Introduction of practical pathology Flashcards
How to process tissue for light microscopy
Specimen must be thin enough for light penetration
Thinner than dimensions of typical cell
How to preserve tissue/cell structure
Treat specimen with fixative Halt metabolism Render cellular macromolecules insoluble through denaturation and cross linking Inactivate degradative enzymes Neutral buffered formaldehyde
How to cut tissue sections thinner than typical nucleated mammalian cell
Support tissue by freezing water
Can also replace water with more supportive medium (wax)
Dehydrate tissue gradually in increasing con of ethanol to prevent severe shrinkage
Immerse in solvent common to ethanol and wax to remove lipid (histoclear)
Wax infiltrates cells and spaces between cells
How to cut wax embedded tissue on microtome
Cooled and solid wax embedded tissue cut into 5-15um slices with steel knife from microtome
Wrinkled section flattened by floating in warm water, picked up on glass slide
How to differentiate between different cellular components
Treat section with stains that bind selectively to different structures making them different colours
How to stain tissues in wax
Stains are aqueous, immiscible with wax
Rehydrate as wax no longer needed as section cut and supported on glass slide
Remove wax with xylene/toluene
Rehydrate section with ethanol solution of decreasing concentration to prevent severe distortion
How to preserve stained section so stain won’t leach out
Dehydrate with graded alcohols
Remove ethanol with xylene/toulene
Use DPX as adhesive, secure coverslip
Hematoxylin
Basic dye
Binds to -ve charged structures (DNA/RNA)
Eosin
Acidic dye
Binds to +ve charged structures (most cellular proteins)
Trichrome
Shows nuclei and cytoplasm
Differentiations between collagen and smooth muscle
Different colors of organelles in hematoxylin and eosin
Nuclei blue Collagen pale pink Cytoplasm pink Muscle fibres red/deep pink Erythrocytes orange
Different colors of organelles in trichrome
Nuclei blue/grey Cytoplasm blue/green Cytoplasm blue Muscle fibres red Erythrocytes red
