intro to histology

Question 1

what is histology

Answer 1

The study of the microscopic structure of biological material and the ways in which individual components are structurally and functionally related

Question 2

diagnostics

Answer 2

Whole cells can be viewed if they can be obtained in suspension
e.g. blood smears, cytology of an effusion (pleural/pericardial/peritoneal), bone marrow aspirate, fine needle aspirate of a breast lump

This is called cytology

Question 3

fixation

Answer 3

Fixatives chemically stabilise the tissue (often by crosslinking proteins) e.g. ethanol or formalin

Question 4

cutting a thin slice of the section

Answer 4

To get the wax into all the spaces in the tissue we need to replace the water with wax

We do this by dehydrating the sample (gradually) to remove the water and replace it with something that is miscible with both alcohol and wax

We can then use a microtome (slicer) to cut the sections

Question 5

sectioning

Answer 5

Sections are prepared using a microtome

Compression of sections during cutting is rectified by ‘floating out’ in a warm water bath

Sections are collected on glass slides and these are dried

The slides may be coated to prevent the sections from dropping off during staining

De-waxed in xylene to allow penetration of stains

Question 6

cell staining

Answer 6

The most common stain is Haematoxylin and Eosin (H&E); it is a general stain Haematoxylin - basic dye  stains ACID components of cell a purple-blue – BASOPHILIC eg nuclei
Eosin - acidic dye  stains BASIC (alkaline) components of cell pink – ACIDOPHILIC – most of cytoplasm

Question 7

freezing as an alternative

Answer 7

If tissue can be obtained and frozen then the tissue processing steps are not necessary

Tissue is instead mounted in a supportive gel called OCT (Optimal Cutting Temperature compound) and sectioned using a cryostat. This is similar to a wax microtome, but in a freezer

Sections are collected on coated slides as before and kept frozen until needed for staining
They are then defrosted, lightly fixed and stained as in wax embedded tissue

Question 8

what level of detain is seen in the mictoscope?

Answer 8

The resolution of a light microscope is 0.2um

Microscope objective lenses are a maximum of 100x, the eyepiece has a magnification of (usually) 10x. Therefore the maximum magnification achieved is approx. 1000x

You can see the nucleus, cytoplasm, cytoplasmic granules, cell outline

You can’t see organelles, cell junctions or surface specialisations like microvilli (more later) with general tissue stains

You can’t see what type of receptors there are unless you use antibodies to label them

Question 9

Transmission Electron Microscopy

Answer 9

TEM uses a high accelerating voltage (80-200kV) to pass a beam of electrons through a thin section of a specimen to give fine utrastructural detail

Preparation has the same steps, but using different chemicals and a copper grid instead of a glass slide

Used in diagnosing renal disease, cancer, some mitochondrial diseases, some identifying some infectious agents, etc

Question 10

trichome stain

Answer 10

to highlight e.g. collagen (green/blue)

Question 11

Periodic Acid Schiff

Answer 11

PAS: for carbohydrates like mucin, glycogen and glycosaminoglycans (magenta)

Question 12

Weigert’s elastin

Answer 12

elastin (black)