Staining/microscopy

Question 1

Define histology and pathology

Answer 1

-the microscopic study of normal cells and tissues

-the microscopic study of diseased cells and tissues

Question 2

4 main tissue types and functions

Answer 2

Epithelial tissue - Barrier and lining tissue (outer layer of skin)

Connective tissue - Provides structural and functional support (cartilage, bone and blood*)

Muscle tissue - Specialised for contraction (cardiac cells)

Nervous tissue - Carries info throughout the body via electrical impulses (nerves)

Question 3

Light and Electron microscopes

Answer 3

Light microscopes reveal basic cellular structure​

Electron microscopes can reveal ultrastructure​

Light - resolution is 0.2 μM and Electron - is 1 nm (200-fold greater)​

Light - most commonly used for routine histopathology ​

**Higher magnification allowing visualization of ultra structures​

Question 4

Steps to collect and examine tissue 1

Answer 4

Specimen collection:
Incision or punch biopsy – skin/oral surfaces.​

Needle biopsy – organs or lumps below the skin.​

Imaging techniques (X-ray, MRI) are often used to guide biopsy. ​

Endoscopic biopsy – flexible tube with light and camera. Cutting tools can be used to collect tissue specimens.​

Question 5

step 2 in collecting and examining tissue

Answer 5

Fixation:
Preserves the structural arrangement between cells and extracellular components.​

Terminates all biochemical reactions and so prevents tissue decomposition.​

Common fixatives include formaldehyde and glutaraldehyde. ​

Question 6

step 3 in collecting and examining tissue

Answer 6

Dehydration:
Paraffin embedding not compatible with water.​

Water removed from tissue using series of graded alcohols.​

Water removed gradually to prevent distortion.​

Question 7

step 4 in collecting and examining tissue

Answer 7

Embedding:
Tissues must be supported to allow thin sections to be cut.​
Resins and paraffin wax most commonly used. ​
Paraffin is not compatible with alcohol​
Alcohol replaced by xylene​

Question 8

step 5 collecting and examining tissue

Answer 8

Sectioning:
Tissue sections must be thin and flat, ideally a single layer of cells.​
Tissue sections should be transparent.​
Embedded specimens are sectioned using a microtome.​
Sections approx 7 μM thick.​
Sections are mounted on glass slides. ​

Question 9

step 6 collecting and examining tissue

Answer 9

Staining:
Staining makes cells and their components visible. ​
Allow identification of different cell and tissue features.

Most stains for LM are aqueous​
Not compatible with paraffin​
Reversal of dehydration steps to remove paraffin wax and replace with water​

Question 10

Types of Staining

Answer 10

H&E (most common)
-Haematoxylin is basic >Stains acidic structures blue/purple (nucleic acids)
-Eosin is acidic >Stains basic structures red/pink (cytoplasmic proteins)

PAS
Stains complex carbohydrate magenta such as mucins produced by goblet cells, brush borders and basement membranes

Masson Tichrome
Stains connective tissues
-nuclei blue/purple
-collagen green/blue
-cytoplasm, muscle, RBC’s red

Other protocols:
Alcain blue, Van Gieson, Reticulin stain (often combined with H&E)