Unit 1: Specimen collection and fixation
Histotechnology-I notes · PTU syllabus (BMLS504-18)
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Unit summary
Histotechnology turns tissue into thin stained sections a pathologist can read. This unit covers the scope of histotechnology, care of equipment, safety, routine methods of examining tissue, collection and transport of specimens, the basics of fixation, and simple, compound and special fixatives.
After this unit you can
- Describe the scope, equipment care and safety in histotechnology
- Describe methods of tissue examination and specimen handling
- Explain the principles and aims of fixation
- Classify fixatives and describe common ones
PTU syllabus topics
- Introduction to histotechnology
- care and maintenance of laboratory equipment
- safety measures
- routine methods of tissue examination
- collection and transportation of specimens
- basic concepts of fixation
- simple/compound/special fixatives
10% neutral buffered formalin
Simple, routine
Most tissues: the standard fixative
Bouin's fluid
Compound (picric acid)
Testis, embryo, biopsies
Zenker's fluid
Compound (mercuric chloride)
Bone marrow, spleen
Carnoy's fluid
Alcohol-based
Glycogen, nucleic acids
Topic 1
Introduction to histotechnology
- Histotechnology: preparing tissues for microscopic examination — reception, grossing, fixation, processing, embedding, sectioning, staining and mounting — so that pathologists can diagnose disease, especially cancer.
- Microtome
- Clean wax debris, oil moving parts, lock the wheel, guard the blade
- Tissue processor
- Change reagents on schedule, check timers and leaks
- Embedding station and oven
- Check wax temperature (2–4 °C above melting point)
- Water bath
- Clean daily; correct temperature
- Cryostat
- Defrost and disinfect regularly
Topic 2
Safety measures
Formaldehyde
Carcinogen, irritant
Fume hoods, ventilation, exposure monitoring, spill kits
Xylene and solvents
Toxic, flammable
Fume hood, substitutes, fire precautions
Sharps
Microtome blades, glass
Blade guards, forceps, sharps bins
Biological
Unfixed tissue (TB, prions)
PPE; fix before handling; prion protocols
Acids and picric acid
Corrosive; dry picric acid is explosive
Keep picric acid wet; acid storage
Topic 3
Routine methods of tissue examination
Paraffin sections
Fixed, processed, wax-embedded, 3–5 µm sections
Routine diagnosis
Frozen sections
Rapid freezing and cryostat cutting
Intraoperative diagnosis in 10–15 minutes; lipids, enzymes
Smears and imprints
Cells spread or touched onto slides
Cytology, rapid diagnosis
Teased preparations
Tissue pulled apart
Nerve and muscle fibres
Celloidin and resin
Special embedding
Eye, bone, electron microscopy
Topic 4
Collection and transportation of specimens
- Place tissue immediately in 10 times its volume of fixative in a leak-proof, labelled container; complete request form with clinical history, site and time of collection (cold ischaemia time — under 1 hour for breast specimens for receptor testing). Fresh tissue is sent unfixed only for frozen sections, cultures, cytogenetics or special studies.
- Reception: match labels and forms, assign accession numbers, record time, reject unlabelled specimens.
Topic 5
Basic concepts of fixation
- Fixation: preserving tissue as close to its living state as possible by stopping autolysis and putrefaction and stabilising proteins.
- Prevent autolysis
- Stops enzyme self-digestion
- Prevent putrefaction
- Kills bacteria and fungi
- Preserve structure
- Hardens tissue for sectioning
- Improve staining
- Mordanting effect
- Make safe
- Inactivates many pathogens
- Volume
- Fixative 10–20 times tissue volume
- Thickness
- Slices 3–5 mm; formalin penetrates about 1 mm per hour
- Time
- 6–48 hours for formalin (at least 6 hours for receptor testing)
- Temperature
- Room temperature routinely; heat speeds it
- pH and osmolality
- Buffered, near neutral
Topic 6
Simple, compound and special fixatives
Simple fixatives
Formaldehyde, glutaraldehyde, alcohol, acetic acid, picric acid, mercuric chloride, osmium tetroxide
Single chemical
Compound (mixed) fixatives
Bouin's (picric, formalin, acetic acid), Zenker's (mercuric chloride, dichromate, acetic acid), Helly's, Carnoy's
Combine advantages
Special fixatives
Glutaraldehyde for electron microscopy; alcohol for glycogen; formal calcium for lipids
Specific purposes
10% neutral buffered formalin
40% formaldehyde 100 mL, water 900 mL, phosphate buffer
Routine; immunohistochemistry
Formal saline
Formalin in 0.9% saline
Routine (older)
Bouin's fluid
Picric acid, formalin, acetic acid
Testis and GI biopsies; yellow colour must be washed out
Zenker's fluid
Mercuric chloride, potassium dichromate, acetic acid
Bone marrow, nuclear detail; mercury pigment removed with iodine
Carnoy's fluid
Ethanol, chloroform, acetic acid
Rapid fixation; glycogen, nucleic acids
Glutaraldehyde 2.5%
Buffered
Electron microscopy
- Formalin pigment (acid formaldehyde haematin) forms in acidic formalin and blood-rich tissue — prevented by buffering, removed with alcoholic picric acid.
Key terms
- Histotechnology
- Preparation of tissues for microscopic study
- Fixation
- Preservation of tissue structure
- Autolysis
- Self-digestion of tissue by its own enzymes
- Compound fixative
- Mixture of fixing chemicals
- Neutral buffered formalin
- Standard routine fixative
Quick revision
- Histology workflow; equipment care; hazards and controls.
- Paraffin, frozen, smear, teased, special embedding methods.
- Collection in 10 volumes of fixative; labelling; cold ischaemia time.
- Aims and factors of fixation.
- Simple, compound, special fixatives; NBF, Bouin's, Zenker's, Carnoy's, glutaraldehyde; formalin pigment.
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.Define fixation.
- Q2.Why is formaldehyde a hazard?
- Q3.What volume of fixative should be used?
- Q4.Give the composition of Bouin's fluid.
- Q5.Which fixative is used for electron microscopy?
- Q6.How is formalin pigment prevented?
Long-answer questions
- Q1.Describe the collection, transport and reception of histology specimens.
- Q2.Explain the aims of fixation and factors affecting it.
- Q3.Classify fixatives and describe commonly used ones.
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