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HISTOLOGIC TECHNIQUES
GERALD V. TEJADA, MD
 OVERVIEW
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 Fixation
 Tissue Processing
 Staining
 TISSUE PREPARATION
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 1. FIXATION
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 The aim of fixation:

1. Terminate cell metabolism
2. Prevent enzymatic degradation of cells and tissues
by autolysis (self-digestion)
3. Kill pathogenic microorganisms such as bacteria,
fungi, and viruses
4. Harden the tissue as a result of either cross-linking
or denaturing protein molecules
 1. FIXATION
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 Factors affecting fixation:

 pH.

 Temperature.

 Penetration of fixative.
 Volume of tissue.
 1. FIXATION
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 Examples of fixatives
 Acetic
acid, Formaldehyde, Ethanol, Glutaraldehyde,
Methanol and Picric acid.
 1. FIXATION
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 Formalin
 37% aqueous solution of formaldehyde, at various dilutions
and in combination with other chemicals and buffers, is the
most commonly used fixative.
 Preserves the general structure of the cell and extracellular
components by reacting with the amino groups of proteins
(most often cross-linked lysine residues).
 Does not significantly alter their three-dimensional structure,
proteins maintain their ability to react with specific
antibodies, important in immunocytochemical staining
methods.
 2. TISSUE PROCESSESSING
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 Aim:
 To embed the tissue in a solid medium firm enough to
support the tissue and give it sufficient rigidity to
enable thin sections to be cut, and yet soft enough not
to damage the knife or tissue
 2. TISSUE PROCESSING
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 Steps:
 Dehydration

 Clearing

 Embedding
 2. TISSUE PROCESSING
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 DEHYDRATION:
 Specimen is washed after fixation and dehydrated in a
series of alcohol solutions of ascending concentration to
remove water and minimize tissue distortion
 Example of dehydrating agents: Ethanol, Methanol,
Acetone
 2. TISSUE PROCESSING
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 CLEARING:
 Organic solvents which are miscible in both alcohol and
paraffin, are used to remove the alcohol before
infiltration of the specimen with melted paraffin
 Examples of clearing agents: Zylene, Toluene,
Chloroform, Benzene, Petrol
 2. TISSUE PROCESSING
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Automated Tissue Processor

 2. TISSUE PROCESSING
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 EMBEDDING
 Process by which tissues are surrounded by a medium
such as agar, gelatin, or paraffin wax which when
solidified will provide sufficient external support during
sectioning
 2. TISSUE PROCESSING
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Tissue Embedding Machine

 2. TISSUE PROCESSING
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 2. TISSUE PROCESSING
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 CUTTING/SECTIONING
 Microtome

 Ribbons of Tissue
 2. TISSUE PROCESSING
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 2. TISSUE PROCESSING
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 2. TISSUE PROCESSING
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Water Bath
 2. TISSUE PROCESSING
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 Station 1: 1 hour 30 mins 10% BNF (neutral buffered

formalin)
 Station 2: 1 hour 30 mins 10% BNF
 Station 3: 1 hour 50 ethyl alcohol
 Station 4: 1 hour 70 ethyl alcohol
 Station 5: 1 hour 80 ethyl alcohol
 Station 6: 1 hour 95 ethyl alcohol
 Station 7: 1 hour 100% ethyl alcohol (absolute alcohol)
 Station 8: 1 hour 100% ethyl alcohol
 2. TISSUE PROCESSING
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 Station 9: 1 hour 30 mins Xylene

 Station 10: 1 hour 30 mins Xylene
 Station 11: 1 hour paraffin
 Station 12: 1 hour paraffin
 Station 13: 1 hour paraffin
 Station 14: 1 hour paraffin
 3. STAINING
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 Hematoxylin and Eosin Staining

 Charge-based, general purpose stain
 Hematoxylin stains acidic molecules shades of blue.

 Eosin stains basic materials shades of red, pink and

orange.
 3. STAINING
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 3. STAINING
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 Basis of Acid and Basic Dyes

 An acidic dye, such as eosin, carries a net negative
charge on its colored portion
 A basic dye carries a net positive charge on its colored
portion
◼ Hematoxylin is not, strictly speaking, a basic dye. It is used
with a mordant (i.e., an intermediate link between the tissue
component and the dye). The mordant causes it to resemble
a basic dye.
 3. STAINING
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 Basic dyes react with anionic components of cells

and tissue (components that carry a net negative
charge).
 Anionic components include the phosphate groups of
nucleic acids, the sulfate groups of
glycosaminoglycans, and the carboxyl groups of
proteins.
 The ability of such anionic groups to react with a
basic dye is called basophilia.
 3. STAINING
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 Acidic dyes react with cationic groups in cells and

tissues, particularly with the ionized amino groups
of proteins.
 The reaction of cationic groups with an acidic dye is
called acidophilia.
 3. STAINING
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 Substances within cells and the extracellular matrix

display basophilia include:
 Heterochromatin and nucleoli of the nucleus (chiefly
because of ionized phosphate groups in nucleic acids of
both)
 Cytoplasmic components such as the ergastoplasm/ER
(also because of ionized phosphate groups in ribosomal
RNA)
 Extracellular materials such as the complex
carbohydrates of the matrix of cartilage (because of
ionized sulfate groups).
 3. STAINING
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 Substances within cells and the extracellular matrix

exhibit acidophilia include:
 Most cytoplasmic filaments, especially those of muscle
cells
 Most intracellular membranous components and much of
the otherwise unspecialized cytoplasm, and
 Most extracellular fibers (primarily because of ionized
amino groups).
 3. STAINING
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 3. STAINING
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 3. STAINING
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 Metachromasia
 Certain basic dyes (ex. toluidine blue) react with tissue
components that shift their normal color from blue to
red or purple
 Underlying mechanism is the presence of polyanions
within the tissue.
◼ When these tissues are stained with a concentrated basic
dye solution, the dye molecules are close enough to form
dimeric and polymeric aggregates. The absorption
properties of these aggregations differ from those of the
individual nonaggregated dye molecules.
 3. STAINING
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 Metachromasia
 Cell and tissue structures that have high concentrations
of ionized sulfate and phosphate groups—such as the
ground substance of cartilage, heparin-containing
granules of mast cells, and rough endoplasmic reticulum
of plasma cells—exhibit metachromasia.
◼ Therefore, toluidine blue will appear purple to red when it
stains these components
 3. STAINING
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 /end.
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