Fresh Tissue
Examination
• Method in examination of tissue:
• According to the structural and chemical components
of the cells to be studied
• Nature and amount of the tissue to be evaluated
• Need for an immediate examination of a tissue
structure
• Examination:
• Fresh
• Advantage of being examined on living state
• Allowing protoplasmic activities such as motion, mitosis,
phagocytosis and pinocytosis
• Disadvantage
• Not permanent, and therefore are liable to develop the changes
that have usually been observe after death
• Preserve tissue
Method of Fresh Tissue Examination
Teasing or
Dissociation
• Whereby a selected tissue specimen
is immersed in a watch glass
containing isotonic salt solution
• Carefully dissected or separated
• Examined under the microscope
• Stained with different dyes
• Unstained by Phase Contrast or Bright
Field microscope
Squash Preparation (Crushing)
• Whereby small pieces of tissue not
more than one mm. in diameter are
placed in a microscopic slide and
forcibly compressed with another
slide or with a cover glass.
• Vital stained may be placed at the
junction of the slide and the cover glass,
and allowed to be absorbed by the
tissue through capillary interaction.
Smear Preparation
• Whereby cellular materials are spread lightly over a slide by means of
a wire loop or applicator, or by making an apposition smear with
another slide
• Useful in cytological examinations, particularly for cancer diagnosis.
• Three type of Smear preparation:
• Streaking
• Spreading
• Pull-apart
• Touch preparation
• Streaking:
• Applicator stick or a platinum loop
• Material is rapidly and gently applied in a direct or zigzag line throughout the slide
• Obtaining a relatively uniform distribution of secretion
• Too thin or to thick smears have to be avoided
• Tissue is unsuitable for examination
• Spreading:
• Selected portion of material is
transferred to a clean slide
• Gently spreading into a moderately
thick film by teasing the mucous
strands apart with an applicator stick
• More tedious than streaking
• Advantage of maintaining cellular
interrelationship of the material to
be examined
• Recommended for smear
preparation of fresh sputum and
bronchial aspirate, and also for thick
mucoid secretion
• Pull-Apart
• Done by placing a drop of secretion or
sediment upon on slide and facing it to
another clean slide
• Material disperses evenly over the
surface of the two slides
• Slight movement of the two slides in
opposite directions may be necessary to
initiate the flow of materials
• The two slides are then pulled apart with a
single uninterrupted motion
• Specimen then place under the
microscope for immediate examination,
or applied with vital stains
• Useful for thick secretion, serous fluid,
concentrated sputum, enzymatic lavage
sample from the GIT and blood smear
• Touch Preparation (Impression Smear):
• The surface of a freshly cut piece of tissue is brought into contact and pressed
on the surface of a clean glass
• Allow the cells to be transferred directly to the slide of examination by Phase Contrast
microscopy or stained for light microscope
• Advantage of this method:
• The cells may be examined without destroying their actual intercellular relationship, and
without separating them from their normal surrounding
• Frozen Section:
• Normally utilized when a rapid diagnosis of the tissue in question is required
• Especially recommended when lipids and nervous tissue elements are to be
demonstrated
• Procedure:
• Very thin slices
• 10-15um in thickness
• Cut from fresh tissue frozen from microtome with CO2 or on a cryostat
• Cold chamber kept at atmospheric temperature of -10 to -20 C
• Frozen sections, both fixed and unfixed, have many applications in histotechnology, and are
commonly used:
• Rapid pathologic diagnosis during surgery
• Diagnostic and research enzyme histochemistry
• Diagnostic and research demonstration of soluble substances such as lipids and
carbohydrates
• Immunofluorescent and immunohistochemical staining
• Some specialized silver stain, particularly in neuropathology
• Frozen cont.
• Tissue for freezing should be fresh, and freezing should be done as quickly as
possible.
• Slow freezing should be done as quickly as possible.
• Slow freezing can cause distortion of tissue due to ice crystal artifacts
• More commonly used method of freezing:
• Liquid Nitrogen
• Isopentane cooled by liquid nitrogen
• Carbon dioxide gas
• Aerosol sprays
• Liquid nitrogen:
• Generally used in histochemistry and during operative procedures, and is the
most rapid of the commonly available freezing agents.
• Disadvantage:
• Soft tissue is liable to crack due to the rapid expansion of the ice within the tissue
• Produce ice crystal or freeze artifacts
• Overcools urgent biopsy blocks:
• Cause damage to both block and blade if sectioning is done at minus 70C or below
• The tissue snap-frozen in liquid nitrogen must
• Therefore be allowed to equilibrate to cryostat chamber temperature before sectioning is
attempted.
• The majority of non-fatty unfixed tissues are sectioned well at temperatures between -
10C and -25C
• Causes a vapor phase to form around the tissue
• It will act as insulator that causes uneven cooling of tissue, particularly of muscle biopsies, and
making diagnostic interpretation difficult
• Can be overcome by freezing the tissue in isopentae, OCT or Freon 2.2 that has a high
thermal conductivity
• Isopentane
• Liquid at room temperature
• Pyrex glass beaker is usually suspended in a flask of liquid nitrogen until half-liquid and
half-solid stage reached.
• The beaker is removed from the liquid nitrogen when small crystal start forming on the
side of the beaker (approximately -170C)
• The tissue to be frozen (affixed on a cork disc, aluminum foil or cryostat chuck) is
dropped into the cooled liquid isopentane
• Tissue block can be frozen
• Adapting a conventional freezing microtome gas supply of carbon dioxide a
CO2 cylinder
• Using a specially made piece of equipment
• The use of aerosol sprays has become increasingly popular in recent
years, and adequate for freezing small pieces of tissue except muscle.
PROCESSING OF TISSUE
• Fresh tissue are usually examined when there is an immediate need
for evaluation
• A better and more effective means of studying tissue, whether
abnormal or normal
• Examination of their sections and smears which have been
permanently preserved, stained for demonstration of specific
structures, and mounted of the glass slides with coverslips for
permenent keeping.
• Solid structure and tissue must be preserved and carefully
processed in the following order.
• Fixation
• Dehydration
• Clearing
• Infiltration (Impregnation)
• Embedding
• Trimming
• Section-cutting
• Staining
• Mounting
• Labeling
