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.
How can you differentiate a serous secreting and a mucus secreting cell?
• If serous it will be a little better stained.
• If mucus secreting acinus, nuclei will be pushed up against the basal membrane and flattened and the cytoplasm will appear very pale, perhaps a little fuzzy/flocculent.
Describe the tissue composition of the tongue.
- Muscular organ covered by epithelium, ventral and dorsal surfaces being very different.
- Surface has obvious papillae, which depending on what the animal eats
- May be keratinised for protection.
- There is glandular tissue very often inside the tongue
- Lots of muscle running in different directions.
- Taste buds are embedded in stratified squamous epithelium. Is a cluster of up to 100 neuroepithelial cells that detect chemicals in food.
State 4 types of papillae.
Fungiform
Filiform
Circumvallate
Foliate
What is the lyssa?
A structural modification of the dog tongue, which is a rod-like structure. Made of adipose tissue.
State the 10 layers of the oesophageal wall.
Stratified squamous epithelium
Lamina propria
Muscularis mucosae
Submucosa
Glandular tissue
Muscular externa (circular)
Muscular externa (longitudinal)
Muscular externa (both circular and longitudinal)
Myenteric plexus - ganglia of enteric nervous system
Adventitia
What are the 4 layers of the GI tract wall?
• Mucosa – has epithelium, lamina poropria and ascularis mucosa
• Submucosa
• Muscularis externa – circular and longitudinal
• Serosa/adventitia
What is the structure and function of submucosal plexus?
Nerve cells of myenteric plexus have large rafts of cell bodies. Can appear as islands of tissue.
Submucosal plexus controls glandular tissue.
Describe the surface of the stomach.
Columnar epithelium cells and gastric pits, which disappear deep into the mucosa. Gastric pits is where acid and pepsin emerge.
- Mucous neck cells, hardest to fund due to crescenteric nuclei – secrete mucus to protect the stomach epithelium as the acid and enzymes emerge
- Parietal cells, large and pyramid shaped – secrete acid
- Peptic/chief cells, most numerous type
What are the 2 main states of parietal cells?
Resting - lot of membrane structure within the cell with tubular vesicles in order to separate the different components of the secretory mechanism. This is one way of turning the cell’s machinery off.
Active and secreting HCl - cell allows all the tubular vesicles to fuse together to form these finger-like projections, which massively increase the surface area fir secretion. Isotonic HCl emerges from apical membrane. Basolateral membrane is in contact with the capillaries in the area.
What are canaliculi?
The luminal plasma membrane forms deep, branching canaliculi that extend throughout the cytoplasm. Numerous microvilli project into canaliculi to increase surface area. In a secreting cell, the tubulovesicles fuse with the canaliculi and secrete HCl. Protons are actively pumped and chloride ions follow passively. Active process requires numerous mitochondria.
Describe the appearance of peptic cells.
- The nucleus is very conspicuous and is basal
- Majority of the rest of the cell is filled with these zymogen granules, which gives the cells foamy appearance
- Lots and lots of endoplasmic reticulums and many ribosomes on the surface of the Golgi apparatus
What are 5 characteristics of intestinal villi?
• Shape – cylindrical to increase surface area
• Epithelial renewal – from crypts for 5 day lifetime. As they mature, they move up almost like a conveyer belt, moving the cells up the crypts. Eventually they emerge out of the crypts on to the surface and up the villi. This process takes about 5 days.
• Microvasculature – small arteries and venules
• Lymphatics – central lacteal
• Smooth muscle – extension from muscularis mucosae. Therefore muscularis mucosae is able to wave finger-like projections in a wave to move them around. This helps bring villi into contact with more of the contents of the gut to improve absorption.
What are crypt cells?
Their function in the crypts is mainly to secrete fluid but increasingly they become matured and adapted for absorption as they move up.
The crypt is the glandular invagination of the mucosa.
What are Paneth cells?
At the base of crypts there are Paneth cells, which produce a variety of substances that bathe the rapidly dividing cells and molecules that effectively kill microbes. Any area of the body which has rapidly dividing cells is a soft spot for invasion of microorganisms, so Paneth cells effectively bathe them in antiseptic.
They have a protective role and are found in region of rapid cell division which is a weak spot for the body’s defences.
Name 4 functions of the liver.
• Metabolic – glucostatic function, protein synthesis, urea formation, lipid metabolism
• Excretory – bile pigments excretion, elimination of drugs
• Secretory – bile acids
• Functional reserve and great regenerative capacity
Describe the structure of hepatic lobules.
Hepatic lobules are the functional units of the liver. Regions of the poles of these hexagonal cells are called portal regions and they have branch of hepatic portal vein, lymphatic vessel, branch of the hepatic artery, bile ductule. There are also central veins where blood drains. Blood flow goes from portal regions to central vein and bile flow is from the central vein to the bile ductules in the portal regions.
Describe the structure of the bile ducts.
Bile is pulled out of the hepatic blood and into the hepatocytes and into an intercellular bile canaliculi. They drain into tiny ducts and then into larger interlobar ducts.
What do the vesicles of of peptic/chief cells contain and look like?
Digestive enzyme pepsin
Pale-staining foamy appearance of apical cytoplasm
Why are there many mitochondria in granular basal cells?
Protein synthesis
What function of parietal cells requires a high density of mitochondria?
HCl formation
What is the connection between parietal cells and vitamin B12 in many mammals?
Parietal cells secrete both HCl formation and intrinsic factor, which is essential for absorption of vitamin B12.
What is the function of Brunner’s glands?
Secrete bicarbonate to raise pH towards pH7
In the renal cortex, what are the 2 distinct profiles of tubule?
- Dark stained profiles of tubule are the proximal tubules, which are much more metabolically active than other parts of the tubule. They are packed with mitochondria and endoplasmic reticulum.
- Paler sections are distal tubules, which are less metabolically active.
There are many peritubular capillaries.
In the kidneys, what is the function of mesangial cells?
Secrete extracellular matrix that supports this network of capillaries.
What is the renal and vascular pole?
Renal pole – where filtrate drains into the proximal tubule from the glomerulus and Bowman’s space to become urine.
Vascular pole – where the afferent and efferent arterioles enter and leave. Where the juxtaglomerular apparatus.
Describe the epithelial cells of the collecting duct.
Collecting duct epithelial cells go from cuboidal at the cortex and more columnar at the renal pelvis. General size of the collecting duct becomes much bigger too.
Describe the epithelium of the ureter and how this adapts the ureter for its function.
Transitional epithelium is stratified and has a shape that allows it to expand to accommodate boluses of urine. Shape of ureter allows it to expand without damaging the underlying tissue to allow a bolus of urine along the ureter by peristalsis.
Describe the structure of the bladder.
- The rugae of the bladder can flatten out and allow the volume of the bladder to increase as it fills. Bladder lumen is lined by transitional epithelium again and is specialised diamond shaped cells that allow the bladder to expand, which flatten but are not damaged.
- There is thick muscular coat of detrusor muscle that will contract and micturition to void urine from the bladder. Muscle is made of bands of muscle that are arranged in a spiral fashion around the wall of the bladder.
- There is a layer of outer conncetive tissue surrounding the outside
What are the similarities and differences between the ureter and bladder?
- Lining and topography of lumen
- Transitional epithelium lining the lumen
- Smooth muscle in the wall
But more connective tissue and less muscle in the ureter than in the bladder.
What does stimulation of the sympathetic nerve fibres to the kidney have on afferent arterioles and urine production?
Vasoconstriction and so reduced filtration and urine production
Describe the histological features of the proximal convoluted tubule.
- More PCT than DCT, as PCT is longer
- Dark blue/purple = basophilic cytoplasmic stain
- Simple cuboidal
- Taller epithelium than DCT
- Has a brush border
Describe the histological features of the distal convoluted tubule.
- Less DCT than PCT, as PCT is longer
- Pale stain, less basophilic cytoplasmic staining than PCT due to less RER
- Simple cuboidal
- Shorter epithelium than PCT
- No brush border
What is renin secreted in response to?
- Reduced blood pressure
- Low sodium activating macula densa
- Sympathetic stimulation
What histological features of the bladder enable it to expand to accommodate urine?
Rugae in the walls flatten to increase volume and transitional epithelium can flatten and expand without being damaged – diamond epithelium.
What type of muscle is found within the muscularis layer of the ureteral and bladder walls?
Smooth
Name the general features of endocrine cells.
• Islands of secretory epithelial cells with supporting tissue
• Simple structure – clusters or branching cords of cells
• Globular follicles – stored secretions
• Solitary cells – C cells in the thyroid
• Do not have ducts
• Rich capillary and lymphatic network
Describe the appearance of the thyroid gland under a microscope.
- Characteristic follicles containing acidophilic colloid
- Lined with epithelial cells
- Many capillaries cab be hard to see in H&E
- C cells scattered or in small clumps
- Species differences
How does the appearance of the thyroid gland change when stimulated by TSH?
Upon TSH stimulation, cuboidal epithelium becomes columnar and the follicles full of colloid are depleted/collapsed. There is hypertrophy of the follicle cells, increased colloid uptake for T3 and T4 production.
Describe the appearance of the parathyroid gland under a microscope.
- Lots of chief cells
- Branching cords – carry blood and lymphatic vessels and nerves
- Rich capillary network
- Oxyphil cells – number increases with age
Describe the appearance of the adrenal gland under a microscope.
- Connective tissue capsule, cortex and a medulla
- Cortex is glandular in 3 layers – zona glomerulosa, zona fasciculata, zone reticularis
- Medulla – large rounded cells in groups/cords
Describe the appearance of the posterior and anterior lobes of the pituitary gland under a microscope.
Posterior:
- Neuroendocrine cells
- Mostly unmyelinated
- Have herring bodies (red)
Anterior:
- Glandular cells in clusters
- Chromophobes
- Acidophils
- Basophils
What epithelium is in the wall of the thyroid follicles?
Cubiodal
Why does the parathyroid gland possess so many capillaries?
So that parathyroid hormones can be easily and quickly secreted into the circulatory system.
What are the characteristics of the thyroid gland?
- Follicles containing acidophilic colloid
- Lined with epithelial cells
- Many capillaries can be hard to see in H&E
- C cells scattered or in small clumps
What happens to the thyroid epithelium upon TSH stimulation?
Cuboidal epithelium becomes columnar and the follicles full of colloid are depleted/collapsed. There is hypertrophy of the follicle cells, increased colloid uptake for T3 and T4 production.
What are the characteristics of the parathyroid gland?
- Lots of chief cells
- Branching cords – carry blood and lymphatic vessels and nerves
- Rich capillary network
- Oxyphil cells – number increases with age
What are the characteristics of the adrenal gland?
- Connective tissue capsule, cortex and a medulla
- Cortex is glandular in 3 layers: zona glomerulosa, zona fasciculata, zone reticularis
- Medulla – large, rounded cells in groups/cords
What are the characteristics of the posterior lobe of the pituitary gland?
- Neuroendocrine cells
- Mostly unmyelinated
- Have herring bodies (red)
What are the characteristics of the anterior lobe of the pituitary gland?
- Glandular cells in clusters
- Chromophobes
- Acidophils
- Basophils
What are the characteristics of the pancreas?
- Exocrine and endocrine
- Endocrine cells grouped in islets of Langerhans – alpha in pale red pink and beta in slate grey blue
