HISTOLOGY

Scientific discipline studying structure of cells and tissues of

multicellular organisms
1/ cytology
2/ general histology
3/ microscopic anatomy

MUDr. Andrea Felšöová

[Link]@[Link]
EMBRYOLOGY
 smallest dimension
Largest cells 150 μm distinguishable by
the naked eye
Average human cells 10 –
20 μm

Largest organelles 1 – 2 μm

200 nm - smallest
Ribosomes 20x30 nm
dimension
distinguishable by
Cytoskeleton 5 – 24 nm
the light microscope

Membrane 7,5 nm

smallest dimension
distinguishable by
the electron
microscope
Light microscope
Transmission electron microscope
(TEM)
Light Electron
microscope microscope
BASIC HISTOLOGICAL TECHNIQUES
LIGHT MICROSCOPY ELECTRON MICROSCOPY
•Sampling •Sampling
•Fixation •Fixation
•Dehydration •Dehydration
•Clearing
•Clearing
•Embedding
•Embedding
•Sectioning
•Sectioning
•Staining
•Mounting •Contrasting
•Observation •Observation
 FIXATION OF TISSUE
• artificially evoked interruption of all
vital processes in the collected
specimen of tissue
• coagulation of tissue proteins by
chemical substances or physical
factors
• formaldehyde
 PARAFFIN EMBEDDING
• DEHYDRATION
– increasing series of alcohol = 70 - 96% alcohol –
consecutive replacement of water with alcohol
– absolute (100%) alcohol (completing dehydration)
• CLEARING
– cedar oil, xylene – replacement of the absolute alcohol
with a paraffin solvent
• PARAFFIN INFILTRATION
paraffin at 56 - 58°C – three baths – consecutive
replacement of the clearing medium with melted paraffin

• EMBEDDING
– submersion of the paraffin-infiltrated specimen with
melted paraffin
tissue processor
embedding centre with cooling plate
 RESIN EMBEDDING
• Dehydration
– increasing series of alcohol = 50 - 96% alcohol –
consecutive replacement of water with alcohol
– alcohol can be diluted by 1% uranylacetate
(precontrasting)
– absolute (100%) alcohol (completing dehydration)
• Clearing (if necessary, e.g. in epoxy resins)
– propylene oxide - replacement of the absolute alcohol
with a resin solvent
• Infiltration with embedding medium
– replacement of the clearing agent with liquid resin
• Embedding with resin in gelatine capsules
• Polymerization - hardening
– heat (60°C) – most resins
– UV light at room temperature or in freezer – some acrylic resins
resin-embedding tissue processor
 SECTIONING
• MICROTOME
– sliding or rotary
– steel knives or blades
– for paraffin (celoidine, celodal, gelatine) blocks
• ULTRAMICROTOME
– glass or diamond knives
– for resin blocks
• CRYOSTAT AND FREEZING MICROTOME
– steel knives or blades
– for frozen specimens
sliding microtome
rotary microtome
Ultramicrotome
 USING OF FREEZING TECHNIQUES
• rapid intraoperative biopsies
– mainly evaluation of
tumors
• lipid staining
• lipid histochemistry
• enzyme histochemistry
• immunohistochemistry –
vulnerable antigens
• some fluorescence methods
• some impregnation methods
 STAINING
Interaction between a dye (staining agent) and
a certain tissue component.
•1/ synoptic staining – all components of the
specimen are stained, based on the acid-base
affinity
− Basic dyes – haematoxylin (various types), thionine, azocarmine, toluidine blue,
nuclear red, methylene green,……
− Acidic dyes – eosin, erythrosin, light green, acid fuchsin, orange G, aniline blue,
picric acid,……
2/ special staining - highlighting of assessed
structures by a dye with specific affinity
Basic staining procedure
(of paraffin section)

•slide deparaffination
•slide rehydration
•staining
•slide dehydration
•slide clearing before mounting
 MAKING A PERMANENT HISTOLOGICAL SLIDE
STAINED WITH HAEMATOXYLIN AND EOSIN (H&E)
• DEPARAFFINATION AND REHYDRATION
– xylene (2 baths) - dissolving of paraffin
– 100% alcohol - washing off the dissolved paraffin and dissolvent
– 96% - 70% alcohol - slow rehydration
– water - washing off the alcohol
• STAINING
– haematoxylin (basic dye) - staining of acid (basophilic) components of
tissue (nuclei, ribosomes)
– washing
– eosin (acid dye) - staining of basic (acidophilic, eosinophilic)
components of tissue (proteins)
– washing (water)
• DEHYDRATION
– 70% - 100% alcohol - gradual replacement of water
• CLEARING
– xylene (2 baths) - replacement of alcohol
with dissolvent of mounting medium
• MOUNTING
– Solacryl BMX - covering of the slide with the coverslip
 basophilic

light (optically empty)

eosinophilic
(acidophilic)
 OTHER EXAMPLES OF SYNOPTIC STAININGS
Weigert - van Gieson iron haematoxylin + nuclei brown,
acid fuchsin + picric collagen red,
acid muscle yellow

Masson’s trichromes haematoxylin + nuclei blue,

-yellow erythrosin + saffron collagen yellow,
muscle red
-blue iron haematoxylin + nuclei brown to black,
acid fuchsin + aniline collagen blue,
blue muscle red
-green iron haematoxylin + nuclei brown to black,
acid fuchsin + orange collagen green, muscle
G + light green red
AZAN azocarmine + aniline nuclei red,
blue + orange G collagen blue,
muscle red
Weigert - van Gieson
blue trichrome
green trichrome
 SPECIAL STAINING

-highlighting of searched structures with a

dye of specific affinity
-no colour chemical reaction takes place
(dye does not change its original colour)

-cytological stainings – staining of selected intacellular

structures
-selective stainings – staining of selected structures or
substances regardless of their location
-impregnation methods – reduction of metals (Ag, Au,
Os) on selected structures
toluidine blue Nissl substance
 EXAMPLES OF SELECTIVE STAININGS
elastics - orcein, aldehyde fuchsin, resorcine fuchsin
mucus, glycogen, glycoaminoglycans (GAG) - alcian blue, mucicarmine, Best
carmine
neutral lipids – Oil red O, Sudan black B, Sudan III, scarlet – avoidance of
lipid dissolvents (frozen sections)
myelin (phospholipids) – luxol blue, Spielmeyer haematoxylin
amyloid - Congo red, methylene violet, Saturn red
fibrin – Weigert staining
Etc. …
resorcine fuchsin
elastics
alcian blue pH 2.5 acidic mucopolysaccharides
mucicarmine mucins
Oil red O neutral lipids
EXAMPLES OF IMPREGNATION

Gömöri, Foot reticular fibres

Hortega, Cajal astrocytes

Penfield oligodendrocytes
Bielschowski nerve fibres

Grimelius cells of DNES

Grocott hyphes of fungi

neuron impregnation
Mycosis (Aspergilus)- Grocott
 MOUNTING
- cover glass

- xylene-soluble media
- acrylic resins (solacryl BMX)
- Canada balsam

- water-soluble media
- glycerol-gelatine
- glycerol
 TEM - GRIDS

• grids can be naked or

• coated with a support film (Formvar –
polyvinylformaldehyde) and evaporated
carbon layer
 CONTRASTING
(for TEM)

• binding of atoms of heavy metals to

structures of an observed object
• grids are laid on drops of metal salts
solutions
• mostly used salts:
– uranyl acetate
– lead citrate
 electron
dense

electron
lucent
 OBSERVATION
• Direct observation
• Photography
• Morphometry
• Stereology
• 3D reconstructions
• Image analysis
 cca 1670

eyepiece

objective
specimen

condenser

light source

Transmission (bright-field)
light microscope
 Fluorescence microscopy
• Fluorescence – ability of some substances(fluorochromes)
to respond to the absorption of excitation light by emission
of light having a longer wavelength
 electron gun (cathode)

condenser lens

specimen

objective lens Ernst Ruska

1931
projector lens

fluorescent screen
or photographic
emulsion

Transmission electron
microscope (TEM)
 electron gun

condenser lens
scanning generator
beam deflector

objective lens

detector secondary and

screen
back-scattered
specimen electrons

Scanning electron microscope (SEM)

SEM TEM
Tracheal ciliary border (SEM)
Tracheal ciliary border (TEM)
 HISTOCHEMISTRY
formation of
coloured reaction product
in situ
(reagent originally colourless)
Detection of - elements (ions)
- nucleic acids
- lipids
- saccharides
- pigments
- proteins (aminoacids) – currently unusual,
immunohistochemistry used instead
 PAS reaction (periodic acid – Schiff)
incompletely specific oxidative method detecting complex
carbohydrate in cells and tissues
1 oxidation of free vic glycol groups 2 detection of just formed aldehyde groups
by periodic acid by Schiff reagent
(Malaprade reaction) Schiff reagent – leukoform of basic fuchsin (colourless)

having reacted with aldehydes,

a new magenta compound arises
PAS reaction neutral mucins
 IMMUNOHISTOCHEMISTRY
1/ Bond of antigen and antibody

2/ Visualization of this complex

CELL = basic structural and functional unit of
a living matter.
Protoplasm surrounded by a cell membrane.

prokaryotic cells (bacteria, blue-green algae,

archea)
protoplasm is not further divided

eukaryotic cells (protists, animals, plants,

fungi)
protoplasm divided by membranes to
compartments
 Prokaryotic Electron
microscope
Light cell
microscope
Eukaryotic
cell

Electron
Light microscope
microscope
General features of cells

• shape
• size
• lifespan
• degree of differentiation
Flat shape (squamous)
Cuboidal shape
Columnar shape
Spindle
shape
 Stellate
(star-like)
shape
Pyramidal shape
Special
shapes
Size of cells
average human cell
10-20 μm

largest – oocyte 150 μm

150 mm
smallest – chief cells of
parathyroid glands
or small granular neurons
of cerebellum
4-5 μm

standard – erythrocyte 20 mm
7.5 μm
Lifespan of cells

1 – 4 days

120 days

whole postnatal life of the individual

 COMPONENTS OF EUCARYOTIC CELL
protoplasm = karyoplasm + cytoplasm

karyoplasm = content of nucleus

cytoplasm = matrix (cytosol) + cytoplasmic structures

1/ organelles – membranous
– non-membranous
2/ cytoplasmic inclusions
3/ elements of cytoskeleton
 mitochondria
(organelles)
Karyoplasm

centriole
(cytoskeleton)

Cytoplasm