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Unit 2 of 4 · B.Sc MLS Sem 5

Unit 2: Decalcification and tissue processing

Histotechnology-I notes · PTU syllabus (BMLS504-18)

3 min read4 topics9 exam questions
On this page
  1. Unit summary
  2. Decalcification
  3. Tissue processing: overview
  4. Dehydration, clearing and infiltration
  5. Manual and automatic tissue processors
  6. Key terms
  7. Quick revision
  8. Important questions

Unit summary

Bone must be softened and tissue must be infiltrated with wax before it can be cut. This unit covers decalcification — criteria, technique and agents — and tissue processing: embedding media, dehydration, clearing, infiltration and tissue processors.

After this unit you can

  • Describe criteria and methods of decalcification
  • Test the end point of decalcification
  • Explain dehydration, clearing and infiltration
  • Describe embedding media and tissue processors

PTU syllabus topics

  • Criteria of a good decalcification agent
  • decalcification technique and workflow
  • types of decalcifying fluids (organic/inorganic acid, chelating agents, ion-exchange resins, electrophoretic decalcification)
  • embedding media
  • dehydration
  • clearing and infiltration
  • manual and automatic tissue processors
ProcessTissue processing
  1. 1Fixation

    Preserve the tissue

  2. 2Dehydration

    Graded alcohols remove water

  3. 3Clearing

    Xylene removes alcohol

  4. 4Infiltration

    Molten paraffin wax

  5. 5Embedding

    Wax block cast in a mould

1

Topic 1

Decalcification

  • Decalcification removes calcium salts from bone and calcified tissue so it can be sectioned, after thorough fixation.
Key termsCriteria of a good decalcifying agent
Complete removal of calcium
Without damaging tissue
Speed
Reasonable time
Preservation
Of cells, fibres and staining
Compatibility
With later special stains and molecular tests
ComparisonDecalcifying agents
Examples
Features

Strong inorganic acids

5–10% nitric acid, hydrochloric acid

Fast; damage staining if prolonged

Organic acids

Formic acid (5–10%), trichloroacetic acid

Gentler; good for routine and marrow

Chelating agents

EDTA (10–14%, neutral pH)

Slow (days to weeks); best morphology, IHC and DNA

Ion-exchange resins

Resin layered under formic acid

Faster acid action

Electrophoretic (electrolytic) method

Current drives calcium ions to the cathode

Rapid

ProcessDecalcification workflow
  1. 1Fix thoroughly
  2. 2Cut thin slices (bone saw)
  3. 3Immerse in a large volume of decalcifier, changing regularly
  4. 4Test the end point
  5. 5Wash, neutralise and process
Key termsEnd-point tests
Physical
Bending, needle probing — damaging, imprecise
Chemical
Add ammonium oxalate to used fluid — no precipitate means no calcium left
Radiography
X-ray shows remaining calcium — most accurate
2

Topic 2

Tissue processing: overview

ProcessParaffin processing
  1. 1Fixation
  2. 2Dehydration — graded alcohols (70%, 90%, absolute) remove water
  3. 3Clearing — xylene or substitutes remove alcohol and make tissue translucent
  4. 4Infiltration — molten paraffin wax (56–58 °C) replaces clearing agent
  5. 5Embedding — tissue oriented in a mould with fresh wax to form a block
  • Embedding media: paraffin wax (routine), paraffin with additives (Paraplast), celloidin (eye, large brain), gelatine (frozen sections), resins (epoxy for electron microscopy; methacrylate for undecalcified bone).
3

Topic 3

Dehydration, clearing and infiltration

ComparisonProcessing steps
Agents
Notes

Dehydration

Ethanol, isopropanol, acetone

Start at 70% to avoid shrinkage; incomplete dehydration gives soft, mushy blocks

Clearing

Xylene, toluene, chloroform, cedarwood oil, xylene substitutes

Miscible with alcohol and wax; prolonged xylene makes tissue brittle

Infiltration

Paraffin wax, 2–3 changes, under vacuum

Overheated wax shrinks and hardens tissue

4

Topic 4

Manual and automatic tissue processors

ComparisonTissue processors
Manual
Automatic

Method

Cassettes moved by hand between containers

Machine moves tissue or pumps reagents on a timed programme

Types

—

Carousel (basket moves between jars) and enclosed (fluid-transfer, vacuum and pressure)

Advantages

Cheap, flexible

Overnight runs, consistent, less fume exposure

Drawbacks

Labour, variability

Cost; malfunction can ruin all specimens — alarms and backups needed

  • Typical overnight schedule: formalin → 70% → 90% → absolute alcohol (3 changes) → xylene (2–3 changes) → wax (2–3 changes), about 12–16 hours.

Key terms

Decalcification
Removal of calcium from tissue
Chelating agent
Chemical binding calcium, such as EDTA
Dehydration
Removal of water by graded alcohols
Clearing
Replacing alcohol with a wax-miscible solvent
Infiltration
Permeation of tissue by molten wax

Quick revision

  • Criteria; acids, organic acids, EDTA, resins, electrolytic; end-point tests.
  • Fixation → dehydration → clearing → infiltration → embedding.
  • Embedding media.
  • Agents and errors at each step.
  • Manual vs automatic processors; schedule.

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

  1. Q1.Why must bone be fixed before decalcification?
  2. Q2.Name a chelating decalcifier.
  3. Q3.Describe the chemical end-point test.
  4. Q4.Why is dehydration graded?
  5. Q5.What is the purpose of clearing?
  6. Q6.Name two types of automatic processor.

Long-answer questions

  1. Q1.Describe decalcifying agents and the decalcification technique.
  2. Q2.Describe the steps of tissue processing.
  3. Q3.Compare manual and automatic tissue processors.

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