Histology Flashcards
What is the order of preparing a histology slide?
Fixation
Embedding
Sectioning
Frozen sectioning
Staining
What is fixation of tissue and how is it done?
To preserve tissue and maintain ‘life-like’ structure:
- Take fresh or perfused tissue and cut into 1cm cube.
- Place into fixative, such as 10% formalin.
- Leave for 24-28 hours.
How is embedding carried out?
In dehydration, specimen is immersed in ascending grades of alcohol to remove water and formalin.
In clearing, organic solvent removes alcohol to allow infiltration with paraffin wax.
In infiltration, the specimen is infused with molten paraffin wax.
How is sectioning carried out?
- Use rotary microtome to cut tissue sections 3-10 microns (/3-5microns?) thick.
- Float tissue section in warm water bath to flatten.
- Pick up sections onto glass microscope slide.
- Allow to dry at 37˚ to ensure sections adhere to slide.
- The tissue is now ready for staining.
What is frozen sectioning used for and how is it carried out?
- Freeze specimen in cryostat.
- Section frozen specimen with rotary microtome within freezer cabinet.
- Mount section on warm glass slide (room temperature).
- Dry slide in air.
- Tissue morphology can be compromised but process is quick, like for biopsy diagnosis.
How is basic staining technique carried out?
- Remove wax with a citrus oil based solvent and rehydrate sections through descending alcohols.
- Place sections in tap water.
- Use buffered stain solutions to highlight different structures, like haematoxylin, which stains blue.
- Stain tissue with haematoxylin for 10 minutes.
- Rinse slides in tap water between stages.
- Stain is differentiated by placing in acid-alcohol until the nuclei are stained blue but all other tissues are colourless.
- Neutralise pH with alkaline tap water.
- Use eosin for 10 seconds to stain the cytoplasm, collagen and muscle fibres red.
- Between each stage, use a microscope to check for correct level of staining.
- Dehydrate sections with ascending grades of alcohol for 10 minutes.
- Clearing agent for 15 minutes and removes all traces of alcohol and raises refractive index to make tissue more transparent.
- Add synthetic moulder, DMX, to a cover slip and then place on top of the section.
- When the adhesive sets, the coverslip will be held in place to protect the stained tissue.
Name basophilic stain examples and describe what cellular components they are used on.
Basic stains, such as toluidine blue and methylene blue.
Used on acidic cellular components, such as:
- DNA in nucleus
- RNA in nucleolus
- RNA in the free ribosomes and RER
Name acidophilic stain examples and what cellular components they are used on.
Acidic stains, such as eosin, orange G and fast green.
Used on basic cellular components, such as:
- Red blood cells
- Most proteins
State the colours that haematoxylin and eosin (H&E) stains in different cellular components.
Blue = nuclei, cytoplasmic RNA
Red = muscle, keratin, course elastic fibres
Pink = collagen, reticulum, nerve fibres
Orange = red blood cells
State the colours that trichromes stain in different cellular components.
Blue, black or red = nuclei
Red = muscle, fibrin
Blue or green = connective tissue
Red or yellow = red blood cells
State the colours that Verhoeffs (V.H) stains in different cellular components.
Black = nuclei, elastic fibres
State the colours that Ven Giesons (V.G) stains in different cellular components.
Black = nuclei
Red = mature collagen
Yellow = muscle, red blood cells
State the colours that osmium tetroxide stains in different cellular components.
Black
Not a stain but used to fix/stain cell membranes
State the colours that Periodic acid Schiff stains in different cellular components.
Pink to deep purple = polysaccharide, glycoproteins, mucopolysaccharides
State the colours that Alcian blue stains in different cellular components.
Blue = mucopolysaccharides, and hence mucin
Name the 3 classes of epithelial cells
Simple
Stratified
Pseudo-stratified
How are simple epithelial cells further classified?
Squamous
Cuboidal
Columnar > ciliated and non-ciliated
Relate the structure of simple squamous epithelium to its function.
Epithelia are one cell thick and are flat. They line vessels where they are called endothelium. Epithelia allow a shorter diffusion pathway for the exchange of substances to be more efficient.
Relate the structure of simple cuboidal epithelia to its function.
Simple cuboidal epithelia are found in the tubules of the kidney, the ductal systems of the salivary glands, thyroid gland and pancreas. They regulate the transport of specific substances across them. Being taller than the simple squamous epithelium means that they have more control over substances that diffuse through them.
Relate the structure of simple columnar epithelia to its function.
Can be ciliated or non-ciliated. Make up villi and microvilli in the small intestine, for example. Acts to increase surface area for absorption of substances.
Relate the structure of stratified squamous epithelia to its function.
More than one layer of cells. A stratified squamous epithelium provides protection to the underneath tissues by:
- Being thicker than a simple epithelium
- Being keratinised (keratin strengthens epithelial surfaces)
- Having a high cellular turnover – damaged surface cells are quickly replaced from below
Why is the level of keratinisation in stratified squamous epithelium different in the footpad of a dog and the dog’s oesophagus?
Very high in footpad, as the skin is subject to a high degree of friction and potential dehydration.
Low in oesophagus, as the risk of hydration is lower as it it moist within the oesophagus and the level of abrasion is lower.
Why are keratin’s strengthening properties not utilised within the corneal epithelium?
Keratin would prevent light entering through the cornea.
Relate the structure of stratified transitional epithelium to its function.
Is several cells thick and the surface cells can change shape, from cuboidal to squamous, according to the degree of stretch. Only found in the urinary system, to allow the lumen of the bladder to expand in volume without an increase in pressure.
Relate the structure of pseudostratified epithelia to its function.
Appears to be several cells thick but is only 1, as each cell is attached to the basement membrane. Has varying locations.
In airways, they protect airways and allow movement of air. They do this using goblet cells to produce mucus to trap irritants and have cilia on epithelia cell surfaces to waft mucus in mucociliary escalator.
How could you tell the difference between the epithelium found in the trachea from the epithelium of duodenum?
Presence of cilia and location of nuclei in the cells.
What are serous secretory glands?
Serous secreting cells secrete a watery secretion with an ionic composition very close to blood plasma.
What is serous demilune?
Produce watery secretion to help flush any mucous through the ducts that empty the mucus secreting cells.
Describe the structure of a major salivary gland.
Intralobular: striated ducts branch into intercalated ducts and to secretory cells
Interlobular: main duct branches into collecting duct
What are acinar cells?
Acinar cells are under enteric control and when they contract around myoepithelial cells, they squeeze these cells to pressurise them and squeeze out the fluid that’s within the duct system, to eject saliva into the mouth rapidly.
What is the function of striated ducts?
Altering the composition of saliva
How can you differentiate acinus from an intercalated duct?
Intercalated duct shows a perfect ring of little nuclei in a tight ring, sometimes with a lumen, but this is not always seen/obvious.
Serous acinous – nuclei are more pr less central within the cell.