HISTOPATHOLOGY LABORATORY
PREPARATION OF FRESH TISSUE
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
OUTLINE
I. Introduction
II. Fresh Tissue Sample Techniques
A. Teasing & Dissociation
B. Squash Preparation
C. Smear Preparation
a. Streaking
b. Spreading
c. Pull-Apart
d. Touch or Impression Smear
D. Frozen Section
INTRODUCTION
Histology is the microscopic study of the normal
tissues of the body while histopathology is the microscopic
study of tissues affected by disease. The procedures
adopted for the preparation of material for such studies are
known as histologic or histopathologic techniques. The
tissues are usually obtained during surgery, biopsy, or
autopsy. They range from very large specimens or whole
organs to tiny fragments of tissue. The following surgical
procedures are usually performed to obtain the
specific-types of tissue that are submitted to a histology
laboratory for processing:
● Fine needle aspiration is the simplest, least
invasive test and uses the smallest needle to
simply remove cells from the area of abnormality.
This is not always adequate to obtain a diagnosis,
depending on the area to be biopsied.
● A core needle biopsy removes not only cells, but
also a small amount of the surrounding tissue. This
provides additional information to assist in the
examination of the lesion.
● An incisional biopsy takes out even more
surrounding tissue. It takes out some of the
abnormality, but not all. The doctor will slice into
the lesion and remove only a portion of it. If the
lesion is found to be cancerous, further surgery
may be needed to remove or excise the entire
lesion.
● An excisional biopsy generally removes the entire
area in question.
● Punch biopsy is considered the primary technique
for obtaining diagnostic full-thickness skin
specimens. It requires basic general surgical and
suture-tying skills and is easy to learn. The
technique involves the use of a circular blade that
is rotated down through the epidermis and dermis,
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and into the subcutaneous fat, yielding a 3- to 4-
mm cylindrical core of tissue sample.
● Shave biopsy - where small fragments of tissue
are “shaved” from a surface (usually skin).
● Curettings - where tissue is scooped or spooned
to remove tissue or growths from body cavity such
as endometrium or cervical canal.
(From the book)
FRESH TISSUE SAMPLE TECHNIQUES
TEASING AND DISSOCIATION
● A process whereby a selected tissue specimen is
immersed in isotonic salt solution such as normal
saline or Ringer’s solution in a petri dish or watch
glass, carefully dissected with a needle and separated
by direct or zigzag spread using an applicator stick.
Selected pieces of the tissue are transferred carefully
to a microscope slide and mounted as a wet
preparation underneath a cover glass, careful to avoid
forming bubbles. It is either stained with a supravital
dye or examined unstained by Phase Contrast or
Bright Field microscopy. It has the advantage of
permitting the cells to be examined in the living state.
● Procedure for 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.
SQUASH PREPARATION (CRUSHING)
● A process 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. If necessary, a
supravital stain may be placed at the junction of the
slide and the cover glass, and allowed to be absorbed
by the tissue through capillary attraction.
HISTOPATHOLOGY LABORATORY
PREPARATION OF FRESH TISSUE
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

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 your slide with your name, group number and
section and submit it to your instructor
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 your slide with your name, group number
and section and submit it to your instructor.

SPREADING
SMEAR PREPARATION
1. Transfer a small amount of the sediments on
● The method of preparing the smear differs
one end of a clean glass slide.
depending on the nature of the material to be
2. Using an applicator stick, spread the sediments
examined. As a general rule, smears are made
throughout the slide (similar to how butter is
either by spreading the selected portion of the
spread on bread). Another technique is to place the
specimen over the surface of the slide with a
drop of sediments at the center of the slide. Then
platinum loop. Smears may be examined either as
with a circular motion, gently spread it into a
fresh preparations similar to that described for
moderately thick film.
teased preparations, or by using a supravital
3. Label your slide with your name, group number
staining technique. Smear preparations can be
and section and submit it to your instructor.
made permanent by fixing them while still wet,
staining them to demonstrate specific structures
and inclusions, and mounting the cleared specimen
beneath a cover glass with a suitable mounting
medium. This is useful for preparing smears of
thick secretions such as serous fluids,
concentrated sputum, enzymatic lavage sample
from the gastrointestinal tract, and blood smears.
This technique is especially useful in cytological ● It is a little more tedious than streaking, but has the
examinations, particularly for cancer diagnosis. advantage of maintaining cellular interrelationships
(from the book) of the material to be examined. It is especially
recommended for smear preparations of fresh
● Note: Centrifuge body fluid samples and decant the sputum and bronchial aspirates, and also for thick
supernatant. Save the sediments for the mucoid secretions. (from the book)
procedures below. Avoid making smears that are
too thin or too thick since this will render your
PULL-APART
smears unsuitable for examination. 1. Place a drop of the sediments at one end of a
glass slide.
STREAKING 2. Cover it with the end of another glass slide.
1. Pour a small amount of the sediments on one Allow the sediments to spread evenly between the
end of a clean glass slide. attached surfaces of the two slides.
2. Using an applicator stick, streak the sediments in
a straight or zigzag line throughout the remaining

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HISTOPATHOLOGY LABORATORY
PREPARATION OF FRESH TISSUE
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

3. With a single, uninterrupted motion, pull the two technique (around 10 minutes vs 16 hours). However, the
slides apart in opposite directions. Cover the technical quality of the sections is much lower.
smear with a cover slip.
4. Label your slide with your name, group number
and section and submit it to your instructor.

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 your slide with your name, group number
and section and submit it to your instructor.

Frozen Section
The technical name for this procedure is cryosection.
With the cryostat, the usual histology slice is cut at 5 to 10 µm.
The surgical specimen is placed on a metal tissue disc which
is then secured in a chuck and frozen rapidly to about –20 to
–30°C. The specimen is embedded in a gel like medium
consisting of polyethylene glycol and polyvinyl alcohol; this
compound is known by many names and when frozen has the
same density as frozen tissue. Subsequently it is cut frozen
with the microtome portion of the cryostat, the section is
picked up on a glass slide and stained. The preparation of the
sample is much more rapid than with traditional histology

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