Steps for preparing tissue for microscopic assesment (after surgical excision)
- Fixation: preserve cell structure
- Dehydration: Remove water = harden sample
- Support: Infiltration (wax)
- Microtomy: thin sections
- Microscopy: differential staining (nucleus & cytoplasm contrast)
- Seal the preparation: preserve stain
Purpose of fixation
Preserve tissue architecture (life-like). Stop degeneration
Causes of degeneration once tiss. removed from body
- Anoxia (no O2)
- Autolysis (self digest of cells bc release enzymes)
- Putrefaction (bact. decomposition)
- Dehydration
- Pressure (e.g. bruise)
- Osmotic injury
What fixation does (to stop post mortem changes)
- Stops autolysis
- Prevent bact. decomposition
- Hardens tiss.
- Permanent preservation
- Stabilise tiss. to allow further treatment
- Enhance avidity for dyes
- Min. loss soluble cytoplasmic components
Factors involved in fixation
- Temp.
- Size of specimen
- Penetration (depend on SA)
- pH (~6-8)
- Osmolality (slightly hypertonic)
- Duration: preserved ASAP
List 3-5 features of ideal fixatives
- quick & even penetration
- life-like preservation
- not add artefact material
- not swell/shrink tiss.
- safe for user & enviro.
- convenient shelf-storage
- economical
Cell structures that need to be stabilised
- lipoproteins of plasmalemma
- cytoskeleton fibrous proteins
- fibrous glycoproteins (collagen, basement memb.)
- globular proteins of cytoplasm & xtra cell. fluid
- nucleic acids
- mucosubstances (e.g. HA)
4 chemical fixatives
- Aldehydes: formaldehyde, glutaraldehyde
- Oxidising agents: K2Cr2O7 (dichrom.), CH3COOH
- Protein coagulants: eth+methanol, mercuric chloride
- Compound fixatives: combo w/ above
Dis+Advantage of formalin
Adv: preserve biomarkers, stable specimens long periods, inexpensive
Dis: oxidises when stored -> formic acid, Irritant
Principles of Tiss. processing (6 steps)
- Fixation
- Dehydration: remove H2O (w/ ethanol)
- Clearing: remove dehydrating agent
- Impregnation: remove clearing agent
- Embedding: support for sectioning
- Microtomy: thin sections
Purpose of staining
contrast diff. elements of tiss.
5 chemical bonds dyes bind onto elements
Ionic, covalent, H bond, van der waals, hydrophobic
Describe some common DEHYDRATION agents and their properties
Alcohol: *Ethanol (50, 70, 95, 100%), methanol, isopropanol
Other: Acetone (fat)
» Rapidly removes water
Describe some common CLEARING agents and their properties
Hydrocarbons: xylene, chloroform, benzene, toluene
|»_space; remove dehydrating agent
How pH alter dye binding?
Acid pH (from proteins) favours anionic dyes: Hi [H+] favours ionisation of amino groups (NH2, NH3+)
Components of zenker’s fluid
- Distilled water
- potassium dichromate
- Mercuric chloride
- Glacial acetic acid
Melting point of paraffin wax
~39º-69ºC but we use 56º-57ºC
Embedding media properties
- elasticity, density, plasticity, viscosity
