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Methods in histology. Microscopy. Organization of the practices How microscopic slides are made Microscop e Staining Observation of slides. Observation of the live cells. Unicellular organisms Metazoas : germ cell s, blood cells, cells in tissue cultures
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Methods in histology Microscopy
Organization of the practices • How microscopic slides are made • Microscope • Staining • Observation of slides
Observation of the live cells • Unicellular organisms • Metazoas: germ cells,blood cells, cells in tissue cultures • Observation is possible by special microscope (phase contrast microscopy) or using supravital staining
Sampling • Sampling of tissue and cells : • From the live organism (BIOPSY) • From the corpse (NECROPSY) • Fixation must be made otherwise enzymes and germs (bacterias) destroy the cells (AUTOLYZE) • Tissue block for fixation must not exceed (be bigger than)1cm3 ( for light microscopy)
Fixation • Fixation stops the metabolic events in the cell either by their reduction or by denaturation (destruction) of enzymes. • Physical methods: • Heat (microwave oven) • Freezing (in liquid nitrogen; -170oC) • Chemical methods: • Immersion (into fixative) • Perfusion (into vessels)
Embedding and cutting • Tissue have to be harden or stabilize for cutting by embedding in special medias (paraffin, celloidin). • These medias are not mixable with water therefore we remove water from the tissue by alcohols (dehydratation) and then we replete it by solvent of embedding medium (xylene, toluene, acetone), which procedure is named „clearing“
Embedding • Tissue can be proceed in beakers in thermostat (in small laboratory) • Automatic embedding machines serve for the pathologic department running
Cutting • Tissue is cutin slides of one cell layer, it means 4-10mm. Tissue is translucent and „well- readable“ in this case • Devices that are used for cutting are called microtomes. • Tissue slices are put on slide.They are stretched out by heat, and stick by egg white-glycerin
Staining Staining facilitates to distinguish tissue and cell components The majority of dyes are water-soluble, therefore we have to remove paraffin(dewax). Slide is covered by cover slip after the staining. Resins (Canadian or synthetic resins) are used as glue. The slide is long lasting.
Staining • For staining we use jars or automatic machines.
Permanent slide • Water is removed from tissue after staining • Cover slip is stick by resin • Permanent slide is made
Microscope • Stative • Adjustment knob • Optic system: • Oculars • Objectives • Condensor • Light
Resolution • Resolving power is the smallest distance between two particles at which we are able to distinguish them as two separate objects • Resolving power for light microscopy is 0,2 mm. • Magnification – 1000-1500 times
Staining • General staining • Haematoxylin - eosin • Masson trichrome • Weigert - van Gieson • Heidenhain iron haematoxylin • Selective • Weigert resorcin fuchsin • Silver methods
Haematoxylin - eosin • Haematoxylin stains acidic components of cell (basophilic structures) – DNA, RNA,ie. nucleus,nucleolus, ribosomes a rough endoplasmatic reticulum • Eosin stains basic structures of cell (acidophilic, eosinophilic) – that are predominantly proteins, ie. cytoplasma, mitochondrias, smooth endoplasmatic reticulum, and collagen in extracellular matrix
AZAN Azocarmine stains nuclei (red) Aniline blue stains collagen fibres and mucus (blue) Orange G stains cytoplasma, muscles (orange) Red blood cells are red - erythrocytes
Weigert van Gieson Weigert haematoxylin nucleus is brown Saturn red stainscollagen fibres and mucus (red) Trinitrofenol stains cytoplasma and muscles (yellow) Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen
Weigert - van Gieson Nalepení řezů na podložní sklo
Yellow Masson trichrome Haematoxylin stains nucleus blue to black Erythrosin stains cytoplasma and muscles red Saffron stains collagen fibres yellow Red blood cells are red
Weigert resorcin - fuchsin • Resorcin –fuchsin stains only elastic fibres • Elective staining for elastic fibres
Heidenhain iron haematoxylin • Heidenhain iron hematoxylin stains nucleus as well as cytoplasma gray-black. • It is used for staining of muscles; and in parasitology for detection of worms in tissue.
Silver methods • Silver stains reticular and collagen fibres in brown to black. • Silver methods are used for staining of neurons in neurohistology.
Cresyl violet • Cresyl violet stains DNA and RNA, ei. nucleus, nucleolus and rough endoplasmatic reticulum • Staining is used in neurohistology
What is necessary to know • What is fixation? Why it is performed? • How we make slide? Overview. • Basic staining. Why we stain tissues by various staining methods? • Haematoxylin –eosin staining • Light microscopy resolution (0,2 mm)