AGRC1041: Cell Ultrastructure & Histology Quiz

Study Notes

Examining cells and tissues requires preparation for light microscopy.
Preparation steps include fixation, processing, embedding, sectioning, and staining.

Fixatives preserve cellular structure by halting autolysis and decay and stabilizing proteins.
Fixatives must be buffered to match the pH and osmolarity of the tissue.
Tissue is cut into small pieces.
Use a 10x volume of 10% neutral buffered formalin (NBF).

Tissue is kept rigid for sectioning by infiltration with paraffin wax (including plasticising polymers).
Wax is not miscible with water, so tissue is dehydrated in increasing alcohol concentrations.
Tissue is cleared of alcohol using xylene (or similar), which is miscible with wax.
Tissue is infiltrated with paraffin wax under a vacuum.

Using a microtome, 3-10 µm thick sections are cut from the block and mounted onto glass slides.

Wax is removed by reversing the prior steps: xylene → decreasing alcohol series → water.
Sections are stained to identify cell types and components (e.g., nucleus and cytoplasm).
Examples of stains include haematoxylin & eosin (H&E), Masson's trichrome, and Wright Giemsa.
Haematoxylin binds to acids, giving a blue/purple color.
Eosin binds to alkali, producing a pink/orange color.

Transmission electron microscopy (TEM) magnifies up to 1.5 million times, with a resolution of 0.14 nm (5x diameter of H atom).
TEM uses a beam of electrons instead of light.
Electron beam is produced by heating a tungsten filament.
TEM optical system must be evacuated of air to prevent distortion.
TEM uses multiple lenses to magnify the image produced by electrons interacting with the tissue section.
Tissue is fixed with formaldehyde + glutaraldehyde + osmium tetroxide.
Tissue is dehydrated.
Tissue is embedded in resin and sectioned at 60-500 nm using an ultramicrotome with glass or diamond knife with water trough.
Sections are mounted onto copper grids.
Stained with electron-absorbing heavy metal salts (e.g., uranyl acetate & lead citrate) for contrast.

Epithelial Tissue: sheets of cells forming linings (externally – skin, internally – digestive, urogenital, and respiratory tracts, blood vessels); forms glands.
Connective Tissue: provides support (physically – cartilage and bone, physiologically – blood); packing between tissues (adipose, fascia, ligaments, tendons). Types include: Connective Tissue Proper (ligaments, tendons, adipose tissue), Cartilage & Bone, and Blood.
Muscle Tissue: three main types: skeletal (striated), smooth, and cardiac.
Nervous Tissue: brain, spinal cord, nerves, and sensory receptors; enables information processing and responses from external and internal environments; consists of neurons (transmit impulses) and support cells.

After fertilization and multiple cell divisions, gastrulation occurs (cell rearrangements).
Germ layers: ectoderm (outer), mesoderm (middle), and endoderm (inner).
Ectoderm gives rise to epidermis, nervous system, and other structures.
Mesoderm gives rise to skeletal, muscular systems, and other structures.
Endoderm gives rise to lining of digestive tract and other structures.

Veterinary Practice: determine tissue health (disease), fine-needle aspirates and biopsies (suspicious lumps), and pathology.
Research: study development, gene function, and regenerative medicine.