UNIVERSITY OF CEBU-BANILAD

COLLEGE OF MEDICAL TECHNOLOGY/

MEDICAL LABORATORY SCIENCE
Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000
Telephone # 032-342-0613
Email Address: medtech@uc.edu.ph
HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES

NAME: MARIA KRISTINA D. GAQUIT DATE SUBMITTED: December 8, 2020

PRECEPTOR: _________________________________________ SECTION: A

ACTIVITY 2

MICROTOMY TO H&E STAINING

1. OBJECTIVES

At the end of the laboratory exercise, the students should be able to:

1.1 Familiarize the proper procedure in using rotary microtome.

1.2 Identify the parts of a rotary microtome along with their functions.

1.3 Describe the proper procedure in H & E stains for liver tissue.

INSTRUCTION:

1. Using the Histopathologic technique book by Bruce-Gregorios, fill-up or answer the following
laboratory exercise for routine tissue processing.

I. Draw and Label the parts of a Rotary Microtome:

II. Enumerate the parts of the microtome & give its uses / functions.

1. Microtome base plate or stage: This serves as a platform consisting of rails that secures the
knife holder base and the instrument.

2. Knife holder: Sets the clearance angle. The knife holder is composed of different components
which includes a blade lamp that holds the blade, the knife tilt used to adjust the knife angle,
and the faceplate that guides the ribbon away from the blade and towards the operator
 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY/
MEDICAL LABORATORY SCIENCE
Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000
Telephone # 032-342-0613
Email Address: medtech@uc.edu.ph
HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES

3. Knife holder base: The part that anchors the knife holder to the microtome base plate. It can
be moved towards or away from the block and must be stationary and locked during
microtomy.

4. Block holder or Cassette clamp: This is responsible for holding the paraffin block in place. It
moves up and down with each revolution while the blade is stationary. Users can manipulate
the block face in various directions using the knobs to bring the tissue in alignment with the
blade.

5. Coarse Handwheel / Pawl ratchet feedwheel: Moves the block holder either toward or away
to the knife

6. Micron Adjustment or Thickness adjustment knob: used for adjusting section thickness to
microns ranging from 1 to 60; Sets tissue sectioning to 5 micra

7. Advancement handwheel: Turns in one direction and advances the block toward the knife at
specified microns. Most handwheels have safety lock to prevent the wheel from releasing nd
having the block holder come down towards the blade while a block is inserted or removed.

III. What are the three (3) different microtome knives available for cutting? Describe each.

The three different microtome knives used for cutting, includes:

a.) Plane concave knife (usually 25 mm in length)  from the name itself one side of the knife
is flat while the other side is concave. It is recommended that less concave sides are used for
cutting celloidin-embedded tissue blocks on a sliding microtome. For cutting paraffin sections
on base sledge, rotary or rocking microtome, more concave sides are recommended.

b.) Biconcave Knife (usually 120 mm in length)  Both sides are concave; It is recommended for
cutting paraffin-embedded sections on a rotary microtome

c.) Plane-Wedge Knife (usually 100 mm in length)  Both sides are straight; it is recommended
for cutting frozen sections extremely hard and tough specimens embedded in paraffin blocks,
using a base sledge type or sliding microtome

IV. What are the four (4) different types of microtome? For what specific embedding media are
they used?

1. Sliding microtome  simplest microtome that is used for cutting serial sections of large blocks of
paraffin embedded tissues.

2. Sliding microtome  for cutting celloidin embedded sections.

3. Rotary microtome  for cutting paraffin embedded sections.

4. Cryostat or cold microtome  for cutting frozen sections

Other microtomes:

5. Freezing microtome  for cutting unembedded frozen sections

6. Ultrathin microtome –> for cutting sections of electron microscopy

V. What are the two stages of knife sharpening? Give its purpose.

The two stages of knife sharpening are Honing or Hard Sharpening and Stropping.
 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY/
MEDICAL LABORATORY SCIENCE
Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000
Telephone # 032-342-0613
Email Address: medtech@uc.edu.ph
HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES

1. Honing or hard sharpening  refers to the removal of gross nicks on the knife edge (coarse
honing) to remove blemishes and Honing proper/ grinding the cutting edge of the knife on stone
to produce an even edge. This procedure uses a hone, a natural sharpening stone or hard grinding
surface (carborundum) which remove the nicks and irregularities on the knife edges. Different
types of hones include: a. Belgium Yellow, b. Arkansas, c. Fine carborundum

2. Stropping –> the process of removing the burr formed during honing and includes polishing of the
cutting edge. The purpose of this procedure is to sharpen and polish the cutting edge. For delicate
work, the knife is stropped prior to sectioning of objects. If the knife is dull and blunt but is free
from nicks or teeth, only stropping is necessary.

VI. Make a schematic diagram of the procedure for H&E staining with timing.

VII. What is the principle of H and E Staining?

Hematoxylin and Eosin staining depends on the affinity of a component to an acidic or basic dye. The
acidic component of the cells has affinity to basic dye while the basic component of the cells has the
affinity to acidic dye. Hematoxylin a basic dye stains acidic part of the cell such as the nucleus thus it is
commonly referred to as nuclear stain. It imparts a blue-purple color to acidic components of the cell.
While Eosin, an acidic dye binds to the basic parts of the cell such as the cytoplasm staining them pink.
 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY/
MEDICAL LABORATORY SCIENCE
Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000
Telephone # 032-342-0613
Email Address: medtech@uc.edu.ph
HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES

VIII. Give the expected color of the following:

TISSUE COMPONENT END COLOR AFTER H&E STAINING

Nuclei Blue or dark purple

Cytoplasm Pink

RBC Intensely orange red

Muscle Fibers Deep Red

Collagen Fibers Pink

IX. Define chromophore, chromogen and auxochrome in relation to dyes?

A dye should consist of a chromophore and an auxochrome group that is attached to a

hydrocarbon benzene ring.

Chromophores  are substances having definite atomic groupings that are capable of producing
visible colors.

Chromogens  are benzene compounds with chromophores. Before they are called a dye, they
must have the property of retaining its color in the tissue and this property can be acquired by the
presence of an auxochrome. Chromogens are any substances than can become a pigment or
coloring matter.

Auxochrome  is an auxiliary radical or substance imparting to the compound the property of

electrolytic dissociation allowing the alteration of the shade of the dye. This enables the formation
of salts with another compound and leads to the retention of its color.

X. Differentiate progressive staining to regressive staining. Give examples of each.

Progressive staining is the technique of staining where tissue elements are stained following a definite
sequence. The staining solution is applied only for a specific period of time or until the desired
intensity of color imparted to the different tissues elements is achieved. The dye that was taken up by
the tissue, is not washed colored.

Example of Progressive staining: Alum Hematoxylin, H & E staining of Frozen Sections for Rapid
Diagnosis

Regressive staining is another technique in staining where the tissue is first overstained to obliterate
the cellular details, then the excess stain is removed or decolorized from unwanted parts of the tissue
until desired color intensity is obtained

Example of regressive staining: The routine and most common method in microanatomical studies,
Hematoxylin and Eosin (H&E). The nuclei is initially overstained by hematoxylin and followed by the
removal of the superfluous and excessive color of the tissue by acid differentiation. Hematoxylins used
are Ehrlich’s Hematoxylin and Delafield’s hematoxylin