W7- Lecture 35.1- Histology- Tissues And Processing Flashcards
How many cells are there in the body ?
How many diff types of cells ?
10^15
200+ types of cells
Name the 4 main types of tissues
Connective
Nervous
Muscle
Epithelial
Name two complex structures that make the extracellular matrix
Collagen fibrils
Basement membranes
Name the 4 main functions of the extracellular matrix (ECM)
Mechanical support for cells
Transport nutrients to cells
Carrys metabolites and secretory products away from the cell
Attach to receptors/ cell membrane surface helps reaction to stimuli/inhibitors
What are the 5 stages of specimen preparation
Fixation Dehydration - Alcohols Clearing - Xylene Embedding - Paraffin wax Sectioning
How long does a preparation of a specimen normally take ?
16-48 hours
Describe the first step of specimen preparation ? (fixation)
Aim/ desired outcome
prevent autolysis and bacterial attack
Stop enzymatic activity/ breakdown of tissue
Prevent tissues from swelling/shrinking (remain tissue close to true state )
Enable tissue to be clearly stained +
Maintain +stabilises molecular and structural composition
What factors are fixation dependant on ?
pH
Temperature
Fixative penetration
Volume of tissue
Name 6 types of fixative chemicals
+ what they fix
+e.g
Aldehyde- reacts with amine groups of tissue proteins (e.g Formaldehyde and glutaraldehyde- the later is reinforced as it is a dialdehyde and can cross link proteins )
Ketones- e.g acetone
Alcohol fixative - coagulated proteins (e.g methanol )
Zinc fixative- amino, carboxylic andsulphydry groups - REVERSIBLE
Temperature (freezing )
Picric acid aka bousin’s fluid - coagulation fixative changes the charge on the th- disrupt hydrogen bonding
Describe the second step in specimen preparation (dehydration)
- purpose
- with what substances
- technique / why
Purpose to remove fixative and water from the specimen and replace with dehydration fluid
With alcohol e.g ethanol, methanol
Technique via graded alcohol series increasing the alcohol % each time
Why- to reduce tissue distortion/ swelling+ damage to cells
Describe the third step of specimen preparation (clearing)
Purpose
What the clearing agent depends on
Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium
Type of tissue Type of processing Processor system being used Processing conditions (temp, pressure, vacuum) Safety conditions Cost/ convenience Speedy removal of dehydrating agent Ease of removal by molten paraffin wax Minimal tissue damage
Name 7 typical clearing agents
Zylene Toluene Chloroform Benzene Petrol Histo-clear® HISTOCHOICE™
Describe the fourth stages of specimen preparation (embedding )
Purpose
Technique
That tissues are surrounded by a medium which will provide sufficient external support during sectioning.
The wax must be free of clearing agent.
No dust particles must be present.
Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size.
5 types of embedding mediums
+ size of sections (smallest to largest)
Plastic Resin (very thin sections 0.5 - 1 μm)
Paraffin wax (sections ≥ 3 μm)
Polymerizing resin i.e. Epoxy, Aradite
Agar ( 10 - 20 μm fixed)
Cryo-embed medium (sections ≥ 5 – 100 μm)
Agar (sections 100 - 300 μm unfixed)
Describe embedding a sample in paraffin wax
What is paraffin wax ?
Qualities
How it can be altered
polycrystalline mixture of solid hydrocarbons
It is about two thirds the density and slightly more elastic than dried protein
melting points which range from 39°C to 68°C
Can add other substances Improve ribboning Increase hardness Decrease melting point Improve adhesion between specimen and wax
