Histology: A Practical Manual: Prepared By: Dr. Hossein Noyan, PHD 2020

Histology: A Practical Manual
Prepared by:
Dr. Hossein Noyan, PhD
2020
IMPORTANT NOTICE:
The content in this manual is provided solely for your personal, non-commercial use. You are not permitted to copy, re-post, broadcast,
transmit, show or play in public, adapt, or change in any way the content of this manual for any other purpose whatsoever.
What is the Purpose of this Manual?
This manual is a guide to work in the histology laboratory or as a supplement for lectures and
virtual microscopy. For each topic there is a brief introduction (identical to that provided in the
Blackboard content for this module). This is followed by a number of images, with commentary.
To study tissue sections in detail, links to relevant websites are provided. You may examine each
tissue section by zooming in or out (different magnifications) and/or quiz yourself. Note: Please
refer to the lectures and suggested textbooks (if you have one) for additional details.
MODULE 1
SECTION 1.1. Histology and It’s Methods
This is a brief introduction to histology and histological methods. For more in depth information
please use the suggested textbooks and other resources identified in the course outline and
elsewhere in this course.
What is Histology?
Histology or microscopic anatomy is a branch of the anatomical sciences. Histology is the
scientific study of cells and tissues of the body and the ways they are structurally and functionally
related. The term “histology” comes from the Greek words “histos,” meaning tissue and “logia,”
meaning science. A modern histologist combines descriptive aspects of this science with many
aspects of molecular and cell biology to describe organization and function of cells and tissues.
Methods of study. Histologists use extremely diverse methods for their study. One of the common
tools found in any histology lab is light microscope. Light microscopy includes:
 bright-field microscopy,
 fluorescence microscopy,
 phase-contrast microscopy,
 confocal microscopy, and
 polarizing microscopy.
Note: To learn more about different microscopy methods indicated above, read chapter 1 of the
suggested textbooks listed in the reference section of this manual. A newer study method, namely
virtual microscopy is becoming popular in histology labs. Using virtual microscopy students and
scientists can view a digitized microscopic specimen on a computer screen or a mobile device.
More detailed knowledge about the microanatomy of the cells and tissues can be obtained using
electron microscope or EM (transmission EM or Scanning EM), or atomic force microscope.
The following techniques are used for routine histology or for the purpose of research:
IMPORTANT NOTICE:
 histochemistry and cytochemistry,
 immunocytochemistry and hybridization techniques,
 autoradiography,
 organ and tissue culture,
 cell and organelle separation by differential centrifugation, and
 specialized microscopic techniques and microscopes
Note: To learn more about different microscopy methods indicated above, read chapter 1 of the
suggested textbooks listed in the reference section of this manual.
Light Microscopy
What is a microscope? Microscope is an instrument that magnifies an image and allows
visualization of greater detail that is possible with the unaided eye. The human eye can differentiate
two objects as close as 0.2 mm. This is called the resolving power of human eye. This also means
that naked eye cannot differentiate two objects as separate objects when closer that 2 mm. That is
why we need a microscope. The resolving power of a bright-field microscope is 0.2 µM. However,
the resolving power is 2.5 nm for scanning electron microscope, 1.0 nm for transmission electron
microscope and 50.0 pm for atomic force microscope. Thus, resolving power is the ability of a
microscope lens or optical system to produce separate images of closely positioned objects.
Resolution: This is a term that is used to describe the ability of a microscope to distinguish detail.
In other words, this is the minimum distance at which two distinct points of a specimen can still
be seen - either by the observer or the microscope camera - as separate entities.
The Bright-field microscope is used by most students, teachers and researchers. Resolving power
of a bright-field microscope allows clear images magnified 1000-1500 times. Magnification is
defined as apparent “visual” increase in the size of an object. There is no limit to magnification.
Resolution limit is reached when additional magnification does not separate further detail. The
result is simply an enlarged blurry image.
General anatomy of a bright-field microscope is shown in the picture below.
IMPORTANT NOTICE:
From: Anthony L. Mescher - Junqueira’s Basic Histology Text and Atlas (2018, McGraw-Hill Education)
Also watch: Parts & Components of a Light Microscope (Run time 2 min 3 sec)
Basic knowledge of the components of a microscope helps users to understand how images are
generated, magnified and visualized. This helps to understand how the concept of interaction of
light with tissue components is used in order to generate a nice image.
Virtual Microscopy (VM)
Watch this video: Virtual Microscopy (Run time 2 min 51 sec)
Virtual microscopy is an innovative way to improve histology learning compared to classical

microscope-based training. Research shows that virtual microscopy enhances student educational
experiences.
The virtual microscopy method is typically used for study of bright-field microscopic images. VM
makes it possible to study high-resolution digital images prepared from stained tissue preparations
using a computer, tablet, cell phone or any other digital devices. The images produced by this
method are what you will be using during this course.
IMPORTANT NOTICE:
Pawlina and Ross, Histology A Text and Atlas With Correlated Cell and Molecular Biology-8E (2020 Wolters
Kluwer)
Preparation of Tissues for Study
Before reviewing this section of the manual, take some time to watch this video from the
University of Bristol which explains how tissues are prepared for light microscopy.
Histology at Bristol (Run time 11 min 54 sec)
As you learned from the video above, preparation of a microscopic specimen on a glass slides
requires multiple steps shown in the picture below. Those steps include: tissue biopsy or dissection,
fixation, dehydration, clearing, infiltration, embedding, sectioning using a microtome, and
eventually staining before visualization using a microscope.
IMPORTANT NOTICE:
Obtaining samples. Single cells including cells obtained from oral mucosa, vagina and cervix,
blood or tissue fluids need to be processed before microscopic inspection. Tissues can be dissected
from dead animals, cadavers, or obtained surgically and further processed for microscopy.
Fixation: To preserve the tissue structure as it had in the body. In order to fix a tissue (or cells) a
fixative is needed. Fixative is a chemical or a mixture of chemicals that stabilizes or cross-links
compounds within a tissue (or cell). Specimens should be immersed in fixative immediately after
they are removed from the body.
Fixation is used to:
 terminate cell metabolism,
 prevent enzymatic degradation of cells and tissues by autolysis (self-digestion),
 kill pathogenic microorganisms such as bacteria, fungi, and viruses, and
 harden the tissue as a result of either cross-linking or denaturing protein molecules.
One of the most commonly used fixative is Formalin. Formalin is a 37% aqueous solution of
formaldehyde. Different dilutions of formalin alone or in combination with other chemicals are
prepared using variety of buffers.
Embedding and Sectioning: After fixation, it needs to be embedded to permit sectioning.

Embedding a specimen requires an embedding medium. Following embedding, thin sections of a
tissue, typically in the range of 5-15 micrometer (µm; 1λm=1/1000 of a millimetre) can be
prepared. In brief, embedding following fixation requires the following steps: washing,
dehydration, clearing, and infiltration (with paraffin). Following cooling and hardening, the
specimen block will be trimmed and sized for sectioning. A slicing machine called microtome is
used for sectioning. The resulting sections are then mounted on glass slides by a mounting
medium or adhesive.
IMPORTANT NOTICE:
Staining. Since most cells and extracellular materials are completely color-less, they need to be
stained (dyed) in order to study them using microscopy. One of the most common staining methods
is called hematoxylin and eosin (H&E) staining. Hematoxylin stains DNA in the cell nucleus,
portions of the cytoplasm which is RNA-rich, and cartilage matrix, producing a dark blue or
purple color. The counterstain, eosin stains other cytoplasmic structures and collagen pink.
Examples of other staining procedure include periodic acid-Schiff (PAS) method which is good
for staining carbohydrate-rich tissues and cells. PAS reaction stains carbohydrates purple or
magenta. Another technique, i.e. Sudan black, satins lipids in tissues.
Considerations and Challenges in the Interpretation of Structures in Tissue

Sections
One of the most challenging aspects for students using the microscope to study histology is the
ability to mentally reconstruct the “missing” third dimension. One difficulty in the study of
histologic sections is the impossibility of differentially staining all tissue components on one slide.
Another difficulty is to understand that when a structure’s three-dimensional volume is cut into
very thin sections, the sections appear microscopically to have only two dimensions: length and
width. When examining a section under the microscope, the viewer must always keep in mind that
components are missing in front of and behind what is being seen because many tissue structures
are thicker than the section. Round structures seen microscopically may actually be portions of
spheres or tubes. Because structures in a tissue have different orientations, their two-dimensional
(2D) appearance will also vary depending on the plane of section. A single convoluted tube will
appear in a tissue section as many separate rounded or oval structures (see below).
IMPORTANT NOTICE:
Artifacts: Certain steps during tissue preparation may distort the tissues slightly, producing minor
structural abnormalities called artifacts not present in the living tissue. Artifacts may include
minor shrinkage or folding and tear of tissue regions (see picture below) produced by different
technical reasons including the type of fixative or duration of fixation,, by the heat needed for
paraffin embedding, precipitation of dyes on sections. Shrinkage can create artificial spaces
between cells and other tissue components. Such spaces can also result from the loss of lipids or
low-molecular-weight substances not preserved by the fixative or removed by the dehydrating and
clearing fluids. Slight cracks in sections may also appear as large spaces in the tissue. Other
artifacts may include small wrinkles in the section (which the novice may confuse with linear
structures in tissue) and precipitates from the stain (which may be confused with cellular structures
such as cytoplasmic granules). You must be aware of the existence of artifacts before examining
histological samples.
IMPORTANT NOTICE:
This figure shows two common artifacts, tissue folding (A), and tear (B).
Source: Journal of Clinical and Diagnostic Research
SECTION 1.2. THE CELL

Basic understanding of cellular structure and ultrastructure is expected from students enrolled in
this course. Please refer to the lecture notes and your textbooks to review cell structure and function
in details.
Using a simple light microscope for examination of mammalian cells a nucleus and a cytoplasm
which is surrounded by some sort of a border (not exactly a real cell membrane) is visible. Current
microscopic techniques along with the use of various histochemical, immunocytochemical, and
staining techniques made it possible to visualize different cytoplasmic or subcellular components
called organelles. As mentioned before, the resolving power of light microscope is limited.
However, with the advent of transmission electron microscopy superior resolution and higher
magnification of cells and tissue components have been achieved.
Cheek cells, A. unstained, B. stained
Cells from oral region, blood and female reproductive system (vagina and cervix) can be obtained
and stained using several staining methods to examine their morphology in health and disease.
IMPORTANT NOTICE:
Human blood smear showing individual cells
Cervical epithelial cells
Study of cell division (Mitosis): This can be done using regular light microscopy or other
microscopy techniques such as fluorescence- or confocal microscopy.
IMPORTANT NOTICE:
Mitotic division of animal cells using bright-field microscopy
Mitotic division of animal cells using confocal microscopy: Courtesy of McGraw Hill Companies, Inc. 2006
LAB
IMPORTANT NOTICE:
In this section, the links provided below will help you to access and study microscopic slides.
Use the following links to study images of cells using different microscopy techniques:
University of Wisconsin Histology Atlas - Cell Biology (in this website you can see images from cells
using different microscopy techniques. You may test yourself using labelled or unlabelled options)
EXAMPLE: Click on the link above. Choose HA4. You will be able to examine the original, unlabeled and
labeled versions of a microscopic picture. In this case, you can identify different components of a nerve
cell body and other adjacent structures.
For each slide (from HA1 to HA34) navigate from the ‘Unlabeled’ tab, to the ‘Original’ tab, where you will find an
image description along with arrows pointing to key structures on which you can quiz yourself. For solutions to the
arrows, complete each slide by navigating to the ‘Labelled’ tab.
Histology at Yale Cell Lab (in this website you can see images from cells using different microscopy
techniques, you can test yourself by quizzes available. Click on Slides and examine different
pictures from cells and their contents. Click on virtual microscopy and examine cells and tissues
with different magnifications)
SECTION 1.3. THE EPITHELIAL TISSUE

What is a tissue? A tissue is a collection of specialized cells and their surrounding extracellular
matrix (ECM) which collectively perform a specific function.
What is epithelial tissue? Epithelial tissue is a kind of basic tissue that is composed of closely
aggregated cells with similar or different morphologies. Epithelial cells adhering strongly to each
other through specialized intercellular junctions and contain a thing layer or very small amount of
ECM forming cellular sheets. Those cellular sheets cover the body surface and line the cavities of
IMPORTANT NOTICE:
the organs. The basal surface of all epithelia rests on a very thin layer of ECM made of
macromolecules called basement membrane. Using electron microscopy two subregions of the
basement membrane, i.e. basal lamina and reticular lamina can be differentiated
Types of epithelia (Classification)
Please read lecture notes and relevant chapters in the suggested textbook for details.
Epithelia can be classified into two main groups:

 A. Covering epithelia (single or multiple layers that cover the surfaces or line cavities)
 B. Secretory (glandular) epithelia (produce or secret various macromolecules)
A. Covering epithelia can be further classified into the following:
 Simple epithelia (contain one cell layer) which based on the shape of the cells can be
further classified as:
o Simple squamous
o Simple cuboidal, and
o Simple columnar epithelia.
 Stratified epithelia (contain more than one layer) which usually based on the shape of the
cells in the topmost layer can be further classified as:
o Stratified squamous (further subdivided as keratinized or nonkeratinized types)

o Stratified cuboidal, and
o Stratified columnar epithelia.
There are two other types of epithelia as well:
 Transitional epithelium (urothelium) which lines most of the urinary tract, and
 Pseudostratified columnar epithelium (e.g. the epithelium lining the upper respiratory
tract).
B. Secretory or glandular epithelia. This type of epithelium is comprised of specialized organs

called glands. However, sometimes, scattered, unicellular glands are present such as goblet cells
which are abundant in the lining of the intestines and in part of the lining of the respiratory tract.
Glands can be exocrine which are connected with the surface epithelium through tubular ducts.
Those ducts deliver the secreted material. Other types of glands are endocrine. Endocrine glands
lack tubular ducts and release their secretions (such as hormones) into blood which will be
transported to target cells throughout the body. Secretions of the endocrine glands may release into
the ECM and affects neighbor cells as local hormones.
Based on structure several classes of exocrine glands exist which are listed below (see lectures and
textbooks for further details).
 Simple glands
IMPORTANT NOTICE:
o Simple tubular
o Branched tubular
o Coiled tubular
o Acinar (or alveolar)
o Branched acinar
 Compound glands
o Tubular
o Acinar (alveolar)
o Tubuloacinar
LAB
In this section we will examine histologic features of major types of covering epithelia.
This video from University of Wisconsin reviews morphology of the epithelial tissue.
Click on the link below to examine some examples of epithelia:
Histology Guide Virtual Histology Lab
Click on SLIDE BOX.

IMPORTANT NOTICE:
Choose Chapter 2 Epithelium, then scroll down to simple squamous epithelium section and click,
for example on MH 016 Simple epithelia.
(Image adapted from The Histology Guide - Virtual Histology Library)
Here are examples of sections from different tissues which contain a type of simple epithelium in
their structures. Pay attention to the right panel. Click on simple squamous epithelium. You will
see a picture as below with arrows pointing at the epithelium. You can zoom in or out using your
computer mouse. Repeat the same procedure for the other sections in this chapter.
IMPORTANT NOTICE:
The following photographs are selected from the above website that can be used as an atlas.
Simple squamous epithelium (arrows) lining interior of the capsule of a renal corpuscle
IMPORTANT NOTICE:
Simple squamous epithelium (or endothelium, arrows) lining the lumen of an artery
Simple cuboidal epithelium covering the outer surface of the ovary

IMPORTANT NOTICE:
Simple columnar epithelium lining the lumen of the small intestine (thin arrows). Thick arrows
point to goblet cells which is a unicellular gland secreting mucous.

IMPORTANT NOTICE:
Pseudostratified columnar epithelium lining the lumen of the trachea. Thick arrows point to cilia
on the apical surface of this epithelium.

Stratified squamous epithelium
Stratified cuboidal epithelium of a large duct in esophagus tissue
IMPORTANT NOTICE:
Transitional epithelium of the urinary tract. The transitional epithelium has several layers of cells
and large, dome-shaped cells on its surface.
Click on the link below to examine some examples of epithelia:
University of Wisconsin Histology Atlas
 Click on Epithelium in Unit 1.
 Then Select:
 HA2 (for simple cuboidal or columnar epithelia)
 HA14 (for stratified squamous nonkeratinized epithelium)
 HA15 (for stratified squamous keratinized epithelium)
IMPORTANT NOTICE:
IMPORTANT NOTICE:

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Histology: A Practical Manual

General anatomy of a bright-field microscope is shown in the picture below.

Virtual Microscopy (VM)

Watch this video: Virtual Microscopy (Run time 2 min 51 sec)

Virtual microscopy is an innovative way to improve histology learning compared to classical

Preparation of Tissues for Study

Histology at Bristol (Run time 11 min 54 sec)

Embedding and Sectioning: After fixation, it needs to be embedded to permit sectioning.

Considerations and Challenges in the Interpretation of Structures in Tissue

Source: Journal of Clinical and Diagnostic Research

SECTION 1.2. THE CELL

Cheek cells, A. unstained, B. stained

Cervical epithelial cells

SECTION 1.3. THE EPITHELIAL TISSUE

Epithelia can be classified into two main groups:

A. Covering epithelia can be further classified into the following:

o Stratified squamous (further subdivided as keratinized or nonkeratinized types)

There are two other types of epithelia as well:

B. Secretory or glandular epithelia. This type of epithelium is comprised of specialized organs

Click on the link below to examine some examples of epithelia:

Histology Guide Virtual Histology Lab

Click on SLIDE BOX.

(Image adapted from The Histology Guide - Virtual Histology Library)

(Image adapted from The Histology Guide - Virtual Histology Library)

(Image adapted from The Histology Guide - Virtual Histology Library)

Simple cuboidal epithelium covering the outer surface of the ovary

(Image adapted from The Histology Guide - Virtual Histology Library)

(Image adapted from The Histology Guide - Virtual Histology Library)

(Image adapted from The Histology Guide - Virtual Histology Library)

Stratified cuboidal epithelium of a large duct in esophagus tissue

Click on the link below to examine some examples of epithelia:

University of Wisconsin Histology Atlas

 Click on Epithelium in Unit 1.

 HA2 (for simple cuboidal or columnar epithelia)

 HA14 (for stratified squamous nonkeratinized epithelium)

 HA15 (for stratified squamous keratinized epithelium)

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