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)

Chemical fixation

Aldehydes Formaldehyde, glutaraldehyde

Alcohols Methanol, ethanol

Acids Acetic acid, trichloracetic acid, pikric acid

Salts of heavy metals Mercury, osmium, chromium

Fixatives Formaldehyde 4%

Bouin fluid trinitrophenol, formaldehyde, acetic acid

Susa mercuric chloride, natrium chloride,acetic acid, trichloracetic acid, formaldedyde

Zenker fluid Mercuric chloride, potassium bichromate, natrium sulphate, acetic acid

Carnoy ethanol, chloroform, acetic acid

Methacarn methanol, chloroform, acetic acid

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

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Cutting

Tissue is cut in slides of one cell layer, it means m. 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

Mikrotomes

StainingStaining 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

ProcedureParaffin slides Cryostat slides

Fixation Freezing at – 170 oC

Washing

Dehydratation by alcohols

Clearing by solvents

Embedding in paraffin

Cutting Cutting in cryostat

Sticking on slide Sticking on slide

Dewaxing and rehydratation Sometime short fixation

Washing

Staining, histochemical reaction Histochemical reactions predominantly

Dehydratation and clearing Sometime dehydratation and clearing

Resins Resins or glycerin -gelatine

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 m.

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

Haematoxylin - eosin

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

AZAN

Weigert van Gieson

Weigert haematoxylin nucleus is brown

Saturn red stains collagen 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

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Weigert - van Gieson

Haematoxylin - eosin

Weigert-van Gieson

Green Masson trichrome

AZAN

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

Yellow Masson trichrome

Weigert resorcin - fuchsin

Resorcin –fuchsin stains only elastic fibres

Elective staining for elastic fibres

Weigert resorcin - fuchsin

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.

Heidenhain iron haematoxylin

Heidenhain iron haematoxylin

Silver methods

Silver stains reticular and collagen fibres in brown to black.

Silver methods are used for staining of neurons in neurohistology.

Silver method

Cresyl violet

Cresyl violet stains DNA and RNA, ei. nucleus, nucleolus and rough endoplasmatic reticulum

Staining is used in neurohistology

Cresyl violet

Cresyl violet

Results of stainingStaining Dyes Nucleus Collagen Elastic Muscle Notice

Haematoxylin-eosin HaematoxylinEosin

Blue to blac

pink pink

Weigert – van Gieson

Weigert haematoxylin

Saturn redTrinitrofenol

Brown red yellow Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen

AZAN Azocarmine aniline blue

Orange G

red blue Orange – red Red - erythrocytesblue- mucus

Blue Masson trichrome

HaematoxylinAcid fuchsinAnilin blue

blue to black

blue red Red- erythrocytesBlue - mucus

Yellow Masson trichrome

HaematoxylinErythrosinsaffron

blue to black

yellow red Red – erythocytes

Green Masson trichrome

HaematoxylinAcid fuchsinLight green

blue to black

green red red - erythrocytes

Weigert resorcin-fuchsin

ResorcinFuchsin

violet

Silver AgNO3 brown grey-black Reticular fibres- black

Heidenhain iron haematoxylin HIH

Heidenhain ironhaematoxylin

brown to black

grey- black

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 m)