Fixation, Fixatives, and Tissue Processing

A comprehensive set of question-and-answer flashcards covering definitions, classifications, formulas, advantages, disadvantages, safety, and procedural steps related to fixation, fixatives, and routine paraffin tissue processing.

What is fixation in microscopy?

A process that preserves the internal and external structures of cells/micro-organisms, inactivates enzymes, and firmly attaches the specimen to the slide for staining and observation.

Name the two fundamental types of fixation and their typical use.

1. Heat (flame) fixation – for bacterial smears, preserves general morphology.
2. Chemical fixation – for delicate or larger specimens, preserves fine sub-structure.

How does heat fixation preserve bacterial smears?

By gently passing an air-dried smear through a flame, killing the bacteria and coagulating proteins to keep cells attached without major structural detail preservation.

Why is chemical fixation preferred for fine cellular substructure?

Chemical fixatives penetrate cells and react with proteins/lipids, rendering them insoluble and immobile, thus maintaining delicate internal architecture.

List five common chemical fixatives.

Formaldehyde, glutaraldehyde, mercuric chloride, osmium tetroxide, acetic acid (others include ethanol, picric acid, chromic acid).

What are post-mortem changes?

Destructive alterations (putrefaction or autolysis) that begin shortly after death or tissue removal.

Define putrefaction.

Tissue decomposition caused by invasion and proliferation of bacteria, usually originating from the alimentary tract.

Define autolysis.

Self-digestion of dead cells by their own released enzymes, especially severe in CNS and endocrine tissues.

Give four essential requirements of a good fixative.

(Any four) Rapidly kills without distortion, penetrates evenly, renders cell components insoluble, inhibits bacteria/autolysis, hardens tissue, allows later staining, permits long storage, restores colour, economical/simple.

How does temperature influence fixation and autolysis?

Low temperature slows both fixation and autolysis; high temperature speeds fixation but accelerates autolysis. Prolonged cold fixation is preferred when immediate processing is impossible.

What are simple fixatives?

Individual chemical substances that can fix tissue on their own (e.g., formaldehyde, glutaraldehyde, osmium tetroxide).

What are compound fixatives?

Mixtures of two or more simple fixatives combined to exploit their complementary actions on tissue.

Differentiate micro-anatomical and cytological fixatives.

Micro-anatomical fixatives preserve overall tissue relationships; cytological fixatives target specific cell parts and are subdivided into nuclear and cytoplasmic types.

State the composition of 10 % formol-saline.

4 % formaldehyde (from 40 % formalin) + 0.85 % sodium chloride in distilled water to 100 mL.

Give two advantages of 10 % formol-saline.

Even fixation with minimal shrinkage; compatible with most stains and excellent for CNS work (also restores natural colour for museum specimens).

Mention one disadvantage of 10 % formol-saline.

Slow fixation; tissues may shrink during dehydration, and formalin vapour is irritating.

To what concentration is formaldehyde gas soluble in water (commercial formalin)?

Approximately 40 % (w/w).

What precipitate can form in stored formaldehyde and how is it avoided?

White para-formaldehyde; minimize by storing at room temperature and stirring before use.

What is the main histological action of mercuric chloride?

Rapid protein precipitation and tissue hardening, enhancing colour contrast for trichrome stains.

Why must tissues fixed with mercuric chloride receive post-treatment?

To remove black mercuric deposits that can obscure microscopic detail.

State one major safety hazard associated with osmium tetroxide.

Its volatile vapour can deposit black oxide (OsO₂) in the cornea, causing blindness—must be handled in a fume hood with dark bottles.

What must be done to chromic-acid-fixed tissue before dehydration?

Thorough washing in running water to remove acid and prevent insoluble sub-oxide formation.

Which tissues are best fixed with Zenker-formal (Helly’s) and for how long?

Pituitary and bone-marrow specimens; 12–24 hours.

What is Flemming’s fluid primarily used for?

Preservation of nuclear structures, especially chromosomes.

List the components of Flemming’s fluid.

15 mL 1 % chromic acid, 4 mL 2 % osmium tetroxide, 1 mL glacial acetic acid.

Give one disadvantage of Flemming’s fluid.

Poor penetration limits use to small tissue pieces; solution deteriorates quickly and must be freshly prepared.

For what purpose is Carnoy’s fluid recommended?

Fixing chromosomes, lymph glands, and urgent biopsies requiring rapid dehydration.

State the formula of Carnoy’s fluid.

60 mL absolute alcohol + 30 mL chloroform + 10 mL glacial acetic acid.

Why is Carnoy’s fluid unsuitable for large tissue blocks?

Causes excessive shrinkage and haemolysis of red blood cells; best for small samples.

Which cytoplasmic fixative is identical to Flemming’s fluid minus acetic acid?

Flemming’s fluid without acetic acid (used for mitochondria).

What is the primary purpose of tissue processing?

To infiltrate tissue with a solid support medium so thin, even sections can be cut for microscopy.

Arrange the main steps of paraffin tissue processing.

Fixation → Dehydration (graded alcohol) → Clearing (xylene/toluene) → Infiltration with molten paraffin → Embedding → Sectioning.

Why must tissues be dehydrated before paraffin infiltration?

Paraffin is immiscible with water; residual water prevents clearing and wax penetration, leading to poor sections.

Define clearing and give a common clearing agent.

Replacement of alcohol with a solvent miscible with both alcohol and wax; xylene is the most common agent.

What is infiltration (impregnation) in tissue processing?

Replacement of the clearing agent with molten paraffin wax, filling all tissue spaces.

How does vacuum impregnation accelerate infiltration?

Reduced pressure removes trapped air and speeds replacement of clearing agent by wax, useful for urgent biopsies.

Define embedding in histology.

Burying the infiltrated tissue in molten wax within a mould; on cooling it forms a solid block that supports cutting.

Name two common types of embedding moulds.

Leuckhart (L-piece) boxes and Tissue-Tek metal base moulds (others: plastic ice-cube trays, Petri dishes).

Briefly describe the standard water-bath method for mounting paraffin sections.

Float cut sections on 20 % alcohol, transfer to a 45 °C water bath, pick up on a clean adhesive-coated slide, drain, and dry on a hot plate 3–5 °C above wax melting point.

State two properties of a good paraffin wax.

Clear, colourless, water-free; free of staining-interfering impurities; no crystal formation on cooling; good tissue penetration.

Give one advantage and one disadvantage of paraffin wax.

Advantage – gives durable serial ribbon sections quickly. Disadvantage – high melting point (50–60 °C) may destroy enzymes and requires complete dehydration/clearing.

Why are section adhesives used?

To prevent sections lifting or floating off slides during staining procedures.

Which slide adhesive should be avoided when demonstrating carbohydrates and why?

Starch or celloidin adhesives, because they are carbohydrates themselves and may give false-positive reactions or artefacts.

List the reagent sequence in routine manual tissue processing (approximate times).

70 % alcohol 1–6 h → 90 % alcohol 1–6 h → 95 % alcohol 1–16 h → Absolute alcohol (2 changes, 2.5 h each) → Chloroform/cedarwood oil overnight → Xylene 1 h → Paraffin wax (3 changes, 1 h each) → Embed.