Methods In Cytology And Histology

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Used routinely in the lab to study tissue sections

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Light (bright field) Microscope

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Used to study cytology or internal structures of cells; electron micrographs

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Transmission electron microscope (TEM)

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Used to study the surface features of cells and tissues; 3D

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Scanning electron microscope

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Determines whether biological materials have different refractive indicies along different optical axes

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Polarizing Mircroscope

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Used to study living tissue

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Phase Microscope

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Modification of the phase microscope

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Interference microscope

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Uses UV light

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Fluorescence Microscope

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Uses laser energy beam

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Confocal scanning microscope

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Purpose of Fixation

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to preserve tissue morphology and chemical composition. Accomplished by rendering tissue insoluble by ppting proteins and carbs

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Dehydration

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To remove water from tissues so that the tissue is miscible with clearing agent. Alcohol commonly used.

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Clearing

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To replace alcohol with an agent miscible with paraffin. Toluene, xylen, benzene

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Infiltration and Embedding

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To replace clearing agent with embedding material

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Paraffin, methacrylate, celloidin, gelatin

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Infiltration and Embedding agents

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Sectioning

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to produce thin sections through which light will pass

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Done on a microtome

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Sectioning

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Hematoxylin and Eosin (H&E)

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Staining agents

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Where are frozen tissue sections used?

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Surgical Biopsies and in research studies for the localization of enzymes

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Refers to any features evident in tissue sections that are a result of imperfect technique

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Artifacts

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Artifact due to lysosomal digestion of the cells

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Post-mortem degeneration

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Artifact due frequently to reagents used in preparing paraffin sections, resulting in empty to clear spaces which during life were occupied by tissue components

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Shrinkage

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Artifact due to defect in paraffin section

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Wrinkles and Folds

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Artifact due to defect in knife, resulting in the tearing or scraping of the tissue when the section is cut

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Nick in the microtome knife

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Artifact frequently due to pinching of the tissue when removing tissue from body

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Mishandling of the tissue

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10 % buffered formalin, glutaraldehyde, alcohol, osmic acid

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Fixation agents

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Toluene, xylene,benzene

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Dehydration Agents

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Dye capable of forming a salt linkage with a _____ charged tissue group; therefore, the dye molecule is ____ charged. (Anionic)

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+, -, Acid Stain

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Dye capable of forming a salt linkage with a _____ charged tissue group; therefore, the dye molecule is ____ charged. (Cationic)

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-, +, Basic Stain

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Atomic groups upon which color of a stain depends (-N=N-)

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Chromophore

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Blue to Purple stain associated with the basic radicle (cation, +)

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Hematoxylin, Basic

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Structures in the cell or tissue that love basic stains, e.g. nucleus - large number of PO4 (3-) radicles (acid radicles)

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Basophilic Substances

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Red to Pink stain associated with the acid radicle (anions, -)

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Eosin, Acid

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Structures in the cell or tissue that love acid stains e.g. proteins (large number of basic groups associated with side chains)

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Acidophilic Substances

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Stain for connective tissue (collagen) rather than cells

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Trichrome

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Ex) Masson's or Mallory's

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Trichrome Stain

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Stain for the elastic fibers or elastic tissue in connective tissue

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Elastic Stain

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Ex) aldehyde fuchsin, orcein, resorcin-fuchsin

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Elastic Stains

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Stain used for reticular ribers in connective tissue and for cells of the CNS

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Silver Impregnation

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Argyrophilic

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Love silver and stain black

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Stains fats red

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Oil red O

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Stains fats black

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Sudan Black

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Periodic acid-Schiff Reaction (PAS)

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Carbohydrate

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Feulgen Staining Reaction

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Nucleic Acid Stains

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Permits the cellular or intracellular localization of specific proteins

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Immunocytochemistry

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How big are red blood cells?

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7 um

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How thick are paraffin sections?

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6-7 um

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How thick are plastic sections? e.g methacrylate

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1-3 um