | Correlate with cytogenetic or flow cytometric findings if required |
| Magenta cytoplasmic staining (methyl green-pyronin) | Confirms abundant RNA, e.g. in plasma cells or actively secreting cells | Supports diagnosis of plasma cell-rich or high protein-synthesis lesions |
| Loss of DNA staining with Bouin's-fixed tissue | Bouin's fixative degrades DNA and interferes with the Feulgen reaction | Avoid Bouin's fixative when Feulgen DNA staining is planned |
Common Errors & How to Avoid Them
Cause: Tissue fixed in Bouin's fixative, which is incompatible with the Feulgen technique.
Prevention: Confirm fixation history before staining; use a fixative other than Bouin's for DNA studies.
Cause: Methyl green contaminated with methyl violet (not chloroform washed) reducing specificity for DNA.
Prevention: Always use properly chloroform-washed methyl green to ensure specific DNA binding.
Cause: Sections left in 1M HCl at 60°C for too long, degrading DNA rather than exposing aldehydes.
Prevention: Follow standardised hydrolysis timing carefully and monitor with a control section.
Laboratory Tips from the Bench
Always double-check that methyl green has been chloroform-washed before use — unwashed methyl green containing methyl violet reduces specificity for DNA.
Never use Bouin's fixative when Feulgen DNA staining is planned; Carnoy's fixative is preferred for RNA demonstration.
Remember: 'Pyronin for RNA, Green for DNA' — an easy way to recall the colour-coding in the methyl green-pyronin technique.
Important Notes
The choice of fixative directly affects nucleic acid preservation — Bouin's fixative must be avoided for Feulgen DNA staining, while Carnoy's fixative is preferred (formalin acceptable) for RNA demonstration.
While Feulgen and methyl green-pyronin are useful histochemical methods, in-situ hybridisation remains the most definitive and sensitive technique for nucleic acid demonstration.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeLymph node biopsy shows a population of cells with abundant basophilic cytoplasm and eccentric nuclei suspicious for a plasma cell-rich reactive or neoplastic process.
The strong red cytoplasmic staining on methyl green-pyronin confirms an abundant RNA content consistent with active immunoglobulin-producing plasma cells, supporting the morphological impression from H&E.
- →Methyl green-pyronin confirms RNA-rich cytoplasm typical of plasma cells.
- →Fixative choice affects nucleic acid preservation and staining quality.
- →Histochemical nucleic acid stains complement, but do not replace, immunohistochemistry for definitive diagnosis.
Frequently Asked Questions
Commercial methyl green is often impure and contaminated with methyl violet; washing with chloroform removes the methyl violet, leaving pure methyl green that is specific for DNA.
Yes — because the Feulgen reaction is stoichiometric with DNA content, it can be used in DNA cytophotometry to estimate nuclear DNA content and ploidy.
Champy's fluid is usually recommended for Altman's technique, though Helly's fluid works equally well.
Quick Revision
10-Minute ReviewKey Takeaways
- Nucleic acid demonstration relies on dye-phosphate reactions or aldehyde-Schiff chemistry.
- The Feulgen reaction is specific and stoichiometric for DNA.
- Methyl green-pyronin differentiates DNA and RNA simultaneously by colour.
- Fixative selection critically affects nucleic acid stain quality.
- In-situ hybridisation remains the gold-standard nucleic acid technique.
- Altman's technique remains a simple histological method for mitochondria, though electron microscopy is most definitive.
Competency Checklist
Track Your MasteryReferences
- NIOS Histology and Cytology Practical Manual, Lesson 16: DNA, RNA and Mitochondria Demonstration.
- Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 7th ed.
- Kiernan JA. Histological and Histochemical Methods: Theory and Practice. 5th ed.