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Pt. 1 : histological techniques - Coggle Diagram
Pt. 1 : histological techniques
1. Levels of Biological Organization
Chemical Level: Atoms combine to form molecules (e.g., water, DNA, proteins).
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Organelle Level: Specialized structures inside the cell (e.g., mitochondria, lysosomes) performing specific functions.
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Cellular Level: The basic structural and functional unit of all living things.
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Tissue Level: A group of similar cells working together to perform specific jobs.
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Organ Level: Structures made of different types of tissues working together (e.g., heart, lungs, kidneys, small intestine).
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Organ System Level: Groups of organs collaborating to carry out complex body functions (e.g., digestive, reproductive systems).
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2. Methodological Procedures & Special Staining
Whole Mount Skeletal Staining: * Alcian Blue: Stains cartilage.
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Alizarin Red: Stains bone.
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Your Example: Mouse development slides showing a fetus at 17 days, and embryos at 16 and 14 days.
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In Vitro Cell Cultures: Taking cells out of the organism to grow them in layers (Primary cell lines → Secondary → Tertiary).
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Vital Dyes (Live Cell Staining):
Trypan Blue: An exclusion dye (it is kept out of living cells; only stains dead/damaged cells).
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Thionine: Used to observe cytoplasmic granules in cultured macrophages.
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Methylene Blue: Highlights the axons of nerve cells.
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Brilliant Cresyl Blue: Used for the demonstration of reticulocytes (immature red blood cells).
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3. The 4 Essential Steps of Tissue Processing (Light Microscopy)
Fixation: * Why: Stops cell metabolism, prevents autolysis (self-digestion) and enzymatic degradation, kills pathogens (bacteria, fungi, viruses), and hardens tissue via protein cross-linking or denaturation.
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Methods: Immersion (soaking tissue, 10:1 volume ratio) or Perfusion (flushing fixative through intact blood vessels of experimental animals—the best method).
Types of Fixatives:
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Cross-linking (Additive): Stabilizes native conformation by forming bonds between proteins. Formalin (10% standard, pH 7.3–7.4) is the most common cross-linking agent; it forms hydroxy-methyl groups and methylene bridges.
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Coagulation (Dehydration): Irreversibly alters tertiary structure using heat, strong acids, metal salts, or alcohols.
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Factors: Thickness of the sample matters (to prevent internal rot before the fixative penetrates) ; also depends on pH, temperature, concentration, and osmolarity.
Dehydration & Clearing: * Dehydration: Replacing water inside the cells with increasing concentrations of Alcohol.
Clearing: Using Xylene (referred to as "selena" in your recording) to dissolve the alcohol and allow the wax to mix.
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Embedding: * Infiltrating the tissue with molten paraffin wax to form a solid, hard block suitable for subsequent cutting.
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Sectioning & Mounting:
Sectioning: Slicing the block using a Microtome (Rotary, Sliding, or a Cryostat for frozen sections).
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Mounting: Placing the cut section on a glass slide, adding a drop of synthetic resin, and covering it with a coverslip to make it stable and long-lasting.
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4. Histochemical Staining Dictionary (Light Microscopy)
Hematoxylin (H): A Basic/Cationic dye. Binds to negative/anionic structures (Basophilic structures like heterochromatin, nucleic acids, DNA/RNA, and cartilage matrix). Stains them Blue/Purple.
Eosin (E): An Acidic/Anionic dye. Binds to positive structures (Acidophilic/Eosinophilic structures like cytoplasmic filaments, muscle proteins, membranes, and extracellular collagen fibers). Stains them Pink/Red/Orange.
Toluidine Blue (Metachromasia): A dye that changes its color from blue to Reddish-Purple when reacting with highly concentrated, ionized sulfate and phosphate groups (e.g., mast cell granules containing eparina, cartilage extracellular matrix, and rough endoplasmic reticulum).
PAS (Periodic Acid-Schiff): Used to localize carbohydrates, glycogen, glycoproteins, mucins, and the basement membrane. Stains them Magenta/Red.
Sudan Red: A lipid-soluble dye used to stain fats, phospholipids, lipoproteins, and triglycerides an intense red.
Silver Staining (Impregnation): Uses silver salts to precipitate onto Type III Collagen (Reticular fibers) and nervous fibers, staining them Black/Brown. Also stains diffuse neuroendocrine cells (argentaffin/argyrophilic cells).
Verhoeff / Weigert (Van Gieson): Specialized stains used specifically to stain elastic fibers and elastic lamellae a Dark Blue to Black color.
Wright's / Giemsa Stain: Specifically designed for blood smears. Stains white blood cell nuclei dark blue/purple, red blood cells salmon pink, and differentiates granules in neutrophils, basophils, and platelets.
5. Artifacts and Planes of Section
Artifacts: Unintended distortions you must detect under the microscope, such as being out of focus, tissue folds, ink/dust contamination, marker lines, or air bubbles trapped under the coverslip.
Planes of Section: The appearance, size, and visible internal structures depend entirely on the angle of the cut.
Your Examples: Cutting an orange versus cutting a spherical kidney—different cutting angles yield drastically different shapes on the slide.
Organ Architecture: Parenchymal organs display distinct regions, usually divided into an outer cortex and an inner medulla, covered by an adventitia or capsule.
6. Optical vs. Electron Microscopy (EM)
Light Microscopy (Brightfield): Uses a condenser to focus a cone of light through the tissue stage , objective lenses for primary magnification (4x for large areas, 10x for medium, 40x for high detail) , and an eyepiece (ocular lens) to further magnify the image (usually 10x).
Advanced Light Options: Phase Contrast and Differential Interference Contrast (DIC/Nomarski) microscopes utilize different refractive indices to highlight structural boundaries without needing heavy staining, making them ideal for observing live, growing tissue cultures.
The Frozen Section Technique (Criostat): Crucial during active surgery; allows rapid freezing and slicing of tissue (like a large intestine biopsy) for instant diagnostic answers without waiting days for paraffin embedding.
The Electron Microscopy (EM) Processing Pathway:
Tissue is fixed, washed, and dehydrated through an alcohol gradient.
It is embedded in a hard plastic resin block and polymerized in a beaker.
The hard plastic block is trimmed into a small cube.
An ultramicrotome equipped with a glass or diamond knife cuts ultra-thin slices.
Slices float onto water where the best sections are collected onto a metal grid, dried, stained with heavy metals, and viewed.
TEM vs. SEM:
TEM (Transmission Electron Microscope): The electron beam passes through the slice, generating a 2D flat image showing high internal ultrastructural detail.
SEM (Scanning Electron Microscope): The electron beam scans the surface of a metal-coated sample, generating a 3D threedimensional image displaying surface topography.