MLS 222

Department of Medical Laboratory Science

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 MLS 222
COURSE LEARNING OUTCOMES
At the end of the module, you should
be able to:
1. Explain the different terms and processes
associated with pathology.
2. Determine the different changes (cell injury,
cell adaptation, inflammation, tissue repair
and cell death) that occur related to
pathologic conditions as well as
pathophysiologic mechanisms.
3. List optimum working conditions, standard
operating procedures for an anatomic
laboratory and ethical behaviour in relation
to clinical specimen handling.
4. Determine the different collection methods
for histopathologic and cytological samples
for tissue processing and their proper disposal
of clinical samples for environmental safety.
5. Make use of the different principles of routine
and automated process, and related
equipment/materials/reagents involved in
proper histopathologic processing (fixation,
decalcification, dehydration, clearing,
impregnation, embedding, trimming and
sectioning, staining, mounting and labelling.)
6. Determine the different problems
encountered in histopathologic processing,
GENERAL PATHOLOGY, their probable
recommended solutions.
causes and apply

HISTOPATHOLOGIC AND 7. Utilize different principles of

processes, and
routine
related
CYTOLOGIC TECHNIQUES equipment/materials/reagents involved in
standard cytological processing for cell
(MLS 222 LECTURE)
blocking (fixation, dehydration, clearing,
impregnation, embedding, trimming and
sectioning, staining, mounting and labeling)
and cytological smears for Papaniculao
staining.
8. Explain recent advances in histopathologic
and cytopathologic techniques with
incorporation of rapid tissue processing and
immunohistochemical techniques tor tissue
processing.
9. Discuss terms and general procedures
associated with autopsy.

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 COURSE INTRODUCTION

Dear Future Registered Medical Technologists,

Congratulations for moving up in pursuing your career. As professionals willing to

engage in rendering services for the purpose of aiding the physician in the diagnosis, study
and treatment of diseases, welcome to MLS 222!

This subject is part of the board examination. By doing this course, it will enable you
to be familiar with the basic concepts of disease processes correlating the etiology of
diseases with the course developement of anatomic and clinical changes brough about by
the disease.

You are expected to learn the theories of histopathologic techniques and by doing
so, be able to perform techniques essential I the production of histopathologic slides for the
diagnosis of diseases including special staining procedures and other related techniques. It
will also introduce you to the study and identification of cells in the diagnosis of diseases
using cytological techniques. In like manner, you are expected to perform these techniques
in the future to identify abnormal cells in the diagnosis of diseases using cytological
techniques while complying with quality control.

During this pandemic, we are in a special educational setting. Your utmost discipline,
commitment and perseverance will be the determining factor in your success. Actual life
lessons in the process of learning this course are immeasurable by numbers. Always do your
best for a better future.

Pray. Hope. Do not worry!

Histopath Facilitators

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 MODULE 2: HISTOPATHOLOGIC TECHNIQUES

Congratulations for completing the preliminary modules.

The laboratory techniques in histopathology and cytology are the foundation of diagnostic
pathology. It is extremely important to know the basic and advanced techniques in
laboratory. This module explains the principles, steps and troubleshooting of the essential
laboratory techiniques in the histology laboratory.

It is expected that you read the main references to augment this module. This module
contains eight units on tissue processing. Kindly correlate with the laboratory module to have
a full understanding on the techniques discussed.

To help you keep track of your module tasks for this module, you are provided in the next
page with a self-monitoring form. Take the time to tick on the “Yes” box for each activity that
you finish, and be reminded about pending activities that you are yet to do. Remember that
your success in achieving the module objectives depends entirely on how conscientious you
are of your own progress.

Let us continue learning!

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 MODULE SELF MONITORING FORM
ACTIVITIES DONE?
YES NO
Read the Module Introduction, Module Contents, and Module
☐ ☐
Objectives
Engage
☐ ☐
Units 1, 2, 3, 4, 5, 6, 7, 8
Explore
☐ ☐
Units 1, 2, 3, 4, 5, 6, 7, 8
Explain
☐ ☐
Units 1, 2, 3, 4, 5, 6, 7, 8
Elaborate
☐ ☐
Units 1, 2, 3, 4, 5, 6, 7, 8
Evaluate
☐ ☐
Units 1, 2, 3, 4, 5, 6, 7, 8
Review Unit s ☐ ☐
Review Module ☐ ☐

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 MODULE CONTENTS

Contents
MODULE 2: HISTOPATHOLOGIC TECHNIQUES ..................................................................................4
MODULE SELF MONITORING FORM....................................................................................................5
MODULE CONTENTS .............................................................................................................................6
Unit 1 : Introduction to Tissue Processing ..........................................................................................7
Unit 2 : Fixation .................................................................................................................................. 11
Unit 3 : Decalcification..................................................................................................................... 14
Unit 4 : Dehydration .......................................................................................................................... 16
Unit 5: Clearing/Dealcoholization .................................................................................................. 18
Unit 6 : Impregnation/Infiltration ..................................................................................................... 20
Unit 7 : Embedding and Trimming .................................................................................................. 20
Unit 8 : Microtome, Section Cutting and Adhesives .................................................................... 23
Reference: ......................................................................................................................................... 27

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 Unit 1 : Introduction to Tissue Processing

This unit will enable you to:

1. Explain the mechanism of tissue preservatives
2. Discuss advantages and disadvantages of fresh tissue and preserved tissue examination
3. Explain different methods of fresh tissue examination
4. Identify which method of tissue examination to be performed per specific sample tissue.
5. Explain the steps in tissue processing

ENGAGE :

Case vignette:
JMO is a 23-year old female. She visits her family doctor for a painless right breast
lump that she discovered on self-examination, she is otherwise asymptomatic. Her medical
history is unremarkable. On physical examiantion, a small encapsulated, well-defined,
ruberry, freely movable 3-cm mass in the right lower quadrant of the right breast was noted.
There is no overlying skin changes; no nipple retraction; no lymphadenopathy and the other
breast is normal. All other routine diagnostic laboratory works are normal. Upon due
deliberation, the attending physician referred her to a surgeon for surgical excision of the
breast mass. The excised specimen was noted to be solid with no areas of necrosis or
hemorrhage. On microscopic examiantion, glandular structures with ductal and stromal
proliferation with no cellular atypia were noted. It was signed out as fibroadenoma. This
condition was explained to her as the most common benign breast tumor in young women.
It sometimes enlarges during pregnancy or normal menstrual cycles.

Did you know that in order to get to the diagnosis and treatment of the above case,
histopathologists play a great role? After the surgical procedure, the tissue undergoes series
of processess so that pathologists may be able to study it under the microscope aid in
diagnosing and prognostication.

The contents of this module will inspire you to know the mechanisms behind tissue
processing. As future medical technologists, you play a vital role in the team to deliver
quality health care services by your knowledge and skills in these histopathologic techniques.

EXPLORE :

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 Recall that 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, and into the subcutaneous fat, yielding a 3- to 4- mm cylindrical core
of tissue sample.
Shave biopsy is where small fragments of tissue are “shaved” from a surface (usually
skin).
Curettings are where tissue is scooped or spooned to remove tissue or growths from
body cavity such as endometrium or cervical canal.

Specimens are usually received in fixative (preservative) but sometimes they arrive
fresh and must be immediately fixed. Tissue specimens received in the surgical pathology
laboratory should have a request form that lists the patient information and clinical history
along with a description of the site of origin. The specimens are accessioned by giving them
a number that will identify each specimen for each patient. It is important that specimens
are properly identified to minimize the risk of mislabeling.

Once tissues are removed from the body, their proteins and cells are digested and
broken down by their own enzymes, independent of a bacterial action. This process is known
as autolysis, which is retarded by cold and accelerated at room temperature. It is more
severe in tissues that are rich in enzymes (e.g. liver, brain, and kidney) and less rapid in elastic
and collagen tissues.

Methods of tissue examination may vary according to the structural and chemical
components of the cells to be studied, and depends on the nature and amount of the tissue
to be evaluated. Fresh tissues are usually examined when there is an immediate need for

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 evaluation. On the other hand, a better and more effective means of studying tissues,
whether normal or abnormal, is by examination of adequately preserved sections and
smears that are stained to demonstrate specific structures. The glass slides are then mounted
with coverslips for permanent keeping.

Examination may be done on fresh or preserved tissues, depending on necessity.

Fresh tissues have the advantage of being examined in the living state, thereby allowing
protoplasmic activities such as motion, mitosis, and phagocytosis to be observed. Its use is
limited, however, because of the fact that tissues examined in the fresh state are not
permanent, and therefore, are liable to develop the changes that have usually been
observed after death.

EXPLAIN :

The methods of tissue examination may vary according to the structural and
chemical components of the cells to be studied, the nature and amound of the tissue to be
evaluated, and the need for an immediate examination of a tissue structure.
Examination may be done on fresh or preserved tissues depending upon necessity.
Fresh tissues have the advantage of being examined in a living state, thereby allowing
protoplasmic activities such as motion, mitosis, phagocytosic and pinocytosis to be
observed. Its use in the fresh state are not permanent, and therefore, are liable to develop
the changes that have usually been observed after death.

Methods of Fresh Tissue Examination include the following:

1. Teasing or Dissociation
2. Squash Preparation
3. Smear Preparation
a. Streaking
b. Spreading
c. Pull-Apart
4. Touch Preparation
5. Frozen Section Examination has two methods of preparation
a. Cold Knife Procedure
b. Cryostat Procedure (Cold Microtome)

The tissue for freezing should be fresh, and freezing should be done as fast as possible.
Slow freezing can cause distortion of tissue due to ice crystal artifacts. Commonly used
methods for freezing include:
a. Liquid nitrogen
b. Isopentane cooled by liquid nitrogen

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 c. Carbon dioxide gas
d. Aerosol sprays

For histochemical evaluation involving enzyme studies, tissue needs to be chemically

active, and the important chemical constituents should not have been removed, altered or
displaced. Like fresh frozen sections, these special techniques have the common principle
of rapidly preserving the tissue block by freezing (quenching). The aim is to produce instant
cessation of cellular activity thereby preventing chemical alteration of tissue and
displacement of cellular tissue components. Methods that may be resorted to, if chemical
fixation of tissue oblocks is to avoided are:
1. Freeze-drying
2. Freeze substition

Fresh tissues are usually examined when there is an immediate need for evaluation.
A better and more effective means, however, of studying tissues, whether normal or
abnormal, is examination of their sections and smears which have been permanently
preserved, stained for demonstration of specific structures, and mounted on glass slides with
coverslips for permanent keeping.

Solid structures and tissues must be preserved and carefully processed in the following
order:
1. Fixation
2. Decalcification (optional)
3. Dehydration
4. Clearing
5. Infiltration (Impregnation)
6. Embedding
7. Trimming
8. Section-cutting (Microtomy)
9. Staining
10. Mounting
11. Labelling

Perform a self-check by stating the definitions and/or descriptions of the new and
unfamiliar terms above in your own words or as used within a context of a discussion with
your classmates. Note also of the advantages and disadvantages of using such methods
with their specific uses for particular specimens.

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 ELABORATE :

Activity 2.1.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (20 points)

Fresh Tissue Examination Preserved Tissue Examination

Advantages

Disadvantages

Application (specific
sample tissue)

EVALUATE :

Activity 2.1.2: Make a flow chart on the overview of tissue processing for paraffin sections. It
should include keywords of its purpose. Your output in this part will be submitted and
graded. Use the answer sheet template for your output. (30 points)

Unit 2 : Fixation

This unit will enable you to:

1. Discuss the importance of tissue fixation immediately after removing it from the human
body
2. Explain the significance of using specific fixative for a specific tissue specimen
3. Discuss the characteristics of good fixaative.
4. Identifiy which of the fixative will be used for a given sample.

EXPLAIN

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 Histotechnology is the art and science performed by the histotechnologist to produce
a tissue section of good quality that will enable the pathologist to diagnose the presence or
absence of disease. The first and most critical step in histotechnology involves fixing or
preserving fresh tissue for examination. Fixation is the process that preserves tissues from
decay, thereby preventing autolysis or putrefaction.

The primary aim of fixation is to preserve the morphologic and chemical integrity of
the cell in as life-like manner as possible. Fixation prevents degeneration, decomposition,
putrefaction, and distortion of tissues after removal from the body.
The secondary goal of fixation is to harden and protect the tissue from the trauma of
further handling for it to be easier to cut during gross examination.

Objectives of fixation include to preserve the tissue, to prevent breakdown of cellular

elements, and to coagulate or precipitate protoplasmic substances.

Two basic mechanisms involved in fixation include:

1. Additive fixation
2. Non-additive fixation

Benefits of fixation include allowing thin sectioning of tissue by hardening the tissue;
preventing autolysis and inactivates infectious agents (except prion diseases); and improves
cell avidity for special stains.

Main factors involved in fixation include:

1. Hydrogen Ion Concentration
2. Temperature
3. Thickness of section
4. Osmolality
5. Concentration
6. Duration of Fixation

At this point, practical considerations include speed, penetration, volume and

duration of fixation to optimize fixation of tissue.

Using the main reference, enumerate the effects of fixatives in general:

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________________________________________________________________________________________

Also find out the characteristics of a good fixative:

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 ________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

Secondary fixation is the process of placing an already fixed tissue in a fixative to

facilitate and and improve the demonstration of particular substances; to make special
staining techniques possible (with secondary fixative acting as a mordant); and to ensure
further and complete hardening and preservation of tissues.

Post-Chromatization is a form of secondary fixation whereby a primarily fixed tissue is

placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant
for better staining effects and to aid in cytologic preservation of tissues.

Washing out is the process of removing excess fixative from the tissue after fixation in
order to improve staining and remove artefacts from the tissues. Several solutions may be
used.

Enumerate the general precautions in handling and fixation of specimens:

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________________________________________________________________________________________
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ELABORATE :

Activity 2.2.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (25 points)

Types of Description Classification Advantages Disadvantage Specific Use

Fixatives

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 Activity 2.2.2. Accomplish the task below. Your output for this part will be submitted and
graded. Use the answer sheet template for your output. (35 points)

Describe two (2) special considerations that should be considered, and the reasons for these
considerations, when doing the following:

1. Lipid fixation
2. Carbohydrate fixation
3. Protein fixation
4. Fixation for electron microscopy
5. Fixation for enzyme histochemistry
6. Fixation for immunofluorescence
7. Fixation for immunohistochemistry
8. Acetone fixation
9. Methanol fixation
10. Ethanol fixation
11. Methanol-acetone fixation
12. Methanol-acetone mix fixation
13. Methanol-acetone mix fixation
14. Formalin fixation
15. Paraformaldehyde-triton fixation
16. Paraformaldehyde-methanol fixation
17. Microwave fixation

Unit 3 : Decalcification

This unit will enable you to:

1. Explain the basic concept of decalcification
2. Enumerate the factors that may affect the process of decalcification
3. Identify the best decalcifying agent or method for decalcified tissue.
4. Discuss the other methods of tissue softening process.

EXPLAIN

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 After fixation, selected pieces of tissues are taken from the specimen, properly
labeled and identified, and then subjected to the subsequent steps of processing. However,
specimens such as bones, teeth and calcified tissues like tuberculous lungs, which contain
calcium interfere with accurate evaluation and examination of histologic sections. All
extraneous materials must be removed before proceeding to the next step in the tissue
processing.
Decalcification is the procedure whereby calcium or lime salts are removed from
tissues following fixation. This is usually carried out by using chemical agents, either with acids
to form soluble calcium salts, or with chelating agents that bind to calcium salts.
Decalcificatio should be done after fixation and before impregnation to ensure and
facilitate the normal cutting of sections and to prevent obscuring the microanatomic detail
of such sections by bone dust and other cellular debris. Inadequate decalcification may
result in poor cutting of hard tissues and damage to the knife edge during sectioning.
There are three main types of decalcifying agents:
- Those based on strong mineral acids
- Those based on weaker organic acids
- Those composed of chelating agents.

Concentration, fluid access, size and consistency, agitation, temperature are factors
influencing the rate of decalcification.

Surface decalcification is a method of dealing with small unexpected deposits of

calcium that may be encountered in paraffin blocks. When the paraffin-embedded block
has been trimmed, the tissue surface may reveal small foci of calcification and may cause
resistance or a "grating" sensation when sectioned with a microtome knife.

Unduly hard tissues which are liable to damage the microtome knives may require
tissue softeners, aside from decalcification. Perenyi's fluid may act both as a decalcifying
agent and tissue softener. To soften unduly hard tissues, selected portions are left in the fluid
for 12-24 hours and dehydrated in the usual manner; or the cut surface of the block may be
submerged in the fluid for 1-2 hours before sectioning, to facilitate easier cutting of tissues.
Washing out and immersion of fixed tissues in 4% aqueous phenol solution for 1-3 days
may also cause considerable tissue softening and easier sectioning of blocks without
producing marked deleterious effects and tissue distortion.
Other substances which may be used as tissue softeners are Molliflex, 2% hydrochloric
acid, or 1% hydrochloric acid in 70% alcohol. Tissues immersed in Molliflex may appear
swollen and soapy. This does not, however, affect the normalizing and subsequent staining
of tissue sections.

ELABORATE :

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 Activity 2.3.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (20 points)

Decalcifying Description Mechanism Advantages Disadvantage Specific

agent of Action Use

1 Chelating
agents

2 Ion
Exchange
Resin

3
Electrophoresis
(Electrical
Ionization)

4 Microwave
oven
decalcification

Activity 2.3.2: Accomplish the task below. Your output for this part will be submitted and
graded. Use the answer sheet template for your output. (20 points)

Describe two (2) special considerations that should be considered, and the reasons
for these considerations, when measuring the extent of calcification:
1. physical or mechanical test,
2. x-ray or radiological method
3. chemical method or calcium oxalate test

Unit 4 : Dehydration

This unit will enable you to:

1. Explain the significance of the process of dehydration

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 2. Describe the toxic effects of some dehydrating agents and how to prevent toxicity
3. Describe the best dehydrating agent for given sample tissue

EXPLAIN

The process of removing intercellular and extracellular water from the tissue following
fixation and prior to wax impregnation is known as "dehydration”, and the solutions utilized
to make this possible are called "Dehydrating Agents".

It is important to distinguish between drying and dehydration. Drying is the removal of

water by evaporation from a solid, semi-solid or liquid. Solid tissues should NEVER be allowed
to air dry. Dehydration involves slow substitution of the water in the tissue with an organic
solvent. Most dehydrating agents are strong organic solvents that bring about some
shrinkage and extraction of cell components. To minimize these effects, dehydrating agents
are used in a graded series for short periods of time, and water is gradually replaced so that
violent osmotic changes do not produce distortions.

Enumerate the characteristics of an ideal dehydrating solution:

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________________________________________________________________________________________

ELABORATE :

Activity 2.4.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (30 points)

Dehydrating Description Mechanism Possible Preventive Specific

agent of Action Toxicity measures for Use
toxicity

1 Alcohol

2 Acetone

3 Dioxane
(Diethylene
dioxide)

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 4 Cellosolve

5 Triethyl
phosphate

6
Tetrahydrofuran

Unit 5: Clearing/Dealcoholization

This unit will enable you to:

1. Explain the significance of clearing prior to impregnation
2. Discuss the toxic effects of some clearing agents and how to prevent toxicigy
3. Describe the best clearing agent for a given sample tissue

EXPLAIN

Clearing (de-alcoholization) is the process whereby alcohol or a dehydrating agent

is removed from the tissue and replaced with a substance that will dissolve the wax with
which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the
tissue is to be mounted (e.g. Canada balsam). Aside from removing alcohol, a clearing
agent must also be miscible with Canada balsam and other resins that are used for mounting
sections. This stage in the process is called “clearing” because many (but not all) clearing
agents impart an optical clarity or transparency to the tissue due to their relatively high
refractive index. This change in appearance is often used as an indication of the
effectiveness or completeness of the clearing process.

Enumerate the characteristics of a good clearing agent:

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________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

The choice of a clearing agent depends on several factors. List them down below:

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________________________________________________________________________________________

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 ________________________________________________________________________________________
________________________________________________________________________________________

ELABORATE :

Activity 2.5.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (75 points)

Clearing agent Description Mechanism Possible Preventive Specific

of Action Toxicity measures for Use
toxicity

1 Xylene

2 Toluene

3 Benzene

4 Chloroform

5 Cedar wood

6 Aniline oil

7 Clove oil

8 Carbon
tetrachloride

9
Tetrahydrofuran

10 Dioxane

11 Terpene

12 Limonene

13 Orange oil
based clearing
agent

14 Chlorinated
hydrocarbons

15 Coconut oil

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 Unit 6 : Impregnation/Infiltration

This unit will enable you to:

1. Explain the importance of tissue impregnation
2. Discuss the importance of paraffin wax impregnation
3. Identify other infiltrating media

EXPLAIN

Impregnation (Infiltration) is the process whereby the clearing agent is completely

removed from the tissue and replaced by a medium that will completely fill all the tissue
cavities and give a firm consistency to the specimen. This allows easier handling and cutting
of suitably thin sections without any damage or distortion to the tissue and its cellular
components.

Activity 2.6.1: Accomplish the task below. Your output for this part will be submitted and
graded. Use the answer sheet template for your output. (25 points)

Describe two (2) special considerations that should be considered, and the reasons
for these considerations in:
1 Paraffin wax impregnation
A Manual processing
B Automatic processing
C Vacuum embedding
D Use of Paraplast
E Use of Ester Wax
F Use of water soluble waxes
2 Celloidin Impregnation
A Wet Celloidin method
B Dry Celloidin method
C Nitrocellulose method
3 Gelatin Impregnation

Unit 7 : Embedding and Trimming

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 This unit will enable you to:
1. Discuss the importance of molding the sample tissue prior to sectioning
2. Explain the importance of trimming the tissue prior to sectioning

EXPLAIN

Embedding (Casting or Blocking) is the process by which the impregnated tissue is

placed into a precisely arranged position in a mold containing a medium which is then
allowed to solidify. Ideally, an infiltrating and embedding medium should be:

- soluble in processing fluids

- suitable for sectioning and ribboning
- molten between 30°C and 60°C
- translucent or transparent; colorless
- stable
- homogeneous
- capable of flattening after ribboning
- non-toxic
- odorless
- easy to handle
- inexpensive
The medium used to infiltrate the tissue is usually the same medium utilized for
impregnation, and for general purposes is known as an Embedding Medium. There are
generally four types of impregnation and embedding medium,

namely:

1. Paraffin wax
2. Celloidin (collodion)
3. Gelatin
4. Plastic

After impregnation, the tissue is placed into a mold containing the embedding
medium and this medium is allowed to solidify. Ideally the embedding medium should match
the tissue type in strength and hardness. If the embedding medium is too soft for the material,
the tissue will not be supported and sections will be torn or shredded. If the medium is too
hard for the tissue, sections will be brittle and will shatter. To infiltrate the tissues with
supporting embedding medium, tissues must be free of all water (since usually embedding

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 medium is not miscible with water). Paraffin embedded tissues are arranged at the bottom
of the mold together with their proper labels and immersed in melted paraffin at a
temperature between 5-10°C above its melting point and then cooled rapidly in a
refrigerator at -5°C or immersed in cold water to solidify. This will allow hardening of tissues,
giving them a firmer consistency and better support, thereby facilitating the cutting of
sections.
The process by which a tissue is arranged in precise positions in the mold during
embedding, on the microtome before cutting, and on the slide before staining, is known as
Orientation. Generally speaking, the surface of the section to be cut should be placed
parallel to the bottom of the mold in which itis oriented. Several types of Blocking-out Molds
may be used:
1. Leuckhart’s Embedding Mold - consists of two L-shaped strips of heavy brass or metal
arranged on a flat metal plate and which can be moved to adjust the size of the mold to
the size of the specimen . Blocks produced are even, with parallel sides, and with a fairly
shaped initial setting of the wax. The mold is adjustable to give a wide variety of sizes to fit
the size of the tissue block for casting. It is recommended for routine use, although, too slow
and cumbersome for use in a busy laboratory.
2. Compound Embedding Unit is made up of a series of interlocking plates resting on a flat
metal base, forming several compartments. It has the advantage of embedding more
specimens at a time, thereby reducing the time needed for blocking.
3. Plastic Embedding Rings and Base Mold -consist of a special stainless steel base mold fitted
with a plastic embedding ring, which later serves as the block holder during cutting.
One model, the so-called Tissue Tek, is equipped with a warm plate to manage the
impregnated specimen, and a cold plate at -5°C for rapid solidification of the block. It
consists of a white plastic cassette mold with detachable, perforated stainless steel hinge
and Snap-On lid, used to hold the tissue specimen through-out fixation, dehydration,
clearing and wax impregnation.
4. Disposable Embedding Molds
a. Peel-Away disposable thin plastic embedding molds, available in 3 different sizes, are
simply peeled off one at a time, as soon as the wax has solidified, giving perfect even block
without trimming. It may be placed directly in the chuck or block holder of the microtome.
b. Plastic Ice Trays -such as those used in ordinary refrigerators may be recommended for
busy routine laboratories. Each compartment may be utilized for embedding one tissue
block, which may then be removed by bending the plastic tray once the wax has solidified
or by smearing the inner mold with glycerin or liquid paraffin before embedding.
c. Paper Boats are normally utilized for embedding celloidin blocks but are equally useful for
paraffin wax blocks. They have the advantage of being cheap and easy to make. They
provide easy and accurate identification of specimen, thereby avoiding confusion and
interchange of tissue blocks. Rapid embedding of small or large volume of individual
specimen is possible, since paper molds can be made to suit any size of tissue. Double-
Embedding is the process by which tissues are first embedded or fully infiltrated with a
supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin

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 wax in which they are subsequently embedded. This is used to facilitate cutting of large
blocks of dense firm tissues like the brain. They are also recommended for making small
sections of celloidin blocks. A shortcoming of using agar as the pre-embedding media is that
certain tissues shrink during the embedding process, and the agar-based pre-embedding
media limits tissue expansion during slide mounting, resulting in difficulties with the tissue
sample adhering to the microscope slide. The availability of paraffin waxes containing
different types of resins has made this technique obsolete.

Activity 2.7.1: Accomplish the task below. Your output for this part will be submitted and
graded. Use the answer sheet template for your output. (10 points)

Describe two (5) special considerations that should be considered, and the reasons
for these considerations in trimming of tissue prior to sectioning.

Unit 8 : Microtome, Section Cutting and Adhesives

This unit will enable you to:

1. Differentiate the types and use of each microtome
2. Explain the use of each microtome knife
3. Discuss the importance of honing and stropping
4. Discuss measures to resolve problems encountered during section cutting
5. Explain the significance of the use of adhesives

EXPLAIN

The process by which processed tissue, most commonly a paraffin embedded tissue,
is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the
microscope is known as Microtomy. The basic instrument used is a microtome that is capable
of cutting a section at a predetermined thickness by sliding the block into a cutting tool,
usually a steel knife, glass or diamond blade, which is fixed and attached to the machine.
The microtome consists of three essential parts, namely:

1. Block Holder - where the tissue is held in position.

2. Knife Carrier and Knife - for actual cutting of tissue sections.

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 3. Pawl, Ratchet Feed Wheel and Adjustment Screws - to line up the tissue block in proper
position with the knife, adjusting the proper thickness of the tissue for successive sections.

Badly nicked knives with blunted ends have to undergo sharpening in orderto ensure
optimum sectioning of tissue blocks and prevent gross irregularities on the tissue sections.
Jagged edges, if not corrected, will produce tears or striations in tissue sections. Sharpening
of the knife involves two stages:

Honing (Hard Sharpening) involves the removal of gross nicks on the knife edge
(Coarse Honing) to remove blemishes, and grinding the cutting edge of the knife on a stone
(Honing Proper) to acquire an even edge. The degree of sharpness is proportional to the
fineness of the abrasive used in sharpening. This procedure makes use of a hone, a natural
sharpening stone or hard grinding surface (carborundum), which serves to remove nicks and
irregularities on the knife edges. Several types of hones may be used:

1 Belgium Yellow - for manual sharpening when cutting edge has been rendered blunt or
nicked. This type usually gives the best result.

2. Arkansas - gives more polishing effect than the Belgium Yellow.

3. Fine carborundum - is much coarser than the first two types and is used only for badly
nicked knives followed by either one of the first two knife sharpeners.

The surface of the hone is wiped clean with a soft cloth moistened with xylene in order
to remove the scattered small particles of stones and metal. It is then covered with a thin
film of Mineral and Clove Oil, Xylene, Liquid Paraffin or Soapy Water for lubrication. The knife
is fitted to its corresponding back, placed on one end of the hone, and with the cutting knife
edge first, the "heel" (handle end) is drawn obliquely or diagonally towards the operator on
the stone until the "toe" (head portion) is reached. The knife is then turned over, and the
other surface is again drawn forward, EDGE FIRST, with a HEEL TO TOE direction. Hone is
placed on non-skid surface. A damp cloth may be used-to prevent movement of the hone.
Light lubricating oil or soapy water is used for lubrication.

What are the precautions during honing? Identify them:

________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

Stropping is the process whereby the "burr" formed during honing is removed and the
cutting edge of the knife is polished. The purpose of stropping is to polish and sharpen the
cutting edge, while that of honing is to remove the irregularities from the knife. If the knife
has become dull and blunt, but is free from nicks or teeth, it is usually only necessary to strop
it. For delicate work, the knife is stropped before every object is sectioned.

A paddle strop made up of the best quality horse leather, firmly attached to a solid
back, in order to prevent sagging is preferred. The procedure is the reverse of honing. The

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 knife is first fitted with its appropriate knife back, then laid obliquely on the strop and with the
cutting edge behind, (EDGE LAST) is pushed backward and drawn forward in a TOE TO HEEL
direction. Around 40- 120 double strokes are usually required. In the case of plane-wedge or
Minot knives, the knife is turned around at the end of each stroke so as to sharpen each
surface alternately. For plane concave knives, only the concave surface should be
stropped.

Identify the precautions to be observed in stopping:

________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

In addition to the microtome and the microtome knife, the following items are also
required during the process of sectioning:

Waterbath - The thermostatically controlled type is preferable, but if this is unavailable, water
from a hot water tap can be used although this can give rise to air bubbles which may be
trapped under cut sections. The temperature of the water should be between 5 and 10°C
below the melting point of the paraffin wax. Alcohol or small quantities of detergent may be
added for reducing surface tension and allowing the section to flatten out with greater ease.

Drying oven or hot plate - Small drying ovens are now available, incorporating a fan,
especially designed for drying tissue section on slides. With a temperature setting at the
melting point of the wax no obvious damage is done to the sections and drying is complete
in 30 minutes. A hot plate may also be used instead of a drying oven. For more delicate
tissues such as brain, a lower drying temperature is used to avoid splitting and cracking of
the section due to excessive heat. In such cases, 37oC for 24 hours or longer is
recommended.

Forceps (fine pointed or curved) and squirrel hair brush - These tools are needed for handling
sections during cutting, and for removing folds and creases on the sections during "floating
out" in water bath.

Clean Slides - For routine work, 76 x 25 mm. slides that are 1.0 -1.2 mm thick are usually
preferred because they do not break easily. Frost-ended slides are generally used, where
the identification number of the section can be inscribed with a pencil. Automatic slide
labeling machines are also now available.

Equipment such as a slide rack is made on the assumption that regular slides have been
used. Larger size of slides are used for sections of eyes or CNS tissues when these will not fit
on the regular/

An adhesive is a substance which can be smeared on to the slides so that the sections stick
well to the slides. The choice of slide and adhesive will be influenced by the staining methods

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 to be subsequently applied. Slides must always be grease- and dust- free and stored and
handled correctly.

The quality of sections cut on a microtome suffer badly from several (avoidable)
causes. Things to avoid include: fecal material in intestine, especially in the colon where this
material is very hard; hair is particularly bad - it can be removed using a razor blade or
clippers. Hair can also sometimes be inadvertently included with organs. Please be careful
during dissections; sutures, thread or staples should be removed from the specimen prior to
cutting with the knife.

ELABORATE :

Activity 2.8.1 Fill out the table below. Your output for this part will be submitted and graded.
Use the answer sheet template for your output. (30 points)

Microtome knife Description Special Specific Use

precaution

1 Rocking microtome knife

2 Rotary microtome

3 Sliding microtome

4 Freezing microtome

5 Cryostat or cold microtome

6 Ultrathin microtome

7 Disposable blades

8 Glass knives

9 Diamond knives

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means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 26
 Activity 2.8.2: Accomplish the task below. Your output for this part will be submitted and
graded. Use the answer sheet template for your output. (10 points)

Describe two (5) special considerations that should be considered, and the reasons
for these considerations in the use of adhesives.

Reference:

Bruce-Gregorios, J.H. (2017). Histopathologic Techniques. US Edition. Miani, Florida.: Jocelyn

H. Bruce-Gregorio, MD

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means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 27