Cytology, Histology Flashcards
– routine laboratory microscope used for studying tissue sections
Light (bright field) microscope
– used to study cytology or internal structures of cells; study of electron micrographs
Transmission electron microscope (TEM)
–used to study the surface features of cells and tissues; obtain a 3-dimensional picture of the tissue
Scanning electron microscope (SEM)
– permits one to determine whether biological materials have different refractive indices along different optical axes
Polarizing microscope
– used to study living tissue; works on principal of different refractive indices of cellular and sub-cellular components
Phase microscope
– a modification of the phase microscope used for the study of living tissue
Interference microscope
– uses UV light as the light source; used to examine the presence of fluorescent material in tissue sections
Fluorescence microscope
– uses a laser energy beam; used to optically section a cell and with the appropriate computer equipment can reconstruct a 3-D image of the cell
Confocal scanning microscope
a. Purpose: to preserve tissue morphology and chemical composition.
Accomplished by rendering tissue insoluble by precipitating proteins and carbohydrates (stabilizes the structure)
b. Commonly used fixatives: 10% buffered formalin, glutaraldehyde, alcohol, osmic acid
Fixation
a. Purpose: to remove water from tissues so that tissue is miscible with clearing agent
b. Alcohols commonly used as dehydrating agents
Dehydration
a. Purpose: to replace alcohol with an agent miscible with paraffin
b. Toluene, xylene, benzene
Clearing
a. Purpose to replace clearing agent with embedding material
b. Paraffin, methacrylate, celloidin, gelatin
Infiltration and embedding
a. Purpose: to produce thin sections through which light will pass. Paraffin sections cut from 5-7 μm thick
b. Done on an instrument known as a microtome
Sectioning
a. Purpose: to impart color to a tissue
b. Hematoxylin and eosin (H & E)
Staining
Artifact due to lysosomal digestion of the cells.
Post-mortem degeneration –
Artifact due frequently to reagents used in preparing paraffin sections, resulting in empty to clear spaces which during life were occupied by tissue components.
Shrinkage –
Artifact that occurs when formalin is not properly buffered.
Precipitates –
Artifact due to defect in paraffin section.
Wrinkles and folds –
Artifact due to defect in knife, resulting in the tearing or scraping of the tissue when the section is cut.
Nick in the microtome knife –
Cause of artifacts frequently due to pinching of the tissue when removing tissue from the body.
Mishandling of the tissue –
the atomic groups upon which color of a stain depends (-N=N-)
Chromophore –
Stain that has chromophore associated with the basic radicle (cation; +):
basic stain
structures in the cell or tissue that love basic stains; e.g. nucleus – large number of PO4(3-) radicles (acid radicles)
DNA – chromosomes; heterochromatin RNA – nucleolus and cytoplasmic ribosomes
Basophilic substances –
Blue to purple basic stain:
Hematoxylin
Dye has the chromophore associated with the acid radicle (anions; -):
Acidic or anionic stain
structures in the cell or tissue that love acid stains
e.g. proteins – large number of basic groups (cations; +) associated with the side chains
Acidophilic substances (eosinophilic substances) –
red to pink acid stain?
Eosin
a. a stain for connective tissue (collagen) rather than cells
b. e.g., Masson’s, Mallory’s
Trichrome stain
a. a stain for the elastic fibers or elastic tissue in connective tissue
b. e.g., aldehyde fuchsin; orcein; resorcin-fuchsin
Elastic stains
a. a stain for reticular fibers in connective tissue; also used in staining cells of the central nervous system
b. connective tissue fibers love silver (argyrophilic) and stain black
Silver impregnation stains
Nucleic acids (DNA and RNA) stains:
Feulgen staining reaction
luekofuchsin (shiff reagent) –> aldehyde shiff (product, colored)
Carbohydrate (e.g., glycogen) stain
Periodic acid-Schiff reaction (PAS)
Fat stains:
a. Oil red O (stains fats red) and Sudan black (stains fats black) commonly used
b. soluble in both alcohol and fats
Protein stains?
a. Specific stains for amino acids or groups of amino acids available, but no longer used
b. Immunocytochemistry is used extensively for localizing specific proteins in cells or tissues
