histology and embryology are fundamental branches on medical

schools
origin of the term of histology

greek histos meaning "tissue„ + logos meaning "the study off" (knowledge or science); the term was
primarily used in strict sense of the word as a denotation for study of microscopic parameters of animal and
plant cells and tissues
recently, histology = as a branch of science that treats microscopic and
submicroscopic structure (organization) of animal or plant bodies

- it deals with microscopic and submicroscopic structure of the human body

animal and plant organisms consist of organs, organs of one or more tissues and tissues are
composed of cells that are considered for elementary units of the living substance

in accordance with 3 mentioned organizational levels – cell, tissue, and organ – histology
divides into 3 sections:

cytology - deals with microscopic and ultrastructural organization of cells

histology proper (or histology in strict sense of this word) - describes light microscopic structure
and function of tissues

microscopic anatomy - a section of branch studying microscopic and fine structure of individual
organs

major interdisciplinary branches:

histochemistry, histophysiology, pathological histology, and electron microscopy

Importance of histology:
 a basic subject, on which pathology a pathophysiology are built
is used in diagnosis of diseases (department of pathology)
used in farmaceutical industry (for testing drugs and artificial materials used
for substitutions of natural organs)
 in control of food quality
embryology – its aim and orientation are quite different
the term - of 2 greek words en = in, bryein = to swell

embryology deals with study of individual development

of multicellular organisms

individual development /ontogenetic development = ontogeny/ involves

the period from fertilization of the ovum to the death of respective
individual

the main reason why histology and embryology are teached together is that
all multicellular organisms begin their existence as single cells =
generative cells or gametes

model of simultaneous teaching of both disciplines (H + E) is often in

countries of middle Europe
 Introduction
• The name "Histology" is derived from the Greek word for a
tissue "Histos", and "-logos" = the study of.
• Four fundamental tissues are recognized: epithelial tissue,
connective tissue, muscular tissue, and nervous tissue.
• Tissues are made of cells and extracellular matrix.
• Intense interaction between cells and matrix
• cells and extracellular matrix form a continuum that functions
together and reacts to stimuli and inhibitors together
• The small size of cells and matrix components makes histology
dependent on the use of microscopes
Sample preparation for
histology
How histological sections are
prepared for light microscopy and
electron microscopy
 Introduction to Histology
• How Histology Slides are Made
• Four basic tissue types:
– Epithelial, connective, muscle, nervous
• All animals are composed of ONLY
these four tissue types
• Tissue types are organized to form
organs, which form the functional
systems of the body
 HISTOLOGICAL TECHNIQUE
A. Histology involves the preparation of tissues for examination
with a microscope.

1. Basic methods of histological preparation of tissues.

a. Fix tissue (e.g. 4 % paraformaldehyde + buffer)
b. Dehydrate tissue (alcohol series followed by toluene)
c. Embed tissue in “hard” medium (e.g. wax)
d. Section embedded tissue on a microtome
e. Mount sections on a supportive structure (e.g. slide) that can be
placed on a microscope stage
f. Usually remove the embedding medium
g. Stain tissue (e.g. hematoxylin-eosin)
h. Examine tissue with microscope.
 BASIC TECHNIQUES
• Frozen sections
• Total preparations
• In some cases the tissue to be examined is a very thin
membrane.
• Cell Smears
• blood or bone marrow, epithelial cells (e.g. from the oral cavity,
cervix uteri).
•  STAINING TECHNIQUES
•  1.  Hematoxylin and Eosin (H&E)
– The Hematoxylin is a basic dye that stains acidic components of cells a blue color (basophilia).
Hematoxylin stains the nuclei of cells, and the RER of the cytoplasm
– Eosin is an acidic dye that stains the basic components of the cells a reddish-pink color
(acidophilia). Most of the cytoplasm of cells is stained by eosin. Bone matrix is also stained by
eosin.
•  2.  Periodic acid-Schiff (PAS) staining
– mucus, the basal lamina, glycogen.
•  3.  Orcein
– elastic fibers a dark brown-purple color.
•  4.  Osmium tetroxide
– Osmium is used to stain lipids a dark black color. myelin of myelinated nerves, or lipid
droplets in the liver or steroid-secreting cells.
•  5.  Oil Red O
– Oil Red O is used to stain lipids a red-orange color in unfixed frozen sections.
•  6.  Toluidine blue
– so-called metachromatic stain. It is a blue stain that stains specific components of tissues a
purple color. This change in staining color is known as metachromasia. Metachromasia is seen
in the matrix of hyaline cartilage, or in the granules of mast cells.
•  7.  Impregnation
– Silver impregnation techniques are also widely used to demonstrate reticular fibers.
Preparing a Biological Specimen for
Light Microscopy

• 1. Obtain sample and cut into

workable pieces (1cm cube)
– Biopsy, surgical excision, postmortem
examination
– Taken quickly, use sharp instruments
• 2. Fix the sample (immobilize, kill and
preserve)
– generally in reactive aldehydes like
formaldehyde.
– This cross links macromolecules, particularly
proteins
– Has hardening effect on soft tissues
– Done rapidly to prevent enzymes from live
cells degrading the tissue
2. Why fix tissue?

a. Preserve structure. Essentially to make the structural

components of the tissue more durable so that the tissue
can be manipulated in various ways.

• Fixed material is dead. You want to preserve the

structure (chemical and morphological) of the living
material so that it appears the same as it was in life.

• It will never be exactly the same. Important to choose

fixative that does the best job. Fixative used will depend
on type of tissue to be fixed.

3. Why dehydrate the fixed tissue

a. Most fixatives are water soluble, most embedding media

are non-polar and are not miscible with water. So, you
have to move the tissue from a polar (water-based)
medium to a non-polar medium (e.g. toluene) that is
miscible with the embedding medium.
• 3.Dehydrate the sample by running
through a series of increasing
concentrations of ethanol.
– Paraffin (embedding material) is not water
soluble, so water is removed

• 4. Clear the sample of ethanol and pass

through several changes of xylene
– Paraffin is also insoluble in ethanol
4. Why embed?

a. Tissue will be sectioned. Needs to be durable enough to

withstand the sectioning process. Also, want components
of tissue to remain in their natural positions. Don't want
them to be moved to new positions.

b. Embedding in wax or plastic immobilizes structural

components of tissue. Holds them in place as sectioning
is done.
• 5. Embed the sample in a supporting
medium like wax (paraffin) or resin
– Tissues are soft and fragile
– Pass through several changes of warm
paraffin wax
– Melted wax occupies space formerly
filled with water
– Cooled wax hardens, easier to cut
5. Why section?

a. Allows you to see internal

structure of tissue.

b. Allows stains, or specific markers such as

antibodies to more easily infiltrate the tissues.

c. Allows light to pass through tissue making

structure visible.

d. While sectioning is useful in many instances, in

some cases tissues are stained and examined
without sectioning.
• 6. Section on a
microtome, which
slices like a mini meat
slicer, into sections 1-
10m thick.
• 7. Mount the sections
on microscope slides.

Unstained section
• 8. Stain the tissue sections with stain of
your choice.
– stains are aqueous so need to reverse xylene,
ethanol series

Automatic stainer
 Stains
• A stain imparts a bright color
to certain compounds
• A counter stain imparts a
contrasting color to remainder
• Most common: Hematoxylin
and Eosin (H&E)
6. Why stain the tissue?

a. Creates higher contrast that allows observation of structure that is not

visible in unstained tissue.

b. May reveal differences in chemical nature of regions of the tissue.

http://www.ipass.net/grc/dimpg9.htm
• Hematoxylin has affinity for
negatively charged molecules like
DNA and RNA and certain
proteins.

• Eosin likes positively charged

molecules like most cytosolic
proteins.
 Why is this slide blue?
• If a portion of a tissue or
cell stains blue/purple,
blue/purple it
is said to be basophilic
• Stained by hematoxylin
• Nuclei and ribosomes
generally stain
basophilically
 And…..
• if a portion stains
red/orange/pink,
red/orange/pink it is
said to be acidophilic or
eosinophilic
• Stained by Eosin
• Stains the cytosolic
proteins
 Other stains
• PAS
– For sugar rich substances
– Like mucus
Goblet cell

• Trichrome White adipose

– For connective tissue
– Fibers look blue
• Fibers usually stain pink in White areas are lipid
H&E
• Osmium or Sudan black
– For fats/lipids/myelin
– Lipids do not pick up myelin
aqueous stains

• Silver or gold stains

– For delicate fibers and cell
processes Nerve cells

Rbcs and neutrophils

• Giemsa
– for blood
– Similar to H&E
What about the “ clear” areas?
• Tissue fluid or interstitial
fluid won’t stain in H&E
– Blood, lymph, other fluids
• Recognized by wide
unstained spaces
• Also, lipids and fats don’t
stain mesenchyme
 View your sample
• Use a good quality
light microscope
• Take a picture
• Can take
measurements
Alternately, use a
red blood cell to
estimate sizes of
structures
-8 microns
 Interpret your sample
• Must think in 3 dimensions
• For best interpretation, must have serial
sectioning and reconstruction
Serial reconstruction
 “ Artifacts”
• Shrinkage
– Leaves extra spaces
– Dehydration and
fixatives

• Nick in cutting edge

– Clear, straight “cut”
– From dull microtome
knife
• Precipitate of stain
– Blobs of stain or
peppered look

• Pinched tissue
– Dull excision
instrument
– During or after
sectioning
• Fold
– After sectioning
MICROSCOPY
A. Histologically prepared specimens of tissue are examined with a microscope.
B. A microscope is a device that not only magnifies (enlarges) the specimen for
examination, it also increases resolution such that it is possible to distinguish
the presence and morphology of very small structures within the tissue.
C. What is resolution?
- the ability to distinguish 2 objects as separate. That is, when viewing
something through a microscope, how close together can two objects be such that you
can still see some space between them?
* * * * *** *
Simply using a magnifying lens to make something appear bigger does not
necessarily increase resolution. Resolution depends on

1. the properties (shape, quality, refractive index, number of lenses) of the

lens or lenses
2. the properties (refractive index) of the substance the specimen is
mounted in
3. the properties (refractive index) of the substance that lies between the
specimen and the lens
4. the properties of the radiation (e.g. wavelength of the light beam) used to
image the specimen

See the Web Notes on

Microscopy and
Histochemistry for information
on how resolution is
determined.
Since resolution is determined by physical properties as just outlined, it turns
out that there are limits to the maximum possible resolution of any given type of
microscope.

Thus, even though it is possible to to design lens systems that would give a
light microscope very high magnifications (e.g. 4000X, 6000X), the resolutions
at these magnifications would be no greater than the best that can be
achieved at about 1200X.

As a result, useful magnification on a light microscope is limited to about

1200X in most cases.

Higher magnifications make the object appear bigger, but no new information
is added since resolution does not increase (i.e. You would not be able to
resolve smaller structures than what you can see at 1200X.).
C. Histochemical reactions must meet 5 criteria

1. During fixation, dehydration embedding, sectioning and

histochemical staining, the substance being analyzed must not
diffuse out of its original site.

2. Procedures must not block or inactivate reactive components

being studied.

3. Appropriate fixative, treatments, and embedding media must be

used such that substance to be identified is not soluble in it. i.e.
lipids - no non-polar solvents.

4. Stain or reaction product must be colored, opaque or electron

scattering so that it can be visualized.

5. Method employed should be specific for substance being studied. If

method is not totally specific, their must be controls that can be run
that will eliminate other possible sites of reaction.
D. Other important factors

1. Reaction product must be insoluble in the media used during the test so
that it will not diffuse away from the original site where the substance being
tested for was located.

2. The histochemical test used must not destroy the structure of the tissue.
145 Fundic stomach (H&E)
243 Fundic stomach, monkey (PAS)
244 Surface mucus cells of Fundic stomach,
rabbit (toluidine blue)
 Toluidine Blue
Eosin (H&E) of retina
Examples of histochemical stains: Identification of lipids in tissues

Osmium tetroxide staining

Identification of lipids in tissues

Sudan IV staining
IMMUNOCYTOCHEMISTRY
A. Immunological techniques are becoming increasingly important in histology.
B. Technique takes advantage of the fact that vertebrate animals have immune
systems that will produce antibodies that react with a specific molecule
(usually protein, but sometimes carbohydrate or lipid component of protein).
C. Antibodies may also bind to peptides and even single amino acids if an
appropriate antigenic substance is used to produce them.
D. These antibodies can be used to identify and localize specific molecules within
tissues, cells, or sub-cellular structures.
E. The antibodies themselves do not allow us to visualize the cell components,
rather, a marker such as a fluorescent compound, enzyme, or electron
scattering particle is linked to an antibody. So where the antibody binds will be
where this marker or its reaction products appear in the sectioned tissue.
F. Two approaches to using antibodies in immunohistochemical methods.

1. Direct method - marker conjugated directly to the antibody that binds to the
molecule we are interested in.
2. Indirect method - marker bound to antibody that will bind to the antibody that
binds to the molecule we are interested in (i.e. GAM - IgG).
Indirect method

Blue light
488 nm
EXAMPLES OF IMMUNOCYTOCHEMICAL PREPARATIONS
Melibe leonina
Apical ganglion
Ciliary tuft

Sensory end of
dendrite

Ampullary neurons
Dendrites

Serotonergic neurons 5
0
Serotonin labeling of specific CNS neurons - Molluscs
Phase-Contrast Microscopy
Phase Contrast
Dead stained cells
Bright Field

Live unstained cells

Phase Contrast

Nomarksi
“differential interference
contrast”

Dark Field
19709 Transparency of unstained tissue
Polarizing Microscopy
Fluorescence Microscopy
Confocal Microscopy
TEM gives higher
resolution, can see
structures not seen at light
microscope level

Resolution of eye:
eye .
2mm=200m
Resolution of LM:
LM
2,000 Angstroms=200nm
Resolution of TEM:
TEM Pinocytosis in capillary
2 Angstroms

1mm=1000m
1m=1000nm
1nm=10 Angstroms
Electron Microscopy
 Transmission Electron
Microscopy
• Electrons have short
wavelength
– Can get better resolution
• Need very thin sections
and special electron
dense stains
• Electrons pass through
sample
 Preparing Biological Samples for
TEM
• 1. Obtain a sample and cut into workable
size pieces (<1mm cubes).

• 2. Fix the sample with glutaraldehyde and

then usually osmium tetroxide
– osmium is a heavy metal that binds to lipids,
causing them to appear electron dense (black)
– most LM stains will not bind to lipids, causing
them to appear clear in sections.
3. Dehydrate the
samples in a series
of increasing
concentrations of
ethyl alcohol.
4. Embed the
samples in small
blocks of resin.
5. Section the embedded samples on an
ultra-microtome using a diamond (or
sometimes glass) knife.
– The surface face to be trimmed must be .
2mm.
– The section thickness ranges from 40-
100nm thick (preferably 65-80nm).
6. Pick up the
sections and place
them on a copper
grid.
7. Stain the sections
with Uranyl Nitrate
or Uranyl Acetate
and Lead Citrate.
8. View on the TEM
9. Take pictures,
develop and analyze
Scanning Electron Microscopy
• SEM scans
the surface
of an image
• Nice 3-D
effect
Preparing biological samples for
SEM
• Obtain a sample
– Handle carefully so as not to damage
surface
• Fix and dehydrate
• No embedding
• Place on stud and
coat with a metal
– Gold, carbon,
chromium, palladium
– Needs to be electron
conductive
• Place in scope
• Take pictures
• Can also analyze Sputter coater
chemical components
of sample via x-ray
analysis