PREPARATION OF TISSUES

FOR MICROSCOPIC
EXAMINATION
John Joseph De Castro
Definition of Terms:

■ Histology – came from the Greek word histos meaning “web or tissue” and logos
meaning “study”. Histology is the study of the structure and function of cells, tissues
and organs of the body at the microscopic level
■ Tissues – societies of cells and their associated extracellular substances, which are
specialized to carry out specific functions (types: epithelium, connective, muscle,
nervous)
■ Microscopic examinations – laboratory procedures performed to observe very small
objects with the aid of the microscope
PREPARATION
OF
TISSUES
Preparation of tissues for microscopic
examination
■ Cells, tissues and organs cannot be studied properly unless they are suitably prepared
for microscopic examinations. Two methods are used for tissues preparation:
■ Methods involving direct observation of living cells
■ Methods employed with dead cells
■ Living cells are usually more difficult to handle and
are available for a short period only. But it is important
that one should be aware of the methods by which
living cells may be observed and understand the ways
in which they differ from fixed cells. In living cells,
structure and functions may be studied simultaneously.
Living cells can be seen moving, ingesting foreign
materials, occasionally dividing, and carrying on other
functions.
■ Observation of living cells – unicellular organisms and occasionally free
cells may be studied directly under microscope while they are still alive.
Free cells are colorless and structure within them lack contrast. This
difficulty may be overcome by using phase contrast microscope.
■ Human blood cells are easily obtained and can be studied in films while
surrounded by plasma, their natural environment.
■ Prolonged preservation of cells outside the body can be achieved by a
technique known as tissue culture. Fragments of tissues are removed
aseptically, transferred to a physiological medium and kept at a temperature
normal for the animal from which the tissues were taken. The culture is
placed in thin glass vessel or on a cover glass mounted over a hollow glass
slide. In this way, they are available for observation under microscope. In
such culture, growth, multiplication and in some cases, differentiation of
cells into other cells type can be observed directly. Tissue culture is a
valuable method for the study of cancer and the activity of many viruses.
■ Preparation of dead tissues – the most convenient way
to study histology is to use sections, each of which is
more or less a permanent preparation. A section is
prepared by cutting a thin slice from a small piece of
fixed tissue, which is then stained, mounted in a
medium of suitable refractive index on a slide and
finally covered with a cover slip.
■ Especially useful for educational purposes and for those who do not want
to undertake the laborious process of creating slides, prepared
microscopes slides are available and can be made available in all areas of
science. A variety of tissues can be selected in different fields of science
such as:
– Biology: plants, animals, single-cell organisms
– Human Anatomy: organ samples, tissues, blood, epithelium cells
– Botany: monocot and dicot tissues
– Zoology: samples from various animal species
– Marine biology: bacteria, algae, coral, fish and crustaceans
– Pathology: bacteria, virus, diseased tissue
■ Prepared microscope slides also offer access to bacteria, animal tissue,
marine life, diseased cells and other specimens that may not be easily
available to students or hobbyists. In addition, they can be used as controls
for students and researchers to compare collected samples.
Histo-techniques
■ techniques – preparation of tissue for microscopic examination
■ series of processes
■ ultimate aim – to make tissue “visible” as it is
■ pathology vs. anatomy
■ steps vary depending on the
– types of tissue and microscopy
– structure to be seen
– stains to be used
– time duration etc.
Steps
■ - tissue procurement and preparation
- fixation
- dehydration
- clearing
- impregnation
- embedding
- section cutting
- staining and mounting in slide
Tissue Procurement and Precautions
■ Start fixation as soon as possible
■ Prevent osmotic damage (do not dry, wash with immense in NS)
■ No unnecessary handling
■ Remove excess blood, mucosa etc.
■ Cut with a sharp knife
■ Marking of cutting surface
■ Labelling and putting in specimen tube
■ Instruction for mounting – wall, tube, stained surface etc.
 Fixation
■ Fixation is the rapid preservation of tissue components in order to arrest
cellular processes and to maintain, as close as possible, a resemblance to
the living condition.
■ In histology, a fixative is typically a chemical agent, which may consist of
a simple solution (e.g. Bouin fixative: picric acid, acetic acid, formalin).
■ The end result is a cross-linking and denaturation of tissue components,
particularly proteins.
■ Equally important, fixation also resists tissue degradation by endogenous
(enzymatic autolysis) or exogenous (bacterial action) mechanisms.
■ While many fixatives are aqueous, some are also alcohol-based.
■ The aim of fixation:
– To prevent autolysis and bacterial attack
– To fix the tissues so they will not change their volume and
shape during processing
– To prepare tissue and leave it in a condition which allow
clear staining of sections
– To leave tissue as close as their living state as possible, and
no small molecules should be lost
– Render tissue unaffected to the harmful effects of chemical
to be used in further processing
FACTORS that may affect
FIXATION:
■pH
■temperature
■penetration of fixative
■time
 pH
■ anoxia in tissue - > higher carbon dioxide
content lowers the pH level
■ general purpose: optimal pH 6-8
■ buffers maintain desired pH
 TEMPERATURE
■Room temperature is alright for
fixation. At high temperature, there
may be distortion of tissues.
■Low temperature – slower rate of
decomposition
PENETRATION OF FIXATIVES

■ Rate of penetration varies with various

factors
■ - Glutaraldehyde > formaldehyde
■ Deeper tissue takes longer time, fixed tissue
acts as a barrier for diffusion of fixative
 TIME
■ Formaldehyde
– too short – reversal of fixation
– too long – shrinkage of tissue
– masks the antigen – immunohistochemistry
■ Depends upon
– thickness of tissue
– temperature
■ Optimal 12 hours, maximum 24 hours
■Volume changes – cell volume
changes because of the membrane
permeability and inhibition of
respiration.
SIMPLE FIXATIVES
 Formaldehyde
■ Commercially available solution contains 35%-40% gas
by weight, called as formalin. Formaldehyde is
commonly used as 4% solution, giving 10% formalin
for tissue fixation.
■ Formalin is most commonly used fixative. It is cheap,
penetrates rapidly and does not over-harden the tissues.
The primary action of formalin is to form additive
compounds with proteins without precipitation.
 Absolute alcohol
■ It may be used as a fixative as it coagulates
protein. Due to its dehydrating property, it
removes water too fast from the tissues and
produces shrinkage of cells and distortion of
morphology. It penetrates slowly and over-
hardens the tissues.
 Acetone
■Sometimes it is used for the study
of enzymes especially phosphates
and lipases. Disadvantages are
same with alcohol.
 Mercuric chloride
■ It is a protein precipitant. However, it
causes great shrinkage of tissues hence
seldom used alone. It gives brown color to
the tissues which needs to be removed by
treatment with iodine during dehydration.
 Potassium dichromate
■ It has a binding effect on protein similar to
that of formalin. Following fixation with
potassium dichromate must be well washed
in running water before dehydration.
 Osmic acid
■It is used for fixation of
fatty tissues and nerves.
 Chromic acid
■ It precipitates all proteins and preserves
carbohydrates. Tissues fixed in chromic
acid also require thorough washing with
water before dehydration.
Osmium tetraoxide
■It gives excellent preservation of
cellular details, hence used for
electron microscopy.
 Picric acid
■ It precipitates proteins and combines with them to
form picrates. Owing to its explosive nature when dry;
it must be kept under a layer of water. Tissue fixed in
picric acid also require thorough washing with water to
remove color. Tissue cannot be kept in picric acid
more than 24 hrs.
COMPOUND
FIXATIVES
 Formal saline
■ It is the most widely used fixative. Tissue can be left in this for a long
period of time without excessive hardening or damage. Tissue fixed for
a long time occasionally contain a pigment (formalin pigment). This
may be removed in sections before staining by treatment with picric
alcohol or 10% alcoholic solution of sodium hydroxide. The formation
of this pigment can be prevented by neutralizing or buffering the formal
saline.
■ Fixation time – 24 hours
■ 100 ml of 40% formaldehyde, 9 gm of sodium chloride and 900 ml of
distilled water
 Formal calcium
■ Useful for demonstration of phospholipids.
■ Fixation time – 24 hours at room
temperature.
 Zenker’s fluid
■ It contains mercuric chloride, potassium di-chromate, sodium
sulphate and glacial acetic acid.
■ Advantages – even penetration, rapid fixation
■ Disadvantages – after fixation the tissue must be washed in
running water to remove excess dichromate. Mercury pigment
must be removed with Lugol’s iodine.
■ 950 ml of distilled water, 25 gm of potassium dichromate, 50
gm of mercuric chloride and 50 gm of glacial acetic acid
Zenker’s formal (Helly’s fluid)
■ In stock Zenker’s fluid, formalin is added instead
of acetic acid.
■ Advantages – excellent microanatomical fixative
especially for bone marrow, spleen and kidney.
 Bouin’s fluid
■ It contains picric acid, glacial acetic acid and 40%
formaldehyde.
■ Advantages – rapid and even penetration without any
shrinkage. Brilliant staining by trichrome method. It is
routinely used for preservation of testicular biopsies.
■ 75 ml of saturated picric acid (1.2 gm/100 ml), 25 ml
of 40% formaldehyde and 5 ml of glacial acetic acid
TYPES OF FIXATION
■ Immersion fixation
■ Perfusion fixation
■ Vapor fixation
■ coating/spray fixation
■ Freeze drying
■ Microwave fixation/stabilization
■ Immersion fixation – which is the most routinely
used method, is performed by placing small
pieces of tissue into a relatively large volume of
fixative. This ratio is chosen to facilitate an
interaction between the two that may result in as
rapid and complete fixation as this method will
allow. The rate of diffusion, which is dependent
on the properties of both the fixative and tissue
type, is obviously a limiting factor for optimal
preservation.
■ Perfusion fixation – which is much
more complex and often difficult to
perform, depends on the introduction of
fixative into tissues via the vasculature.
Nevertheless, this method has the
greatest potential for rapid and fine
preservation of histologic architecture.
■ Vapor fixation – provides for tissue fixation by
subjecting the tissue to the vapor of a fixative
while the tissue is unstressed. The tissue may be
rotated during fixation to avoid collecting
condensed fixative at localized areas of the
tissue. Intermittent rinsing of the tissue during
fixation is possible. The concentration of fixative
in the vapor may be varied, increasing after an
initial period. Specific rinsing and fixative
solutions are disclosed.
■Coating/spray fixation – a type
of fixation used when fixing
small animals like insects
(butterflies, bugs etc.) by
spraying a fixative on the
specimen.
■ Freeze drying – is a water removal process
typically used to preserve perishable
materials, to extend shelf life or make the
material more convenient for transport.
Freeze-drying works by freezing the
material, then reducing the pressure and
adding heat to allow the frozen water in the
material to sublimate.
IMPORTANT RULES IN
FIXATION
■Volume of fixative, 10x
■Time – 12-24 hrs. (formalin)
■SIZE OF TISSUE, 2 cm 3