Unit 4: Staining theory and routine procedures
Histotechnology-I notes · PTU syllabus (BMLS504-18)
On this page
- Unit summary
- Theory of staining
- Classification and chemistry of dyes
- Mordants, accentuators and metachromasia
- Stains, buffers and mountants
- Nuclear and cytoplasmic stains
- Haematoxylin and eosin staining
- Manual and automatic staining
- Mallory's phosphotungstic acid haematoxylin (PTAH)
- Key terms
- Quick revision
- Important questions
Unit summary
Staining makes cells and tissues visible and tells them apart. This unit covers the theory of staining, classification and chemistry of dyes, mordants and accentuators, metachromasia, controls, preparation of stains and buffers, mountants, nuclear and cytoplasmic stains, manual and automatic staining, haematoxylin and eosin, and Mallory's PTAH stain.
After this unit you can
- Explain the theory of staining and classify dyes
- Explain mordants, accentuators and metachromasia
- Prepare stains, buffers and mountants
- Perform H&E and PTAH staining
PTU syllabus topics
- Theory of staining
- classification of dyes and dye chemistry
- mordants and accentuators
- metachromasy
- use of controls
- stain and buffer preparation
- mountants
- nuclear and cytoplasmic stains
- manual and automatic staining
- Haematoxylin and Eosin staining
- Mallory's Phosphotungstic Acid Haematoxylin (PTAH)
- 1
Dewax in xylene
- 2
Rehydrate
Through alcohols to water
- 3
Haematoxylin
Nuclei stain blue
- 4
Differentiate and blue
Acid alcohol, then Scott's tap water
- 5
Eosin
Cytoplasm stains pink
- 6
Dehydrate, clear and mount
Topic 1
Theory of staining
- Staining depends on physical factors (absorption, solubility, capillarity) and chemical bonding (ionic attraction between charged dye and tissue groups). Acidic tissue components (nucleic acids, mucins) bind basic dyes; basic components (cytoplasmic proteins) bind acidic dyes.
- Progressive staining
- Stain until the desired intensity (Mayer's haematoxylin)
- Regressive staining
- Overstain, then differentiate (Harris's haematoxylin with acid alcohol)
- Differentiation
- Selective removal of excess stain
- Direct staining
- Dye binds without a mordant
- Indirect staining
- Needs a mordant
Topic 2
Classification and chemistry of dyes
- A dye has a chromophore (gives colour — e.g., azo, quinoid groups) and an auxochrome (ionising group that binds tissue — e.g., amino or hydroxyl).
Natural dyes
From plants or insects
Haematoxylin (logwood tree), carmine (cochineal), orcein
Synthetic (coal tar) dyes
Manufactured
Eosin, methylene blue, safranin
Acidic dyes
Coloured anion; stain cytoplasm
Eosin, orange G, light green
Basic dyes
Coloured cation; stain nuclei
Methylene blue, toluidine blue, crystal violet
Neutral dyes
Both acidic and basic components
Romanowsky stains
Topic 3
Mordants, accentuators and metachromasia
- Mordant
- Metal salt forming a dye–mordant lake that links dye to tissue — alum (aluminium) for haematoxylin, iron for Heidenhain's
- Accentuator
- Increases staining without combining with the dye — phenol in carbol fuchsin
- Accelerator
- Speeds staining (in silver impregnation)
- Metachromasia
- Dye stains certain tissues a different colour from the dye itself — toluidine blue stains mast cell granules, cartilage and mucins purple–red
- Controls: a known positive tissue stained alongside every special stain run to confirm the stain worked.
Topic 4
Stains, buffers and mountants
- Stain preparation: weigh accurately, dissolve in the correct solvent, filter before use, label with date, check with controls; ripen haematoxylin (oxidise to haematein) naturally or chemically (sodium iodate, mercuric oxide).
Resinous (permanent)
Refractive index close to glass (about 1.5); after dehydration and clearing
DPX, Canada balsam
Aqueous
For fat stains and some enzymes; no dehydration
Glycerine jelly, Apathy's medium
Topic 5
Nuclear and cytoplasmic stains
Harris's
Alum; mercuric oxide or sodium iodate oxidised
Regressive; routine and cytology
Mayer's
Alum
Progressive; IHC counterstain
Ehrlich's
Alum
Long-lasting; bone and cartilage
Heidenhain's iron
Iron alum
Muscle striations, mitochondria
Weigert's iron
Iron
Acid-resistant nuclear stain with van Gieson
- Cytoplasmic stains: eosin Y (routine), phloxine, orange G, light green.
Topic 6
Haematoxylin and eosin staining
- 1
Dewax in xylene
- 2
Rehydrate through absolute, 90%, 70% alcohol to water
- 3
Harris's haematoxylin 5–10 minutes
- 4
Wash in water
- 5
Differentiate in 1% acid alcohol briefly
- 6
Blue in Scott's tap water or running tap water
- 7
Eosin 1–3 minutes
- 8
Dehydrate, clear in xylene, mount in DPX
- Results: nuclei blue–black; cytoplasm and collagen pink; red cells orange–red.
Topic 7
Manual and automatic staining
- Manual: racks moved through staining dishes; flexible for special stains. Automatic stainers and coverslippers: programmed, consistent, high throughput; reagents must be monitored and changed on schedule.
Topic 8
Mallory's phosphotungstic acid haematoxylin (PTAH)
- Principle: haematoxylin with phosphotungstic acid (mordant) forms a blue lake binding muscle and fibrin, while collagen takes the reddish-brown phosphotungstic acid. Tissue is often post-mordanted in Zenker's fluid.
- Results: muscle cross-striations, fibrin, neuroglial fibres and nuclei blue; collagen and bone brownish red. Uses: rhabdomyosarcoma (striations), fibrin, gliosis.
Key terms
- Chromophore
- Group giving a dye its colour
- Mordant
- Metal salt linking a dye to tissue
- Metachromasia
- Staining of tissue in a colour different from the dye
- Regressive staining
- Overstaining followed by differentiation
- DPX
- Synthetic resinous mountant
Quick revision
- Physical and chemical theory; progressive vs regressive; differentiation.
- Chromophore and auxochrome; natural and synthetic; acidic, basic, neutral dyes.
- Mordants, accentuators, metachromasia; controls.
- Stain preparation; ripening; resinous and aqueous mountants.
- Haematoxylins; H&E steps and results; manual vs automatic; PTAH.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Distinguish progressive and regressive staining.
- Q2.What is a mordant? Give an example.
- Q3.Define metachromasia.
- Q4.Why is Scott's tap water used?
- Q5.Name a resinous and an aqueous mountant.
- Q6.What does PTAH stain blue?
Long-answer questions
- Q1.Explain the theory of staining and classification of dyes.
- Q2.Explain mordants, accentuators and metachromasia.
- Q3.Describe haematoxylin and eosin staining.
- Q4.Describe Mallory's PTAH stain.
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