Fresh Tissue

Examination
MLS-LAB 410 | BSMLS
Histopathologic Examination
• offers beneficial diagnostic information

• fresh or fixed (preserved) state

• factors affecting the method to be used:

• structures or inclusions
• amount and nature of the tissue
• urgency of investigation
Histopathologic Examination
Fresh Tissue Examination
◦ for rapid microscopic analysis while surgery is
taking place

Fixed Tissue Processing

◦ preferred in many conditions for more accurate
diagnosis
Fresh Tissue Examination
• Advantage:
• tissues are examined in a living state
• protoplasmic activities may be observed (e.g.,
motion, mitosis, phagocytosis, pinocytosis)
• Disadvantage:
• tissues examined in fresh state are not permanent
• tissues develop changes that are usually observed
after death
Methods of
Preparing Fresh
Tissue Mounts
FRESH TISSUE EXAMINATION
Methods of Preparing
Fresh Tissue Mounts
• Teasing or Dissociation

• Squashing or Crushing

• Smearing

• Frozen Section
Methods of Preparing
Fresh Tissue Mounts
• Teasing or Dissociation
• tissue specimen is immersed in a watch glass
containing isotonic solution, carefully dissected or
separated, and examined under the microscope
• done by carefully separating very thin tissue slices
with dissecting needles
Teasing and Dissociation
1. From the frozen block of tissue, cut several
small, thin slices of tissue.
2. Immerse the tissue slices in a petri dish filled
with saline solution.
3. Using a dissecting needle, carefully tease the
tissue slices until they unfold and spread out.
4. Carefully mount a teased slice on a slide and
cover it with a coverslip.
Methods of Preparing
Fresh Tissue Mounts
• Squashing or Crushing
• done by simply crushing small pieces of tissue (1
mm in diameter) between two glass slides or
between a glass slide and a cover slip
Squash Preparation
From the 4th step of the previous procedure:
1. Add 1-2 drops of methylene blue stain directly
on the tissue slice on the glass slide.
2. Cover the stained tissue with a cover slip or
another glass slide.
3. Applying enough pressure to crush the tissue
flat.
4. Label the slide.
Methods of Preparing
Fresh Tissue Mounts
• Smearing
• method of choice for examining sediments
• cellular materials are spread lightly over a slide
with:
• a wire loop or an applicator stick; or
• another glass slide
Smear Preparation
• Centrifuge body fluid samples and decant the
supernatant.

• Avoid making smears that are too thin or too thick

since this will render your smears unsuitable for
examination.
Methods of Preparing Fresh Tissue Mounts
Smearing
• Streaking
• Spreading
• Pull-apart
• Touch preparation (Impression smear)
Methods of Preparing Fresh Tissue Mounts
Smearing
• Streaking
• material is rapidly and gently applied in a direct or
zigzag line throughout the slide, attempting to obtain
a relatively uniform distribution of secretion
Smear Preparation
Streaking
1. Pour a small amount of the sediments on one end
of a clean glass slide.
2. Using an applicator stick, streak the sediments in a
straight or zigzag line throughout the remaining
length of the slide. If a wire loop is used instead,
collect a loopful of the material and smear the
material on the slide. A relatively uniform
distribution of the material should be obtained.
Cover it with a cover slip.
3. Label the slide.
Methods of Preparing Fresh Tissue Mounts
Smearing
• Spreading
• portion of a material is gently spread into a
moderately thick film by teasing the strands apart
with an applicator stick
• recommended for smear prep of fresh sputum,
bronchial aspirates, and thick mucoid secretions
Smear Preparation
Spreading
1. Transfer a small amount of the sediments on
one end of a clean glass slide.
2. Using an applicator stick, spread the sediments
throughout the slide (similar to how butter is
spread on bread). Another technique is to place
the drop of sediments at the center of the slide.
Then with a circular motion, gently spread it into
a moderately thick film.
3. Label the slide.
Methods of Preparing Fresh Tissue Mounts
Smearing
• Pull-apart
• placing a drop or secretion or sediment upon one
slide and facing it to another clean slide
• material disperses evenly over the surface of two
slides, then slides are pulled apart with a single
uninterrupted motion
Smear Preparation
Pull Apart
1. Place a drop of the sediments at one end of a
glass slide.
2. Cover it with the end of another glass slide.
Allow the sediments to spread evenly between
the attached surfaces of the two slides.
3. With a single, uninterrupted motion, pull the two
slides apart in opposite directions. Cover the
smear with a cover slip.
4. Label the slides.
Methods of Preparing Fresh Tissue Mounts
Smearing
• Touch preparation (Impression smear)
• surface of a freshly cut piece of tissue is brought
into contact and pressed into the surface of a clean
glass slide
• cells are transferred directly to the slide for
examination
Smear Preparation
Touch or Impression Smear
1. Retrieve the block of tissue from the first
procedure. Cut the block in half, exposing a
fresher surface.
2. Allow the freshly cut surface to come in contact
with the surface of a clean glass slide. Apply
gentle pressure, allowing the cells to be
transferred directly to the slide. Cover the smear
with a cover slip.
3. Label the slide.
Methods of Preparing
Fresh Tissue Mounts
• Frozen Section (Cryosection)
• rapid diagnosis
• uses freezing microtome or cryostat
• tse should be fresh, and freezing should be done as
quickly as possible
• liquid N2, isopentane, CO2, aerosol sprays
Methods of Preparing
Fresh Tissue Mounts
• Frozen Section (Cryosection)
• commonly used for:
• rapid pathologic dx during surgery
• dx and research enzyme histochemistry
• dx and research demonstration of soluble substances
such as lipids and carbohydrates
• immunofluorescent and immunohistochemical staining
• some specialized silver stains, particularly in
neuropathology
Methods of Preparing
Fresh Tissue Mounts
• Frozen Section (Cryosection)
• cryostat
• 5-10 μm, frozen rapidly to about –20 to –30°C
• embedded in a gel like medium consisting of
polyethylene glycol and polyvinyl alcohol