Practical Guide Cell Biology

MARIANA CORNELIA TILINCA
CELL BIOLOGY
PRACTICAL WORKS
GUIDE
2011
MARIANA CORNELIA TILINCA

Associate Professor
Cellular and Molecular Biology Department
University of Medicine and Pharmacy
Targu Mures
Scientific reviewers:
Prof. Dr. ANCA SIN, University of Medicine and Pharmacy, Targu Mures
Lecturer Dr. SIMONA MARCU, University of Medicine and Pharmacy, Targu Mures
CIP nr.00703 / 10.01.2011

CIP registration number of the National
Library of Romania
TILINCA, MARIANA CORNELIA
Cell Biology Practical Works Guide/ Mariana
Cornelia Tilinca Trgu MureUniversity Press, 2011
Bibliography
Index
ISBN 978-973-169-143-5 576
University Press Publisher - Trgu Mure

Editor in Chief: Prof. Univ. Dr. Alexandru chiopu
Correspondence/orders: U.M.F. Trgu Mure, Romania
Address: Trgu Mure, Str. Gh. Marinescu No. 38, postal cod 540130
Tel. 0744527700, 0265215551 126 Fax: 026521040
PREFACE
This paper presents an overview of the shape and chemical composition of cells, its
components, cell membrane and its changes, cellular organelles, cell division and secretion.
For each theme were given some concrete examples of digital images obtained from
optical microscopy, transmission electronic microscopy respectively.
The material of this book was designed as a guide for practical works that should help
students in first year degree from the medicine faculty in studying and learning the concepts
of cell biology, to acquire knowledge and skills regarding the morphofunctional aspects of the
cell and microscopic techniques as well.
The micrographs are made by light microscopy with the support of the Pathology
Department, Emergency County Hospital, Targu Mures, on the samples from Cell Biology
Department, Faculty of Medicine, University of Medicine and Pharmacy, Targu Mures.
Special thanks for my colleagues who have supported me in publishing this guide, dr.
Simona Marcu, dr. Simona Gurzu, dr. Adrian Florea, prof. dr. Jung Janos and for my
colleagues from Cell Biology Department.
I wish to express my thanks to my teacher prof. dr. Silvia Andreicut who had a major
contribution in my career teaching me cell biology.
My thanks to my family, and in particular for my children who helped me in the
computerized publishing of the whole material.
Mariana Cornelia Tilinca
CONTENTS
PREFACE 3
1. MICROSCOPY TECHNIQUES 6
1.1. LIGHT MICROSCOPY 6
1.2. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY 15
1.3. ELECTRON MICROSCOPY 22
2. SHAPES OF THE CELLS 28
3. CHEMICAL COMPOSITION OF THE CELL 40
4. EXTENSIONS OF THE CELL SURFACE 55
5. EXTRACELLULAR MATRIX 66
6. SHAPES OF THE NUCLEI 77
7. CHROMOSOMES 97
8. CELL DIVISION 102
9. ENDOPLASMIC RETICULUM. RIBOSOMES 118
10. GOLGI APPARATUS AND CELL SECRETION 124
11. LYSOSOMES 140
12. MITOCHONDRIA 144
BIBLIOGRAPHY 151
Cell Biology Practical Works Guide
1. MICROSCOPY TECHNIQUES
1.1. LIGHT MICROSCOPY
Cell biology and light microscopy represent a suitable combination for scientific
studies in experimental cell research. In general, with light microscopes equipped for bright
field only the stained biological specimens can be visualized.
The microscope represents a device used to view objects which are too small to see or
explore with naked eye.

THE OPTICAL MICROSCOPE
The optical microscope, often referred to as the "light microscope", is a type of

microscope which uses visible light and a system of lenses to magnify images of small
samples. Optical microscopes are the oldest and simplest of the microscopes. However, new
designs of digital microscopes are now available which use a camera to examine a sample and
the image is shown directly on a computer screen without the need for expensive optical
components such as eye-pieces. A compound microscope consists of many important
functional components.
Basic optical microscope elements are:
1. ocular lens, or eyepiece
2. objective rotating turret
3. objective lenses
4. coarse adjustment knob
5. fine adjustment knob
6. object holder or stage
7. stage clips
8. arm
9. base
10. mirror or light (illuminator)
11. condenser and iris diaphragm
12. condenser focus knob
Figure 1. The optical microscope
All optical microscopes share the same basic components:

The eyepiece is a cylinder containing two or more lenses to bring the image into focus
for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are
interchangeable and many different eyepieces can be inserted with different degrees of
magnification. Most ocular lenses have a 10x magnification level. Binocular microscopes
allow interpupillary distance adjustment for different distances between the eyes of different
individuals.
Figure 2. The eyepiece
The objective lens is a cylinder containing one or more lenses, typically made of glass
to collect light from the sample. At the lower end of the microscope tube one or more
objective lenses are screwed into a circular nose piece which may be rotated to select the
required objective lens. Typical magnification values of objective lenses are 6x, 10x, 20x, 40x
and 90x. Some high performance objective lenses may require matched eyepieces to deliver
the best optical performance. Most objectives are designed to image specimens with air as the
medium between the objective and the cover glass.
On a typical compound optical microscope there are three objective lenses: a scanning
lens (6x), low power lens (10x) and high power lens (ranging from 20 to 100x). Some
microscopes have a fourth objective lens, called an oil immersion lens. To use this lens, a
drop of immersion oil is placed on top of the cover slip, and the lens is very carefully lowered
until the front objective element is immersed in the oil film. Such immersion lenses are
designed so that the refractive index of the oil and of the cover slip are closely matched and so
the light will be transmitted from the specimen to the outer face of the objective lens with
minimal refraction. An oil immersion lens usually has a magnification of 50 to 100x.
The actual power or magnification of an optical microscope is the product of the
power of the ocular (eyepiece), usually about 10x and the objective lens being used.
Compound optical microscopes can produce a magnified image of a specimen up to 1000x
and at high magnifications they are used to study thin specimens as they have a very limited
depth of field.
Figure 3. The turret with the objective lenses (10x, 20x, 40x)
Figure 4. The immersion lens
The tube connects the eyepiece to the objective lenses.

The arm supports the tube and connects it to the base.
Base: The bottom of the microscope used for support.
Revolving nosepiece or turret: This is the part that holds two or more objective
lenses and can be rotated to easily change power.
The stage is a platform below the objective which supports the specimen being
viewed. In the centre of the stage is a hole through which light passes to illuminate the
specimen. The stage usually has arms to hold slides (rectangular glass plates with typical
dimensions of 25 mm by 75 mm, on which the specimen is mounted). A mechanical stage is
used when working at higher magnifications where delicate movements of the specimen slide
are required.
Figure 5. The stage with the stage clips
Stage clips are used when there is no mechanical stage. The viewer is required to
move the slide manually to view different sections of the specimen.
Condenser is used to collect and focus the light from the illuminator onto the
specimen. It is located under the stage often in conjunction with an iris diaphragm. The
condenser is a very important part of the light transmission system of a microscope.
Iris diaphragm controls the amount of light transmitted through the object.
10
It is located above the condenser and below the stage. Most high quality microscopes
include a condenser with an iris diaphragm. Combined, they control both the focus and
quantity of light applied to the specimen.
Condenser focus knob moves the condenser up or down to control the lighting focus
on the specimen. Coarse and fine focus knobs are used to focus the microscope.
Coarse adjustment knob is a large, round knob on the side of the microscope used
for focusing the specimen. It may move either the stage or the upper part of the microscope.
(Objective 10x)
Fine adjustment knob is a small, round knob on the side of the microscope used to
fine-tune the focus of your specimen after using the coarse adjustment knob. (Objectives 20x,
40x). Both of these move the stage up or down, depending on the direction of turning, and
serve to change the distance between the specimen and the objective lens.
The adjustment of objective lens. In some microscopes coarse and fine focusing
adjustment knobs are provided in order to lower or raise the body tube with lenses for
rendering image clear. This is done by rotation of the knobs. The coarse adjustment is meant
to bring to object into vision whereas the fine adjustment is used for focusing finer details.
These knobs serve to improve the quality of the image.
Figure 6. The condenser with the iris diaphragm, the condenser knob, fine and coarse focus knob
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Figure 7. The light source

The illumination source is localized below the stage. The light is provided and
controlled in a variety of ways. At its simplest, daylight is directed via a mirror. Most
microscopes, however, have their own controllable light source that is focused through an
optical device called a condenser, with diaphragms and filters available to manage the quality
and intensity of the light. The whole of the optical assembly is attached to a rigid arm which
in turn is attached to a robust U shaped foot to provide the necessary rigidity. The arm is
usually able to pivot on its joint with the foot to allow the viewing angle to be adjusted.
Mounted on the arm are controls for focusing, typically a large knurled wheel to adjust coarse
focus, together with a smaller knurled wheel to control fine focus.
How to Focus Your Microscope
The proper way to focus a microscope is to start with the lowest power objective lens
first and while looking from the side, crank the lens down as close to the specimen as possible
without touching it. Now, look through the eyepiece lens and focus upward only until the
image is sharp. If you can't get it in focus, repeat the process again.
Once the image is sharp with the low power lens, you should be able to simply click in
the next power lens and do minor adjustments with the focus knob. If your microscope has a
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fine focus adjustment, turning it a bit should be all that's necessary. Continue with subsequent
objective lenses and fine focus each time.
The basic optical principles of a microscope are quite simple.
The specimen is usually supported by a slide and essentially at the higher powers of
the biological microscope covered by a cover glass. Thus introduced into the image-forming
light path, slide and cover glass become part of the optical system. The light passes through
the specimen.
The thickness of the slide is important to the correction of the substage condenser, and
the thickness of the cover glass is critical to the performance of the objective, especially those
of higher power (20x and greater).
The resolution represents the ability of a lens to distinguish the fine details of the
specimens being viewed. The best resolution of light microscopes is 0.2 m. In other words
the shortest distance between two neighbouring points that can be seen separately with optical
microscope is 0.2 m. The resolution depends on the optical system, wavelength of the light
source and other factors such as specimen thickness and staining intensity.
Figure 8. The position of the slide for examination

The eyepiece lens magnifies the image produced by the objective lens, but it cannot
increase the resolution.
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The essence of a microscope is its ability to magnify a specimen. Total magnification

of a microscope is determined by multiplying the magnification capability of the eyepiece
lens by that of the objective lens. Magnification is generally limited by the properties of the
glass used to make microscope lenses and the physical properties of their light sources. The
generally accepted maximum magnifications in biological uses are between 1000X and
1250X. The microscope requires a fine manipulation being forbidden to any forcing.
Always the slide will be placed with the cover slip up towards the objective lens.
Main rules regarding the examination with an optical microscope
1. Plug in the microscope

2. Check if the 10x objective lens is in the centre of the microscope; this will avoid lens
scratching
3. Lift up the arm using the coarse adjustment knob
4. Open the mobile clip stage and put the slide on
5. Use the stage knob to position the slide over the light
6. Make sure that the iris diaphragm is always open
7. Lower the arm as much as possible
8. Adjust your interpupillary distance until you will see one circle of light instead of two
9. While looking into the microscope, slowly lift up the arm using the coarse adjustment knob
until the image is focused
10. It should be about 1 cm between slide and the 10x objective lens
11. Use the coarse adjustment knob only with 10x objective lens
12. Adjust the light level with the condenser knob if necessary
13. If the image is sharp rotate the 20x objective
14. Focus using fine adjustment knob only
15. Adjust the amount of light
16. Rotate the 40x objective
17. Use the fine knob to the fine focus
18. In the end of examination rotate the 10x objective into position
19. Remove the slide
20. Turn off the power
The final image of the optical microscope is a virtual magnified and reversed image.
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1.2. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY
One of the oldest methods of preparing tissue samples is by making sections on a

microtome. A microtome is an instrument that cuts very thin slices of tissue that can be
examined at the microscope. For this purpose the tissue is first fixed by chemical substances,
embedded in paraffin wax and sliced with the microtome. The paraffin is removed and the
tissue is stained to reveal the cell structures, nuclei, organelles, cell membrane, chromosomes,
mitochondria, endoplasmic reticulum, Golgi apparatus, cell secretion, cell division, lysosomes
and the chemical components of the cell.
Materials: slides, cover slides, piece of tissue, fixative solutions, alcohols, paraffin,
xylene, dyes, microtome.
The slide is a rectangular glass plate on which the specimen is mounted.
The cover slide protects the specimen.
A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm
thick, used to hold objects for examination under a microscope. The object respectively the
specimen is placed on the slide and then is inserted in the microscope for examination. This
arrangement allows several slide-mounted objects to be quickly inserted and removed from
the microscope, labelled, transported, and stored in appropriate slide cases or folders.
Microscope slides are often used together with a cover slip or cover glass. This is a smaller
and thinner sheet of glass that is placed over the specimen. Slides are held on the stage by
slide clips or stage clips.
Figure 9. The slide and the cover slide

15
The Steps of the Specimen Preparation for Light Microscopy
1. Specimen collection
2. Fixation
3. Dehydration
4. Embedding
5. Sectioning
6. Staining
7. Slide mounting
8. Examination at the microscope
1. Specimen collection
The samples are collected from living organisms by biopsy or from dead organisms
during necropsy. These samples should not be bigger than 1cm3.
2. Fixation
The first step in specimen preparation for light microscopy is fixation. The fixation
preserves cell structure. Artefacts are artificial structures produced during fixation that may
appear to be true histological features. Small tissue fragments are immersed in a solution
containing a fixative. In the fields of histology, pathology and cell biology fixation is a
chemical process by which biological tissues are preserved from decay, either through
autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may
also increase the mechanical strength or stability of the treated tissues. Even the most careful
fixation does alter the sample and introduce artefacts (false structures) that can interfere with
interpretation of cellular structure.
Fixation is usually the first stage in a multistep process to prepare a sample of
biological material for microscopy or other analysis. Therefore, the choice of fixative and
fixation protocol may depend on the additional processing steps and final analyses that are
planned. Formalin 10% is usually used for this step.
The sample of tissue is immersed in fixative of volume at least 15 times greater than
the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to
fix. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.
16
Figure 10. Fixation in formalin
3. Dehydration
After fixation specimens are washed and dehydrated by immersion in a series of
solutions containing increasing concentrations of ethanol 70%, 90%, 100% to remove water.
Dehydration is necessary because most embedding media like paraffin are immiscible in
water.
4. Embedding
After dehydration the specimen is immersed in xylene. Then the specimen is treated
with a liquid embedding medium that infiltrates and hardens the specimen so that it can be
sliced into thin sections suitable for staining and microscopic examination. Light microscopy
often uses paraffin, which hardens the specimen and it cools as an embedding medium.
Paraffin is easy to work with, speeds specimen preparation and stains reliably. The paraffin
and the tissue sample form a paraffin block.
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Figure 11. The paraffin block with the tissue sample
5. Sectioning
Light microscopy studies 5-10 m thick paraffin-embedded sections. The block is
mounted in a specially designed slicing machine called microtome. Sections are cut with a.
rotary microtome, a machine that moves the specimen across a sharp metal blade, advancing
the specimen the desired thickness after each pass. Successive sections come off the
microtome in a ribbon. Sections are separated from this ribbon and mounted on glass slides,
with albumin used as an adhesive.
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Figure 12. A microtome for cutting wax embedded biological specimens
Figure 13. Collecting paraffin embedded slices in water bath
19
Figure 14. The section mounted on glass slide
6. Staining
Light microscopy sections usually are stained with dyes.
Figure 15. The staining battery

20
The embedding process must be reversible in order to get the paraffin wax out of the
tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining
can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes)
to alcohols to water (ethanol 100%, 90%, 70%). There are no stains that can be done on
tissues containing paraffin. The staining process makes use of a variety of dyes that have been
chosen for their ability to stain various cellular components of tissue. The routine stain is that
of hematoxylin and eosin (H&E).
Other stains are referred to as "special stains" because they are employed in specific
situations according to the diagnostic need. After staining the specimens are dehydrated using
solutions containing increasing concentrations of ethanol 70%, 90%, 100%.
Specimens are clarified with xylene.
7. Slide mounting. The mounting medium is the solution in which the specimen is
embedded, generally under a cover glass. It is used Canada balsam or synthetic resin
substitutes in xylene. In a dry mount, the simplest kind of mounting, the specimen is placed
on the slide. The specimen is protected by a cover slip. The slides should be marked with the
label code and kept flat and undisturbed in a dust free area.
8. Examination at the microscope. The microscope allows a magnified view of the
sample. Examination of slides at the optical microscope permits us to recognise the shape of
the cells, their components (organelles, nucleus, cytoplasm), the adaptations of the cellular
surface, the extracellular matrix and the chemical composition of the cell.
Figure 16. Microscopic specimen
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1.3. ELECTRON MICROSCOPY
With the light microscope many organelles are poorly revealed, whereas with the
electron microscope the detailed structure or the fine structure of the cell can be seen.
Presently electron microscopy provides a detailed picture of subcellular anatomy, a real map
of the cell components.
The electron microscopes use a beam of electrons instead of a beam of light. They rely
on the interaction of the electrons with a specimen to produce an image. They illuminate
specimens with a short wavelength electrons stream and form images with magnetic lenses.
For this reason electron microscopes have a high resolving power. This is high enough to
reveal fine details of all cell organelles and some macromolecules.
The main components of the electron microscope are in general similar to those of the
light microscope.
The source, the cathode is represented by a tungsten filament which emits electrons
that are attracted by an anode. The electrical difference between the cathode and anode
accelerates the electrons forming the beam. The beam of electrons passes through
electromagnetic lenses, the condenser lens and then through the specimen and objective
lenses. The magnetic lenses form an image that is displayed on a video screen and recorded
permanently.
There are two types of electron microscopes: the transmission electron microscope or
TEM and scanning electron microscope or SEM.
In transmission electron microscopy, are examined thinly sliced, resin embedded
sections stained with heavy metals ions. The electrons are transmitted through the specimen.
The electron micrograph reveals the ultrastructure of the organelles and the fine structure of
the cell elements.
In scanning electron microscopy are examined the surfaces of the specimen. This is
coated with a thin layer of gold and palladium. The surface is scanned by the electrons beam.
The reflected electrons are collected by a detector device that form a three dimensional image
of the specimen.
In the following there are presented some organelles viewed in transmission electron
microscopy: the Golgi apparatus, rough endoplasmic reticulum, mitochondria, lysosomes and
peroxysomes from the personal collection of dr. Adrian Florea, with his permission.
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Image 1. Golgi apparatus, secreting cell, adrenal medulla, TEM
Image 2. Rough endoplasmic reticulum, hepatocyte, liver, TEM

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Image 3. Mitochondrion, neuron, TEM
Image 4. Mitochondria, secreting cell, adrenal cortex, TEM
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Image 5. Mitochondria, hepatocyte, liver, TEM
Image 6. Mitochondria, striated muscle fiber, TEM

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Image 7. Primary lysosomes, nephrocyte, kidney, TEM
Image 8. Secondary lysosome, secreting cell, adrenal cortex, TEM

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Image 9. Peroxysome, hepatocyte, liver, TEM
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2. SHAPES OF THE CELLS
The cells vary in shape and size. They may be oval, spherical, rectangular, cubical,
polygonal, spindle shaped, star shaped, rod-shaped or totally irregular.
The diversity in cells is in accordance with their role or function they have to perform
as part of the tissue or the organ system. In general, there is no typical shape for cells.
H&E stain, HE stain or hematoxylin and eosin stain, is a routine staining method in
cytology and histology. It is the most widely used stain in medical diagnosis.
The staining method involves application of the basic dye hematoxylin, which stains
basophilic structures with blue-purple hue, and alcohol-based acidic eosin Y, which stains
eosinophilic structures bright pink.
The basophilic structures are usually the ones containing nucleic acids, such as the
ribosome and the chromatin-rich cell nucleus, and the cytoplasmic regions rich in RNA.
The eosinophilic structures are generally composed of intracellular or extracellular
proteins. Most of the cytoplasm is eosinophilic. Red blood cells are stained intensely red. The
structures do not have to be acidic or basic to be called basophilic and eosinophilic. The
terminology is based on the affinity to the dyes. Hydrophobic structures also tend to remain
clear; these are usually rich in fats, e.g. adipocytes, myelin around neuron axons, and Golgi
apparatus membranes.
PRACTICAL DEMONSTRATIONS
Slide 1. Syncytium. Elongated cells. Striated muscle fibers. Skeletal muscle. HE Stain
Slide 2. Polygonal cells. Hepatocytes. Liver. HE Stain
Slide 3. Stellate cells. Motor neurons. Spinal cord. HE Stain
Slide 4. Stellate cells. Neurons. Spinal cord. Silver impregnation
Slide 5. Fusiform cells. Fibroblasts. Mandible. HE Stain
Slide 6. Scuamous cells. Mesentery. Silver impregnation
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Slide 1. Syncytium. Elongated cells. Striated muscle fibers. Skeletal muscle. HE

Syncytium represents a mass of cytoplasm containing many nuclei enclosed by a
single plasma membrane. It is typically the result either of cell fusion or of a series of
incomplete division cycles in which the nuclei divide but the cell does not.
This specimen represents a section from striated skeletal muscle stained with
hematoxylin and eosin. Skeletal striated muscle fibers are multinucleated cells. Some of these
fibers are cut longitudinally while others transversally. The cross sectioned fibers have
polygonal shape and those sectioned longitudinally have elongated shape. It can be seen the
parallel alignment of the fibers. Nuclei are located at the peripheral parts of the fibers.
Muscle cells are typically elongated and oriented with their long axes in the same
direction. Skeletal muscle cells are large and easy to see. Their cytoplasm contains contractile
proteins, actin and myosin, which are stained dark pink. Their many nuclei are normally
located under their surface membrane and are stained blue with hematoxylin. The striated
muscle cells contain longitudinal units called myofibrils. Striated fibers are contractile cells.
A fiber consists of a single skeletal muscle cell.
Muscle is classified in two principal types: striated muscle and smooth muscle.
Striated muscle is sub-classified in: skeletal muscle, visceral striated muscle, cardiac muscle.
Skeletal and visceral striated muscle cells are called fibers.
Fibers are long multinucleated protoplasmic units arranged in parallel to their
neighbours. The multiple nuclei are at the periphery of the cell, under plasma membrane
(sarcoplasma). Contractile elements called myofibrils fill the rest of the cell. The highly
ordered arrangement of the myofibrils accounts for the cross-striations characteristic of a
longitudinal section of skeletal muscle fibers. They are stained in dark pink with eosin. These
characteristic cross-striations are seen along the fibers using high magnification objectives
starting with 20x.
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Slide 1 a, 10x
Slide 1 b, 20x
30
Slide 1 c, 40x
Slide 2. Polygonal cells. Hepatocytes. Liver. HE Stain
The parenchyma of the liver is contained within a thin capsule of fibro-connective
tissue. Often the capsule is not distinguishable in light microscopic preparations. The
parenchyma is primarily composed of organized plates of hepatocytes which are normally one
cell thick and are separated by sinusoidal capillaries (vascular channels).
Hepatocytes are large, polygonal cells which constitute about 80% of the cell
population of the liver. Their size is between 20 and 30 m. Their nuclei are large and
spherical and occupy the centre of the cell. Many cells in the adult liver are binuclear.
Heterochromatin is present as scattered granules in the nucleoplasm and as a distinct band
under the nuclear envelope. Two or more well-developed nucleoli are present in each nucleus.
These are stained in blue with hematoxylin. The cytoplasm of the hepatocyte is generally
acidophilic, so it is stained pink with eosin. Liver cells are capable of considerable
regeneration when liver substance is lost to hepatotoxic processes, diseases or surgery. Their
average lifespan is about 5 month.
Vacuolization of hepatocytes resulting from glycogen and/or fat storage can produce
considerable histological variability. Other cell types typically found in liver parenchyma
include hematopoietic tissue and macrophage aggregates.
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Slide 2 a, 10x
Slide 2 b, 20x
32
Slide 2 c, 40x
Slide 3. Stellate cells. Motor neurons. Spinal cord. HE Stain

Neurons are highly specialized for the processing and transmission of cellular signals.
Given the diversity of functions performed by neurons in different parts of the nervous
system, there is a wide variety in the shape, size, and electrochemical properties of neurons.
For instance, the body of a neuron can vary from 4 to 100 micrometers in diameter. The cell
body or perikaryon is the central part of the neuron. It contains a large nucleus with a
prominent nucleolus and surrounding perinuclear cytoplasm. The cytoplasm contains
abundant rough endoplasmic reticulum and free ribosomes. Ribosomal content appears as
small bodies called Nissl bodies that stain intensely with basic dyes (hematoxylin blue).
Each Nissl body corresponds to a stack of RER.
There is an area of the neuron body called axon hillock which is free of large
cytoplasmic organelles and serves as a landmark to distinguish between axons and dendrites
in light microscope specimens. The dendrites (receptor processes) of a neuron are shorter
cellular extensions with many branches that transmit stimuli from the periphery toward the
cell body. The axon (effector process) is a finer, long, cable-like projection which carries
nerve signals away from the cell body.
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Slide 3 a, 10x
Slide 3 b, 20x
34
Slide 3 c, 40x
Slide 4. Stellate cells. Neurons. Spinal cord. Silver impregnation
This preparation of the spinal cord is from the ventral horn highlighted by silver
impregnation method. Cell bodies of neurons and their processes, axons and dendrites, appear
stained in brown.
Slide 3 a, 10x
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Slide 4 b, 20x
Slide 5. Fusiform cells. Fibroblasts. Mandible. HE Stain

The shape of the fibroblasts varies from fusiform to star shape with extensions. Each
cell has a single nucleus with nucleolus located in the middle part of the cytoplasm.
Fibroblasts are involved in extracellular matrix secretion, ground substance, collagen, elastic
and reticular fibers. They intervene in wound healing process. They contain an abundant
rough endoplasmic reticulum, Golgi apparatus and many mitochondria.
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Slide 5 a, 10x
Slide 5 b, 20x
37
Slide 5 c, 40x
Slide 6. Scuamous cells. Mesentery. Silver impregnation

The mesentery is the double layer of peritoneum that surrounds parts of the small
intestine, the jejunum and the ileum. In silver impregnation the cell membranes can be seen,
respectively the limits between the neighboring cells as irregurar lines stained in black. The
nuclei are not highlighted with this method.
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Slide 6 a, 10x
Slide 6 b, 20x
39
3. CHEMICAL COMPOSITION OF THE CELL
All cells have chemical substances in their composition, from both the organic and
inorganic world. They appear in approximately the same proportions and perform the same
general tasks.
Organic substances are: glucose, lipids, proteins, nucleic acids.
Glucose is the principal circulating sugar in the blood and the major energy source of
the body. Glucose is a monosaccharide sugar which assures the energetic support of metabolic
processes. Glucose has a central role as an energy source for cells. It is deposited in the form
of polysaccharides, glycogen, which can be seen in the cytoplasm having the form of a
granule with a diameter of between 15 and 150 nm. It is stored in liver and muscles.
The lipids are generally classified in two main groups: simple lipids and complex
lipids. Simple lipids are: triglycerides, waxes and steroids. Complex lipids include
phospholipids, glycolipids and sphingolipids. Lipids are present in all living cells, but the
proportion varies from tissue to tissue. The triglycerides accumulate in certain areas, such as
adipose tissue in the human being where they represent a form of energy storage. High levels
in the flow blood lead to occurrence of the atherosclerosis a progressive process responsible
for most heart disease. It is characterized by plaque deposits that block the flow of blood.
Proteins are the essential regulators of all cellular processes. Proteins are fundamental
components of all living cells and include many substances, such as enzymes, hormones, and
antibodies that are necessary for the proper functioning of an organism. Proteins are huge,
complex molecules that are carriers through the membranes, carriers of respiratory gases,
have a role of biocatalysis, in contraction processes, in immunity.
Nucleic acids are nucleotide polymers that store and transmit genetic information.
They are macromolecules constructed as a long chain of monomers called nucleotides.
There are two types of nucleic acids found in living organisms, deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA). DNA is localised almost entirely in the nucleus. RNA
can be found mostly in the cytoplasm.
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Slide 7. Glycogen. Hepatocytes. Liver. Periodic Acid Schiff (PAS) Stain

Slide 8. Acid mucopolysaccharides. Goblet cells. Colon. Alcian Blue (AB) Stain
Slide 9. Neutral and acid mucopolysaccharides. Salivary gland. Secreting cells. PAS&AB
Slide 10. Triglycerides. Artery. Sudan III Stain
Slide 11. Triglycerides. Skin. Sudan III Stain
Slide 12. Phospholipids. Artery. Sudan IV (Black) Stain
Slide 13. Cell Proteins. Hepatocytes. Liver. Mazia method with bromophenol blue
Slide 7. Glycogen. Hepatocytes. Liver. Periodic Acid Schiff (PAS) Stain

The liver is the largest exocrine gland in the body. It secretes large amounts of bile.
The liver has several lobes. Each lobe contains many lobules which are the basic functional
units of the liver. Lobules are roughly hexagonal. They are centred on a central vein and have
a portal channel in each corner. Each portal channel contains a hepatic artery, a portal vein
and a hepatic bile duct joined into a unit by connective tissue. Liver parenchymal cells are
polygonal cells arranged in anastomosing plates and cords. The plates radiate from the central
vein and the cords alternate with sinusoids.
The Periodic Acid-Schiff (PAS) reaction is used to identify glycogen in cells, mucus
in various cells, the basement membrane that underlines epithelia and reticular fibers. It stains
carbohydrates and carbohydrate-rich macromolecules. The reaction of periodic acid
selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent
and creates a purple-magenta colour.
PAS staining is mainly used for staining structures containing a high proportion of
carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in
connective tissues, mucus, and basal laminae. It is used to distinguish different types of
glycogen storage diseases. The PAS reaction is used to identify glycogen in liver cells.
Glycogen appears into the cytoplasm as granules stained in purple magenta colour. The nuclei
remain clear.
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Slide 7 a, 10x
Slide 7 b, 20x
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Slide 7 c, 40x
Slide 8. Acid mucopolysaccharides. Goblet cells. Colon. Alcian Blue Stain

Alcian blue (AB) contains copper. The dye stains acid mucopolysaccharides and
glycosaminoglycans. Alcian blue is one of the most widely used cationic dyes and it has many
positive charges on the molecule. It is thought to work by forming reversible electrostatic
bonds between the cationic dye and the negative (anionic) sites on the polysaccharide. The
stained parts are blue to bluish-green.
The specimen represents a section from large intestine or colon. The mucosal
epithelium is composed of columnar absorptive cells with microvilli called enterocytes and
numerous secreting cells named goblet cells. Enterocytes are tall columnar cells which have
an apical brush border of microvilli that are covered by a glycocalyx. The glycocalyx is an
external coat of the cell surface. Enterocytes are not stained. Goblet cells secrete mucus
composed of glycoproteins packed into mucous droplets. The goblet cells store such large
quantities of droplets that they distend the cell apex, creating the goblet shape for which the
cell is named. The stained part of the goblet cell is blue. It emphasizes the acid
mucopoysaccharides from the mucus. The cytoplasm and the nuclei of the cells are not
visible.
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Slide 8 a, 10x
Slide 8 b, 20x
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Slide 8 c, 40x
Slide 9. Neutral and acid mucopolysaccharides. Salivary gland. Secreting cells.
PAS&AB Stain
The secretory unit of the salivary gland is the acinus. The acinus is composed of either
serous cells, mucous cells or both cell types. So, there are three types of acini: serous acini,
mucous acini and mixted acini.
The serous cells have spherical nuclei stained in blue with hematoxylin and a deeply
stained cytoplasm in red with eosin. The mucous cells have flattened nuclei pushed at the
basal pole of the cell by the mucus accumulated into the cytoplasm. Their cytoplasm is
reduced quantitatively and the mucus is transparent.
Mixed acini have the both types of the secreting cells, serous cells and mucous cells.
Serous cells form a cap known as serous demilune on the periphery of the mixed acini.
The secretory cells secrete their product that contain acid and neutral
mucopolysaccharides. Using a combined method with alcian blue and periodic acid Schiff
stains, it can be seen on the same slide both types of mucopolysaccharides. Alcian blue stains
in blue the acid mucopolysaccharides and the PAS stains in magenta color the neutral
mucopolysaccharides secreted by acinous cells.
Being a special staining technique it highlights these chemical components. Thus, the
morphological parts of the cells, the nuclei and the cytoplasm, cannot be identified.
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Slide 9 a, 10x
Slide 9 b, 20x
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Slide 9 c, 40x
Slide 10. Triglycerides. Artery. Cross section. Sudan III Stain

Aorta is a large elastic artery which is composed of three layers. Tunica intima is a
thin layer at the lumen. Tunica media contains many elastic fibers. Tunica adventitia contains
besides elastic fibers and other connective tissue components small blood vessels called vasa
vasorum.
Sudan III is a fat-soluble dye used for staining of triglycerides in frozen sections. It has
the appearance of reddish brown drops.
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Slide 10 a, 10x
Slide 10 b, 20x
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Slide 11. Triglycerides. Skin. Sudan III Stain

Triglycerides can be seen with Sudan II stain as red droplets located in the adipose or
fatty cells, in the sereting cells of the sebaceous gland or in the lipids deposits from the skin.
Using this special techique for highlighting lipids the morphological aspects of the
skin structure, the epidermis, dermis and hypodermis can be recognized, while the cellular
components are not visible.
Slide 11 a, 10x
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Slide 11 b, 20x
Slide 11 c, 40x
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Slide 12. Phospholipids. Artery. Cross section. Sudan Black Stain

Sudan Black (IV) is a fat-soluble dye used for the staining of lipids, phospholipids on
frozen paraffin sections. Phospholipids are shown like small drops in black colour. This
staining is an important biochemical technique, offering the ability to visually qualify the
presence of the fatty compound of interest without isolating it.
Slide 12 a, 10x
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Slide 12 b, 20x
Slide 12 c, 40x
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Slide 13. Cell Proteins. Hepatocytes. Liver. Mazia method with bromophenol blue
Proteins are found in all cellular elements: cell membrane, organelles from the
cytoplasm and nucleus. The plasma membrane contains protein molecules in its structure. All
the intracellular membranes have the same basic structure as the plasma membrane: a lipid
bilayer with proteins inserted among lipids. Endoplasmic reticulum membranes have a higher
protein concentration than plasma membrane. The ribosomes are the protein synthesis sites
where the polypeptide chains grow.
The Mazia method with bromphenol blue allows viewing in blue color the place where
the proteins are located in whole cell: at the level of the cell membrane, at the cytoplasm and
nuclei. The following slide is from the liver and it can be recognize the specific structure of
the liver. The hepatocytes are organized in cords having an ray-like arrangement in relation to
the central vein.
Slide 13 a, 10x
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Slide 13 b, 20x
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4. EXTENSIONS OF THE CELL SURFACE
Cell surface is a dynamic structure which presents some extensions, adaptations in

order to be able to adjust to the given conditions. It presents changes that may have a
transitory or a permanent character. Microvilli are differentiations with a permanent character
of the cell surface, found in cells with absorption functions. Kinocilia represents the
expansion of the cell surface localized at the apical pole of the cell, having mainly a
mechanical role. Stereocilia are imobile, agglutinated cilia present at the apical pole of the
cells of the epithelium from the epididymis and ependymal cells from the central canal of the
spinal cord.
Slide 14. Microvilli. Striated border. Enterocytes. Duodenum. HE Stain

Slide 15. Microvilli. Brush border. Nephrocytes. Proximal renal tubules. HE Stain
Slide 16. Kinocilia. Epithelial cells. Trachea. HE Stain
Slide 17. Stereocilia. Epithelial cells. Epididymis. HE Stain
Slide 18. Stereocilia. Ependimal cells. Central canal. Spinal cord. HE Stain
Slide 14. Microvilli. Striated border. Enterocytes. Duodenum. HE Stain

The intestinal villi are covered by a simple columnar epithelium.
Cells of this epithelium are mainly absorptive cells called enterocytes, that are tall
columnar cells which have an apical striated border of microvilli, covered by a glycocalyx.
The nucleus of these cells tends to be located towards the base of the cell. Between the
enterocytes goblet cells can be found, that secrete mucus, which coats and protects the surface
of the epithelium.
Microvilli of the enterocytes extend from the apical surface of epithelial cells into the
intestinal lumen. They increase the surface area and thereby facilitate absorption. Together,
the microvilli are visible as a light red band along the apical limit of the epithelium. This band
is called the striated border. Thousands of microvilli form a structure called the striated border
located at the apical end of the small intestinal enterocytes and another structure called the
brush border that is found on the apical surface of some epithelial cells, such as nephrocytes
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from the kidney proximal tubule. Microvilli also occur in sensory cells of the inner ear (as
stereocilia), in the cells of taste buds, and in olfactory receptor cells.
Slide 14 a, 10x
Slide 14 b, 20x
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Slide 14 c, 40x
Slide 15. Microvilli. Brush border. Nephrocytes. Proximal renal tubules. HE

Stain
A glomerulus and its surrounding Bowman's capsule constitute a renal corpuscle. The
nephron is the basic structural and functional unit of the kidney. A nephron is composed of a
capillary tuft called the renal glomerulus, Bowmans capsule, a proximal convoluted tubule, a
loop of Henle and a distal convoluted tubule. Proximal convoluted tubules have a simple
cuboidal epithelium with a prominent apical brush border consisting of many long microvilli.
Nephrocytes from the renal tubules (proximal convoluted tubules) present at the apical pole
long, thin microvilli, with different lengths in comparison with microvilli from the
enterocytes. Their contraction is weaker and intervenes in the processes of liquids absorption.
These microvilli form the brush border stained in red with eosin.
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Slide 15 a, 10x
Slide 15 b, 20x
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Slide 15 c, 40x
Slide 15 d, 40x
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Slide 16. Kinocilia. Epithelial cells. Trachea. HE Stain

A ciliated pseudostratified columnar epithelium with goblet cells is a characteristic
feature for some parts of the respiratory system, where it is called respiratory epithelium. All
cells of this type of epithelium rest on a thick basement membrane, but not all of them reach
the surface of the epithelium. Nuclei of the epithelial cells are typically located in the widest
part of the cell. Consequently, the nuclei of cells which do or do not reach the surface of the
epithelium are often located at different heights within the epithelium and give the epithelium
a stratified appearance. The epithelium will look stratified but it is not - hence its name
pseudostratified. Ciliated cells have numerous cilia that project into the mucus and move it
towards the larynx. Each cilium is anchored in the ciliated cell apical cytoplasm. Cilia and the
cytoplasm are stained in red with eosin. Nuclei are stained in blue with hematoxylin.
Slide 16 a, 20x
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Slide 16 b, 40x
Slide 16 c, 40x
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Slide 17. Stereocilia. Epithelial cells. Epididymis. HE Stain

The epididymis is an organ that lies along the superior and posterior surface of the
testis. The duct of the epididymis is a highly coiled tube which stores spermatozoa after they
leave the testes. The epididymis has a pseudostratified epithelium containing numerous tall
columnar cells and scattered shorter pyramidal basal cells. Columnar cells extend from the
basement membrane to the lumen of the epididymal epithelium. These cells have numerous,
long, nonmotile extensions of the membrane called stereocilia. They are located at the apical
pole of the cell and are stained in red with eosin. The position of the nuclei varies and makes
the epithelium to appear stratified. The cytoplasm and the stereocilia are stained in red with
eosin and the nuclei in blue with hematoxylin. The slide shows different shape profiles of the
epididymal duct.
Slide 17 a, 10x
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Slide 17 b, 20x
Slide 17 c, 40x
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Slide 18. Stereocilia. Ependimal cells. Central canal. Spinal cord. HE Stain
The spinal cord consists of gray matter centrally located which is surrounded by the
white matter. The gray matter is organized in anterior horns, intermediate horns and posterior
horns. In the anterior horns the motor neurons are located that conduct impulses from the
spinal cord to skeletal muscles. In the middle of the spinal cord is placed the central canal. It
is lined by ependymal cells. These cells contain large nuclei that occupy most of the cell
volume. With a high power objective we may identify the stereocilia at the apical pole of the
cells. Stereocilia are immobile extensions of the cell surface. Unlike stereocilia from the
epidydymis that are numerous, long projections, those of the ependymal cells are shorter and
discontinuous.
Slide 18 a, 10x
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Slide 18 b, 20x
Slide 18 c, 40x
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5. EXTRACELLULAR MATRIX
Extracellular matrix represents a component of the connective tissue that can be found
between the cells of the connective tissue, blood vessels and nerves and that fulfils many
important functions. It is composed of ground substance and three types of fibers: collagen,
reticular and elastic fibers. The basement membrane is a membrane placed between an
epithelial tissue and the components of the connective tissue, being a product of both of them.
Slide 19. Extracellular matrix. Hyaline cartilage. Chondrocytes. Trachea. HE Stain

Slide 20. Basement membrane. Nephrocytes. Renal tubules. Kidney. PAS Stain
Slide 21. Reticular fibers. Basement membrane. Nephrocytes. Renal tubules. Kidney.
Silver Impregnation
Slide 22. Elastic fibres. Aorta. Cross section. Resorcin fuchsin Stain
Slide 23. Collagen fibers. Skin. HE Stain
Slide 19. Extracellular matrix. Hyaline cartilage. Chondrocytes. Trachea. Cross

section. HE Stain
This slide is of the hyaline cartilage which supports the trachea. We may identify
small cluster of chondrocytes encased within spaces called lacunae. Surrounding the lacunae
is the extracellular matrix stained blue. Though the matrix appears homogenous, it consists
primarily of numerous, fine collagen fibers. Hyaline cartilage is a specialized form of
connective tissue containing chondrocytes which secrete and are surrounded by an extensive
extracellular matrix. Chondrocytes occur singly or in isogenous groups, composed of 2-8 cells
derived by mitosis from a single chondrocyte. Their nuclei are stained in purple-blue with
hematoxylin. The cells are in the lacunae (cavities) within the matrix. There are clear areas
between many of the chondrocytes and the walls of their lacunae because of shrinkage of the
cells brought by fixation, and because some chondrocytes had lipid droplets which dissolved
during specimen preparation. Matrix stains more intensely immediately adjacent to the
lacunae and the dark staining zone is called the capsule. The strength and durability of
cartilage are properties of the matrix, which is an interlaced network of collagenous and/or
elastic fibers in a ground substance, a gel of complex proteoglycans.
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Slide 19 a, 10x
Slide 19 b, 20x
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Slide 19 c, 40x
Slide 20. Basement membrane. Nephrocytes. Renal tubules. Kidney. PAS Stain
The basement membrane is a specialized structure of the extracellular matrix. It is a
thin, flexible support located in the epithelial cells of the tissues structure, of renal glomeruli.
This layer is visible in the light microscope stained in magenta with PAS. Cytoplasm and
nuclei are not stained. The basement membrane performs selective filtration. The glomerular
basement membrane filters wastes from blood passing through the kidneys.
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Slide 20 a, 10x
Slide 20 b, 20x
69
Slide 20 c, 40x
Slide 21. Reticulin fibers. Basement membrane. Nephrocytes. Renal tubules.

Kidney. Silver Impregnation
The cortical labyrinth contains renal corpuscles and tubules. The renal corpuscle is
formed by two structures, an epithelial capsule and a tuft of capillaries, the glomerulus.
Reticular fibers consist of one or more types of very thin collagen fibers, mostly types with
which abundant carbohydrate has been combined. Reticular fibers are impregnated with a
silver salt and appear as sharp black.
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Slide 21 a, 10x
Slide 21 b, 40x
71
Slide 21 c, 40x
Slide 22. Elastic fibres. Aorta. Cross section. Resorcin fuchsin Stain
The vessels which receive blood from the heart, the elastic arteries, have thick, strong
walls to cope with the sudden high pressure produced during diastole. They contain abundant
elastic material to allow stretch so that the vessel lumen may accommodate the change of
volume. The elastic recoil of these elastic arteries is responsible for maintaining a continuous,
though decreased, flow of blood to smaller vessels during systole. Further along the arterial
system, elastic components gradually diminish. Most of the muscle is arranged circularly, in
the middle layer of the vessel wall (the tunica media). These muscular arteries contribute to
the regulation of the amount of blood flowing into a region. The slides contain only one wall
of the vessel. The innermost layer is the tunica intima which can be recognized as having a
smooth more sharply defined free-edge than the opposite surface and no visible blood vessels.
The intima has endothelial cells. The tunica media contains bundles of elastic fibers with gaps
between them lying within a background of very fine collagen. The elastic fibers stain dark
purple-blue and the collagen pink-red. They have wavy shape, which are parallel between
them. The cellular components of this layer consist of smooth muscle cells which are difficult
to see. The outermost layer, the tunica adventitia, consists of connective tissue with thick
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collagen fibers. The adventitia contains numerous blood vessels (vasa vasorum or blood
vessels of the blood vessels) and nerves.
Slide 22 a, 10x
Slide 22 b, 20x
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Slide 22 c, 40x
Slide 23. Collagen fibers. Skin. HE Stain

The skin is the outer covering of the body. Skin is composed of three primary layers:
the epidermis, which provides waterproofing and serves as a barrier to infection; the dermis,
which serves as a location for the appendages of skin and it is rich in collagen fibers; and the
hypodermis (subcutaneous adipose layer). The superficial epidermal layers are formed by
dead, dehydrated cells containing hydrophobic proteins that prevent desiccation of the body.
The lower epidermal layers consist of highly proliferative cells that continuously replace cells
in the upper epidermal layer as they are lost to exfoliation. This stratified epithelium provides
an important protective barrier for the bodys outer surface. The basement membrane is a
barrier that prevents microorganisms from entering the organisms inner domain, and also
prevents the loss of cells and fluids from the body. In the dermis collagen fibers and cells of
the connective tissue can be observed. The randomly arranged collagen fibers, running in all
directions are reddish, while the cells, in general, have a pink-red cytoplasm with eosin and
purple-blue nuclei with hematoxylin.
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Slide 23 a, 10x
Slide 23 b, 20x
75
Slide 23 c, 40x
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6. SHAPES OF THE NUCLEI
The nucleus is an organelle which can be found in eukaryotic cells.

Usually the nucleus, occupies a central position in the cell (hepatocytes, young cells
and blood cells). The nucleus has an eccentric position in the adipose cells and plasmocytes,
or it can be found at the basal pole, like in goblet cells.
The shape of the nucleus varies. It takes the shape of the cell in which it can be found:
spherical in young cells (lymphocytes), ovoid (prismatic cells), fusiform in oblong cells,
oblate (endothelial cells, adipose cells), and lobate (neutrophil leucocytes), reniform
(monocytes), budded in polyploid cells (megakaryocytes, Reed-Sternberg cell), irregular
shape, monstrous in neoplastic cells.
In optical microscopy, the nucleus is highlighted as an intracellular body bounded by
an envelope, having in the interior part a granular network (nuclear chromatin) and a body
(the nucleolus).
Slide 24. Round nucleus (Lymphocyte), Segmented nucleus (Neutrophil), Bilobed

nucleus (Eosinophil), Trilobed nucleus (Basophil), Reniform nucleus (Monocyte).
Peripheral blood smear. May Grunwald Giemsa (MGG) Stain
Slide 25. Lobulated nucleus. Megakaryocyte. Bone marrow. MGG Stain
Slide 26. Flattened nucleus. Adipocytes. Adipose tissue. Skin. HE Stain
Slide 27. Elongated nuclei. Skeletal muscle fibers. Ferrical Hematoxylin (FeH) Stain
Slide 28. Monstruous, irregular nuclei. Neoplastic cells. Malignant tumor. FeH Stain
Slide 24 A. Peripheral blood smear. MGG Stain

The blood consists of a suspension of special cells in liquid called plasma. In an adult
man, the blood is about 5-6 litres. Blood consists of 55 % plasma, and 45 % by cells called
blood cells. In the blood are present special cells, classified in: erythrocytes and leukocytes.
There are also platelets which are not considered real cells. The erythrocytes are the most
numerous blood cells. Leukocytes, or white cells, are responsible for the defence of the
organism.
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Slide 24 A, 10x
A peripheral blood smear is a microscope slide obtained from a drop of blood that
allows the cells to be examined under the microscope. Blood smears are usually done to
investigate hematological disorders. Blood smears are made by placing a drop of blood on one
end of a slide, and using a spreader slide to disperse the blood over the slide's length. The aim
is to get a region where the cells are spaced far enough apart to be counted and differentiated.
The slide is left drying to air, after which the blood is fixed to the slide by immersing it briefly
in methanol. The fixative is essential for good staining and presentation of cellular detail.
After fixation, the slide is stained to distinguish the cells from each other. May Grunwald
Giemsa stain is a classical blood film stain for peripheral blood smears and bone marrow
specimens.
Erythrocytes stain pink, platelets show a light pale pink colour, lymphocyte cytoplasm
stains sky blue, monocyte cytoplasm stains pale blue, leukocytes nuclear chromatin stains
magenta.
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Platelets are not true cells. They develop in the bone marrow from large leukocytes
called megakaryocytes, being than released in the blood stream. They are small sized diskettes
about 3m in diameter. They appear purple coloured and are more intense coloured than red
cells. They remain in the bloodstream for 7-10 days.
Slide 24 B. Erythrocytes. Peripheral blood smear. MGG Stain

Erythrocytes are devoid of a nucleus and have the shape of a biconcave lens.
Red cells do not have mitochondria, endoplasmic reticulum and Golgi apparatus. Usually,
they measure 7-7.5 m in diameter. Under the microscope, they look like pink discs clearer in
the middle. These cells are responsible for providing oxygen to tissues and partly for
recovering carbon dioxide produced as waste. The mean life of red cells is about 120 days.
They are destroyed in the spleen, liver and bone marrow.
Slide 24 B, 60x, immersion
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Slide 24 C. Round nucleus. Lymphocyte. Peripheral blood smear. MGG Stain

Peripheral blood contains two kinds of leukocytes: granulocytes and agranulocytes.
Granulocytes distinguish themselves in neutrophil, eosinophil and basophil. Agranulocytes
comprise lymphocytes and monocytes. The shape of the nucleus helps us in the recognition of
the leukocytes. The term granulocyte is due to the presence of granules in the cytoplasm of
these cells. In the different types of granulocytes, the granules are different and allow us to
recognize them. These granules have a different affinity towards neutral, acid or basic stains
and give the cytoplasm different colours.
Lymphocytes have a compact round nucleus which occupies nearly all the cellular
volume. The cytoplasm is very reduced and transparent. Most lymphocytes circulating in the
blood are in a resting state. Lymphocytes are about 8-10 m in diameter and generally they
are smaller than the other leukocytes but they are still a few larger than red cells.
Lymphocytes are small, medium and large. They are key cells in the immune system.
Slide 24 C a, 20x
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Slide 24 C b, 40x
Slide 24 D. Segmented nucleus. Neutrophil. Peripheral blood smear. MGG Stain

The granulocytes originate from the bone marrow. Their cytoplasm is rich in granules
which take typical colours which help their recognition. The nucleus is condensed in lobes.
In the blood, there are immature cells as well. They distinguish themselves by having a less
segmented nucleus. There are three types of granulocyte: neutrophil, eosinophil, basophil.
Neutrophils (polymorphonuclear leukocytes) are the more common leukocytes. They
have a diameter of 12-15 m. Their nucleus is divided into 2 - 5 lobes connected by a fine
nuclear filament. The cytoplasm is transparent because its granules (azurophilic granules) are
small and faintly pink coloured. They are involved in phagocytosis and destruction of
bacteria.
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Slide 24 D a, 20x
Slide 24 D b, 40x
82
Slide 24 D c, 40x
Slide 24 D d, 60x, immersion
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Slide 24 E. Bilobed nucleus. Eosionophil. Peripheral blood smear. MGG Stain

Eosinophils are quite rare in the blood. They have the same size as the neutrophils.
Generally their nucleus is bilobed. The cytoplasm is full of granules (eosinophilic granules)
which assume a characteristic pink-orange colour. The nucleus is still easily visible. They are
involved in parasitic infections.
Slide 24 E a, 40x
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Slide 24 E b, 40x
Slide 24 E c, 60x immersion

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Slide 24 F. Trilobed nucleus. Basophil. Peripheral blood smear. MGG Stain

Basophils are the rarest leukocytes: less than 1 %. They are quite small: 9-10 m in
diameter. Cytoplasm is very rich in granules (basophilic granules) which take a dark purple
colour. The nucleus is bi- or trilobed, but it is hard to be seen because of the high number of
granules which hide it. They mediate the inflammatory response.
Slide 24 F a, 40x
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Slide 24 F b, 40x
Slide 24 F c, 100x, immersion
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Slide 24 G. Reniform nucleus. Monocyte. Peripheral blood smear. MGG Stain

Monocytes are the precursors of macrophages. Monocytes are the largest leukocytes:
16-20 m. They have a great reniform nucleus, in some cases even bilobed. Their cytoplasm
is transparent, but with an appearance of "ground glass. They do not contain specific
granules. Their main function is phagocytosis.
Slide 24 G a, 20x
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Slide 24 G b, 40x
Slide 25. Multiobulated nucleus. Megakaryocyte. Bone marrow. MGG Stain

The sample of bone marrow is obtained by bone marrow aspiration, stained with
MGG. The bone marrow produces the cellular elements of the blood, including platelets, red
blood cells and white blood cells. In the red marrow hematopoietic cells outnumber fat cells.
The megakaryocyte is a bone marrow cell responsible for the production of
thrombocytes (platelets), which are necessary for normal blood clotting. In general,
megakaryocytes are enormous cells 10 to 15 times larger than a typical red blood cell,
averaging 50-100 m in diameter. As a result, the nucleus of the megakaryocyte can become
very large and lobulated, which, under a light microscope, can give the false impression that
there are several nuclei. The cytoplasm contains abundant rough endoplasmic reticulum, well
developed Golgi apparatus, granules and dense bodies. Megakaryocytes exist only in bone
marrow. They slowly release small cytoplasmic fragments called platelets into the
bloodstream that are continuously replaced every 7-10 days.
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Slide 25 a, 10x
Slide 25 b, 40x
90
Slide 26. Flattened nucleus. Adipocytes. Adipose tissue. HE Stain

The deepest layer of the skin, the hypodermis contains fat cells called adipocytes.
Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose
adipose tissue, specialized in storing energy as fat. White fat cells or monovacuolar cells
contain a large lipid droplet surrounded by a layer of cytoplasm stained in red with eosin. The
nucleus is flattened and located at the periphery being stained in blue with hematoxylin.
Slide 26 a, 10x
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Slide 26 b, 20x
Slide 26 c, 40x
92
Slide 26 d, 40x
Slide 27. Elongated nuclei. Skeletal muscle fibers. Ferric Hematoxylin (FeH)
Stain
The skeletal muscle cells possess some hundreds to a few thousand nuclei located
close to the cell surface. Some of them are several millimetres long. They are stained in dark
blue with ferric hematoxylin (FeH).
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Slide 27 a, 20x
Slide 27 b, 40x
94
Slide 28. Monstruous, irregular nuclei. Neoplastic cells. Malignant tumor. FeH
Stain
Neoplastic cells are characterized by abnormal growing in their volume and
pathological changes of their morphofunctional features. The neoplastic nuclei are increased
in size with an irregular envelope and many nucleoli stained in black with ferrical
hematoxylin. The nuclei contain high amounts of euchromatin that appears in pale grey.
Slide 28 a, 20x
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Slide 28 a, 40x
96
7. CHROMOSOMES
Chromosomes are structures composed of a very long DNA molecule and associated
proteins which carries the hereditary information of an organism. They are formed during
mitosis by condensation of the chromatin.
Slide 29. Metaphasic chromosomes. Bone marrow. Basic Fuchsine Stain

Slide 30. Barr corpuscle. Mouth mucosa cells from females. Smear. Basic Fuchsine Stain
Slide 31. Gender nodule. Neutrophils. Peripheral blood smear from females. MGG Stain
Slide 29. Metaphasic chromosomes. Bone marrow. Basic Fuchsine Stain

The chromatin is a cytological concept representing the biological intranuclear
material which is stained with basophilic dyes (heamatoxylin).
Besides division, it has the appearance of a granulofilamentous network, which has
two distinct tinctorial aspects: euchromatin and heterochromatin, visible in optical
microscopy.
Euchromatin appears as a network of very fine filaments, coloured weakly, and pale.
Heterochromatin is granular and intensely stained. The nuclear chromatin has two different
packaging (condensation). The nuclear chromatin in the interphase represents the unfolded
form of the chromosomes. During division process it represents the compact form of
chromosomes, because of the chromatins packaging and condensation.
The chromosomes represent the hypercondensed form of existence of the chromatin.
They are the carriers of the genetic information. They were described as cellular entities
visible during cell division in the form of rods. The morphology of the chromosomes can be
observed in optimal conditions in the metaphase as it ca be seen in the following pictures.
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Slide 29 a, 10x
Slide 29 b, 20x
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Slide 29 c, 40x
Slide 30. Barr corpuscle. Mouth mucosa cells from females. Smear. Basic
Fuchsine Stain
Barr body is used in genetics as a marker for the cytological recognition of gender.
This is a chromatinian corpuscle present in the somatic cell nuclei at females. It actually
represents the interphase condensation of the second X chromosome. Normally, the female
has one Barr body, depriving the male sex. In mouth mucosa cells it is present in at least 20%
of cells with a size of about 1m. The corpuscle has a triangular or convex shape being
located near the nuclear membrane.
99
Slide 30 a, 20x
Slide 30 b, 40x
100
Slide 31. Gender nodule. Neutrophils. Peripheral blood smear from females. MGG Stain
Another structure with the same significance as Barr body represents leukocyte
drumstick or gender nodule located in some neutrophil polymorphonuclear leukocytes. This
node looks like a body of about 1.5 m, connected by a fine chromatin filament to the nuclear
lobes. In females leukocytes drumstick are present at the rate of 7 to 500 and they are absent
in males.
Slide 31 a, 40x
Slide 31 b, 60x, immersion

101
8. CELL DIVISION
Cell division is a phase of the cell cycle, in which the distribution of genetic material
between the two daughter cells, derived from the parent cell, takes place.
Cell division can be achieved either by direct division, called amitosis or through
indirect division, which includes mitosis and meiosis.
Slide 32. Amitosis. Hepatocytes. Liver. HE Stain

Slide 33. Mitosis. Bone marrow. May Grunwald Giemsa (MGG) Stain
Slide 34. Endomitosis. Megakaryocyte. Bone marrow. MGG Stain
Slide 35. Meiosis. Oogenesis. Ovary. HE Stain
Slide 36. Meiosis. Spermatogenesis. Testis. HE Stain
Slide 32. Amitosis. Hepatocytes. Liver. HE Stain

Amitosis is the direct cell division. During amitosis the cell breaks into two parts by
constriction of the nucleus and cytoplasm. The result is represented by two daughter cells. It is
characterized by the lack of mitotic spindle. It is specific to unicellular organisms being
considered an inferior form of cell division. Amitosis is seen in the human species under
specific conditions, such as tumor and regenerative processes, when the rapid remaking of the
cell mass is necessary.
102
Slide 32 a, 10x
Slide 32 b, 40x
103
Slide 33. Mitosis. Bone marrow. MGG Stain

Mitosis and meiosis are indirect cells divisions. Indirect cell division is a complex
process encountered in superior organisms. Mitosis is seen in all somatic cells. Following
mitosis, two daughter cells appear. They have the same number of chromosomes as the parent
cell. Mitosis comprises four successive phases: prophase, metaphase, anaphase and telophase.
Slide 33, 20x
Slide 33.A. Mitosis. Prophase. Bone marrow. MGG Stain

In prophase the second centrosome emerges. The two centrosomes depart one from
each other reaching the two poles of the cell. Between the centrosomes the microtubules form
a spindle helping the movement of the chromosomes during mitosis. The nucleolus disappears
and the nuclear chromatin begins to condense in the form of thin filaments, arranged in a
shape called bouquet (spirem). The nuclear envelope disappears and the chromosomes
become visible under the form of some rods.
104
Slide 33 A a, 60x, immersion
Slide 33 A b, 100x, immersion

105
Slide 33. B. Mitosis. Metaphase. Bone marrow. MGG Stain

In the metaphase, the chromosomes form the metaphase plaque where the longitudinal
split of chromosomes into two chromatids takes place. By longitudinal chromosome division
results monochromatidic chromosomes. During metaphase, chromosomes are superspiralized,
having a maximum thickness; they are best seen at this stage.
Slide 33 B a, 40x
106
Slide 33 B b, 40x
Slide 33 B c, 60x, immersion

107
Slide 33. C. Mitosis. Anaphase. Bone marrow. MGG Stain

In the anaphase the migration of monochromatidic chromosomes begins towards the
two poles of the cell at each pole reaching 46 monochromatidic chromosomes.
Slide 33 C a, 40x
Slide 33 C b, 40x
108
Slide 33 C c, 100x, immersion
Slide 33 C d, 60x, immersion

109
Slide 33. D. Mitosis. Telophase. Bone marrow. MGG Stain

The telophase starts with the ending of the migration process of chromosomes towards
the cell poles. The chromosomes begin to despiralize, forming a bouquet at each pole. Around
the despiralized chromatin a nuclear membrane appears.
At the end of the mitosis two diploid daughter cells arise, each one having an equal
amount of DNA as the parent cell.
Slide 33 D a, 40x
110
Slide 33 D b, 40x
Slide 34. Endomitosis. Megakaryocyte. Bone marrow. MGG Stain

Endomitosis is a mitosis taking place without dissolution of the nuclear membrane and
not followed by cytoplasmic division, resulting in doubling of the number of chromosomes
within the nucleus.
The megakaryocyte is a bone marrow cell responsible for the production of
thrombocytes which are necessary for normal blood clotting. Megakaryocytes are the largest
cells of the bone marrow, their size being of 50-100 m in diameter. During its maturation,
the megakaryocyte grows in size and replicates its DNA without cytokinesis. The nucleus of
the megakaryocyte can become very large and lobulated. As a result, the number of
chromosomes duplicates with each mitosis, phenomenon known as endopolyploidy.
111
Slide 34, 40x
Slide 35. Meiosis. Oogenesis. Ovary. HE Stain

Meiosis is a specific division of the gametes, spermatozoon and ovum.
It is encountered during spermatogenesis and oogenesis. It is characterized by halving of the
chromosomes number in daughter cells, which are haploids besides the parent cell, which is
diploid. In female, the result of meiosis is an ovum and 3 polar bodies. In male, the result of
meiosis is 4 spermatozoa. Unlike mitosis, when two diploid cells are formed, at the end of
meiosis four haploid cells are formed. The surface of the ovary is covered by a simple
cuboidal epithelium called germinal epithelium. Underneath, the ovarian cortex contains
ovarian follicles in different stages of development. Ovarian follicles consist of an oocyte and
a layer of follicular epithelial cells that varies in thickness. In certain more mature follicles, an
acellular layer called the zona pellucida surrounds the oocyte. Primordial follicles consist of
follicular epithelial cells in a single layer around the oocyte. Primary follicle is formed by
follicular epithelial cells in several layers which surround the oocyte.
112
Slide 35 a, 10x
Slide 35 b, 20x
113
Slide 35 c, 40x
Slide 35 d, 40x
114
Slide 35 e, 40x
Slide 36. Meiosis. Spermatogenesis. Testis. HE Stain

Spermatogenesis occurs in the seminiferous tubules within the testes. Seminiferous
tubules are closed continuous loops. They contain cells of the gamete-producing line and
Sertoli cells which support the gamete-producing cells. Sertoli cells rest on the seminiferous
epithelial basal lamina and reach the lumen. They are bounded by numerous cellular and
acellular barriers, including several layers of myoid cells. Myoid cells have many of the
characteristics of smooth muscle cells. The spermatogenic cell line contains spermatogonia
resting on the basal lamina, primary spermatocytes, secondary spermatocytes, spermatids and
spermatozoa.
115
Slide 36 a, 10x
Slide 36 b, 20x
116
Slide 36 c, 40x
117
9. ENDOPLASMIC RETICULUM. RIBOSOMES
Rough endoplasmic reticulum is the site of the protein synthesis. The endoplasmic
reticulum works closely with the Golgi apparatus and ribososmes in synthesis and packaging
of proteins. Some of these proteins are used by the cell and some are exported through
exocytosis.
Slide 37. RER. Nissl Bodies. Neurons. Spinal cord. Toluidine Blue Stain
Slide 38. RER. Berg Bodies. Hepatocytes. Liver. HE Stain
Slide 39. RER. Ribosomes. Plasmocyte. Bone marrow. MGG Stain
Slide 37. RER. Nissl Bodies. Neurons. Spinal cord. Toluidine Blue Stain
The endoplasmic reticulum and the Golgi complex are fluid-filled compartments
located into the cytoplasm between the nucleus and plasma membrane. They are bounded by
lipoprotein bilayers with the same structure as the plasma membrane. The neurons are the
basic cellular elements of the nervous system. Their shapes and structures vary. The motor
neurons conduct the impulses from the spinal cord to the skeletal muscles. They are situated
in the anterior horns of the spinal cord. Each motor neuron has a cell body and cell extensions
which radiate from the cell body. These are of two types: dendrites and one axon. The
dendrites are shorter extensions that vary in morphology. The axon is a long projection
responsible for the transmission of the impulses to the muscle fibers. The cell body has a star
shape. In the cytoplasm there are many clusters of rough endoplasmic reticulum and free
ribosomes called Nissl bodies or tigroid bodies. These structures determine cytoplasmic
basophilia. They can be seen as irregular granules spread in the vicinity of the nucleus, stained
in blue with alcian blue. Also others basophilic structures are stained in blue like the
nucleolus. Motor neurons are rich in darkly stained Nissl bodies. They are thought to be
involved in the synthesis of neurotransmitters such as acetylcholine
118
Slide 37 a, 10x
Slide 37 b, 20x
119
Slide 37 c, 40x
Slide 38. RER. Berg Bodies. Hepatocytes. Liver. HE Stain

Hepatocytes are organized into plates separated by vascular channels called sinusoids.
The hepatocyte plates are one cell thick in mammals. Sinusoids show a discontinuous,
fenestrated endothelial cell lining. Hepatocytes display an eosinophilic cytoplasm, reflecting
numerous mitochondria, and basophilic granules due to large amounts of rough endoplasmic
reticulum and free ribosomes. These small spherical areas from the cytoplasm are known as
Bergs bodies. They are stained in blue with hematoxylin. The nuclei of the hepatocytes are
round with dispersed chromatin and prominent nucleoli.
120
Slide 38 a, 20x
Slide 38 b, 40x
121
Slide 39. RER. Ribosomes. Plasmocyte. Bone marrow. MGG Stain

Plasmocytes or plasma cells are white blood cells that produce large amounts of
antibodies and have a characteristic appearance on light microscopy. They have an intense
basophilic cytoplasm with a high amount of ribosomes and rough endoplasmic reticulum
stained in blue. The nucleus is eccentrically positioned with heterochromatin in a
characteristic cartwheel arrangement, the heterochromatin alternates with euchromatin. It is
stained in purple. In the perinuclear area exists a pale zone that contains an extensive Golgi
apparatus. Abundant rough endoplasmic reticulum combined with a well-developed Golgi
apparatus makes plasma cells to produce immunoglobulins.
Slide 39 a, 20x
122
Slide 39 b, 40x
Slide 39 c, 60x, immersion
123
10. GOLGI APPARATUS AND CELL SECRETION
The main function of the Golgi apparatus (Golgi complex) consists of its involvement
in the intracellular secretion. So, it has a crucial role in processing and transporting the
products secreted by the ER to the secretory vesicles.
Slide 40. Golgi Apparatus. Secretory neurons. Hypothalamus. Silver Impregnation

Slide 41. Apocrine secretion (exocrine secretion). Goblet cells. Colon. HE Stain
Slide 42. Apocrine secretion. Mammary gland. HE Stain
Slide 43. Merocrine secretion (exocrine secretion). Secretory (serous and mucous) cells.
Serous, mucous and mixted acini. Submaxillary gland. HE Stain
Slide 44. Holocrine secretion (exocrine secretion). Secretory cells. Sebaceous glands.
Hair follicles. Skin. HE Stain
Slide 45. Endocrine secretion. Secretory cells. Islet of Langerhans. Pancreas. HE Stain
Slide 46. Endocrine secretion. Epithelial cells. Thyroid follicles. Thyroid Gland. HE
Stain
Slide 40. Golgi Apparatus. Secretory neurons. Hypothalamus. Silver

Impregnation
The hypothalamus controls body temperature, hunger, thirst, fatigue, sleep, and
circadian cycles. It is a portion of the brain which links the nervous system to the endocrine
system. The hypothalamus contains secretory neurons that synthesize and secrete certain
neurohormones. In silver impregnation, the silver particles will be deposited on the surface of
the Golgi apparatus, present in the secretory neurons. This complex is usually located close to
the cell nucleus between the nucleus and the secretory surface.
The Golgi apparatus has a black laced aspect which encircles the nucleus in an
unbroken ring in light microscopy. The black small vacuoles, fairly uniform, round or oval
bodies are elements of the Golgi apparatus. The nucleus is optical-clear.
124
Slide 40 a, 10x
Slide 40 b, 20x
125
Slide 40 c, 40x
Slide 40 c, 40x
126
CELL SECRETION
The Golgi complex is especially prominent in cells which are specialized for secretion.
Following how the cell removes its secretory product, the secretory cells called
adenocytes can have an endocrine cell secretion mechanism or an exocrine cell secretion
mechanism. Exocrine secretion takes 3 distinct aspects: merocrine, apocrine, holocrine
secretion. Glands can be divided into 2 groups: endocrine and exocrine glands.
Endocrine glands secrete their products through the basal lamina into blood vessels
and lack a duct system.
Exocrine glands secrete their products through a duct or directly onto the apical
surface. These glands can be divided into three groups: apocrine, merocrine and holocrine
glands.
Apocrine glands use the apocrine method of secretion. Thus a portion of the secreting
cell's body more precisely the apical pole is lost during secretion (e.g., goblet cells, fat droplet
secretion by mammary gland).
Holocrine glands use a mechanism by which the entire cell disintegrates to secrete its
substances (e.g., sebaceous glands). Sebaceous glands secrete sebum that coats the hair and
skin surface. They are located near the hair follicles.
Merocrine glands cells secrete their substances by exocytosis (e.g., mucous and serous
glands, submaxilary gland, parotid gland, pancreatic acinar cells). They are also called
eccrine".
The morphofunctional units of the exocrine glands are the secretory acini, composed
of secretory cells and myoepithelial cells. Depending on the secretory product we may
distinguish: serous acini, mucous acini and serous-mucous or mixed acini. Serous acini
consist of serous cells that secrete a watery, often protein-rich product. Mucous acini are
formed by mucous cells that secrete a viscous product, rich in carbohydrates (e.g.,
glycoprotein). Mixed acini, composed of serous and mucous cells, secrete both protein and
mucus.
The serous cell has a basal round nucleus and a large amount of rough endoplasmic
reticulum located in the basal part of the cytoplasm. They are basophilic structures being
stained in blue with hematoxylin. In the upper part of the cell there are many apical granules
containing secretion product. The cytoplasm is highlighted in red with eosin. Serous cells
secrete proteins, often enzymes.
127
Mucous cells secrete mucous droplets. They have a well-developed rough

endoplasmic reticulum and a prominent Golgi complex. The mucous droplets accumulate into
the cytoplasm and push the nucleus at the basal pole of the cell. Their content is unstained.
The nucleus is flattened being pushed at the periphery of the cell at the basal part. It is stained
in blue with hematoxylin.
Mixed acini are composed of mucous acini, having at one side associated in form of a
demilune, a few serous cells.
Myoepithelial cells are located between secretory cells. They have actin
microfilaments in their cytoplasm probably responsible for releasing acinar secretion product.
The secretion from these acini is eliminated through excretory ducts.
Slide 41. Apocrine secretion (exocrine secretion). Goblet cells. Colon. HE Stain
Goblet cells secrete mucus that is eliminated through apocrine secretion mechanism.
They are numerous in the colon. The upper part of the cell contains granules filled with
secretion product that does not stain by eosin. The basal part of the cell contains the nucleus
stained in blue with hematixylin and the cytoplasm which is quantitatively reduced is stained
in red with eosin.
Slide 41 a, 10x
128
Slide 41 b, 20x
Slide 41 c, 40x
129
Slide 42. Apocrine secretion. Mammary gland. HE Stain

In the mammary gland, by apocrine secretion a small part of the apical portion of each
secretory cell is released with the secretion product, the milk. The glandular acini are
composed of cuboidal cells placed in one layer on the surrounding basement membrane.
During lactation, secretory cells become columnar. They discharge their products through
ducts. Among acini there are lactiferous ducts.
Slide 42 a, 10x
130
Slide 42 b, 20x
Slide 42 c, 40x
131
Slide 43. Merocrine secretion (exocrine secretion). Secretory (serous and mucous)
cells. Serous, mucous and mixted acini. Submaxillary gland. HE Stain
The secretory unit of the gland, the acinus is composed of either serous cells, mucous
cells or both cell types. So, there are three types of acini: serous acini, mucous acini and
mixed acini.
The serous cells have spherical nuclei stained in blue with hematoxylin and a deeply
stained cytoplasm in red with eosin. The mucous cells have flattened nuclei pushed at the
basal pole of the cell by the mucous accumulated into the cytoplasm. Their cytoplasm is
reduced quantitatively and the mucus is transparent.
Mixed acini have both types of the secreting cells, serous cells and mucous cells.
Serous cells form a cap known as serous demilune on the periphery of the many mixed acini.
Slide 43 a, 10x
132
Slide 43 b, 20x
Slide 43 c, 40x
133
Slide 44. Holocrine secretion (exocrine secretion). Secretory cells. Sebaceous

glands. Hair follicles. Skin. HE Stain
The skin has three main layers: epidermis, dermis and hypodermis.
The dermis lies beneath the epidermis. The epidermal-dermal boundary is the
basement membrane. The extracellular matrix of dermis is rich in collagen and elastic fibers
synthesized by fibroblasts. Collagen fibers are abundant and they can be seen as elongated
fibers stained in red/pink with eosin. All the nuclei of the cells are stained in blue with
hematoxylin.
The sebaceous gland secretes sebum through holocrine mechanism. This gland is
associated with hair follicle. It is composed of two types of cells: the basal cells and the
central cells which accumulate lipids in their cytoplasm and become full of lipid droplets.
These cells are destroyed and completely eliminated with their secretion product in the
excretory duct. The basal cells constantly proliferate and differentiate into secretory cells,
providing the replacement and cell growth. The sebum contains triglycerides, fatty acids and
cellular debris. The sebaceous gland appears as a cluster of cells containing lipid droplets.
They discharge their secretion into the hair follicle from where it reaches the skin surface. The
sebum has a protective role for the skin.
Slide 44 a, 10x
134
Slide 44 b, 20x
Slide 44 c, 40x
135
Slide 45. Endocrine secretion. Secretory cells. Islet of Langerhans. Pancreas. HE

Stain
The pancreas, located in the abdomen close to the stomach, is both an exocrine and an
endocrine gland.
The endocrine portion is represented by the pancreatic islets or Islets of Langerhans -
the regions of the pancreas that contain its endocrine cells. These cells produce hormones like
insulin and glucagon that are released into the bloodstream. Glucagon, released by alpha ()
cells when glucose levels in blood are low, stimulates the liver to release glucose to the blood.
Insulin is released by beta () cells when blood levels of glucose are rising. It increases the
rate of glucose uptake and metabolism by most body cells. Hyposecretion of insulin results in
diabetes mellitus.
The exocrine portion of the pancreas is formed by secretory acini. The acinus is
composed of 6-8 serous cells. These cells have a nucleus located at the basal pole and large
amount of rough endoplasmic reticulum (ergastoplasma) near the nucleus. They are
basophilic structures being stained in blue with hematoxylin. In the upper part of the
cytoplasm there are many granules containing secretion product. The cytoplasm is stained in
red with eosin. Serous cells secrete proteins.
Slide 45 a, 10x
136
Slide 45 b, 20x
Slide 45 c, 40x
137
Slide 46. Endocrine secretion. Thyroid epithelial cells. Thyroid follicles. Thyroid
Gland. HE Stain
The thyroid gland is located in the anterior throat region. Thyroid follicles store
colloid containing thyroglobulin, a glycoprotein from which thyroid hormone is derived.
The thyroid is composed of spherical follicles that selectively absorb iodine from the
blood for production of thyroid hormones, but also for storage iodine in thyroglobulin. Inside
the follicles, colloid serve as a reservoir of materials for thyroid hormone production and act
as a reservoir for the hormones themselves. Colloid is rich in a protein called thyroglobulin.
The follicles are surrounded by a single layer of thyroid epithelial cells, which secrete thyroid
hormones, thyroxine (T4) and triiodothyronine (T3). When the gland is inactive it does not
secrete hormones, and the epithelial cells become from low columnar to cuboidal cells. These
cells have a central spherical nucleolus. When they are active, the epithelial cells become tall
columnar cells, with oval nuclei located at the lower part of the cytoplasm. All the nuclei are
stained in blue with hematoxylin and the cytoplasm and the colloid in red with eosin.
Slide 46 a, 10x
138
Slide 46 b, 20x
Slide 46 c, 40x
139
11. LYSOSOMES
Lysosomes are cytoplasmic organelles representing the intracellular digestive

apparatus. At present, there is a lysosomal system in the cell which comprises several types of
vesicles, containing acid hydrolases. Lysosomes are common organelles, present in all cell
categories, being more numerous in the cells involved in the degradation of organic
compounds. The size of lysosomes varies between 0.1 - 1.2 m.
Lysosomes are vesicular structures bounded by a simple lipoprotein membrane
containing a fine granular matrix loaded with enzymes, which become active when the
organelles membrane becomes damaged. It contains over 40 types of acid hydrolases:
ribonuclease, dezoxiribonuclease, collagenase, catepsine, acid phosphatase, alpha-
glucosidase, galactosidase, glucuronidase. These enzymes are maintained in an inactive state
because of the lysosomal membranes integrity and acid pH found in the interior of the
lysosome.
Lysosomal enzymes are degradative enzyme which degrades biological molecules.
Slide 47. Lysosomes. Kupffer cells. Hepatocytes. Liver. Acid phosphatase

Slide 48. Primary lysosomes. Azurophilic granules. Neutrophil. Peripheral blood smear.
MGG Stain
Slide 47. Lysosomes. Kupffer cells. Hepatocytes. Liver. Acid phosphatase

It has been used an enzymatic reaction for acid phosphatase to show the places where
the lysosomes are located into the cells. The positive reaction is demonstrated by brown
granules dispersed in the cytoplasm of the hepatocytes and Kupffer cells. The Kupffer cells
are specialized macrophages located in the liver which line the walls of the sinusoids.They
have a phagocytic action containing high amount of lysosomes. The marker enzyme of these
organelles is acid phosphatase.
140
Slide 47 a, 10x
Slide 47 b, 20x
141
Slide 47 c, 40x
Slide 48. Primary lysosomes. Azurophilic granules. Neutrophil. Peripheral blood

smear. MGG Stain
Neutrophils are the more common leukocytes. They have a diameter of 12-15 m.
Their nucleus is divided into 2 - 5 lobes connected by a fine nuclear filament. The cytoplasm
is transparent because its granules (azurophilic granules) are small and faintly pink coloured.
They are primary lysosomes involved in phagocytosis and destruction of bacteria, in defense
processes of the body against infections.
142
Slide 48 a, 40x

Slide 48 b, 60x, immersion
143
12. MITOCHONDRIA
The mitochondria are organelles specialized in producing the necessary energy for all
cellular activities. They are considered cellular energy centrals.
The form and dimensions of the mitochondria vary. They can have an elongated
aspect of approximately 3 m or a filamentous one, up to 10 m. The more a cell is
functionally active, the more the dimensions of the mitochondria are larger. The mitochondria
of inactive cells are small.
Their volume varies in proportion to their degree of hydration. They are dynamic
organelles with a high plasticity showing conformational changes dependent on the degree of
cell differentiation, the level of nutrition or cell hydration.
The number of the mitochondria in the cell varies in relation to the functional activity.
They are numerous in functional active cells and are reduced in cells with reduced activity.
Mitochondria permanently change their position in the cell, being perinuclearly concentrated
in moments of intense activity of cell synthesis. Usually they are dispersed throughout the
cytoplasm. Cell viability is ensured by the energy provided by mitochondria. The
mitochondria may be stained by histochemical procedures that demonstrate some enzymes
such as those involved in electron transport and ATP synthesis.
Succinate dehydrogenase or succinate-coenzyme Q reductase is an enzyme complex,
bound to the inner mitochondrial membrane. It is the only enzyme that participates in both the
Krebs cycle and the electron transport chain.
High amount of mitochondria exist in active organs that need high quantity of energy
to perform their functions as the liver, the myocard and the kidney.
The following picture show sections from the liver and the myocard.
It has been used an enzymatic reaction with nitro blue tetrazolium to reveal the places
where mitochondria are located into the cells. The positive reaction is demonstrated by blue
granules dispersed in the cytoplasm. The nuclei are not highlighted.
144
Slide 49. Mitochondria. Succinate dehydrogenase. Hepatocytes. Liver. Nitro blue

tertrazolium
Slide 50. Mitochondria. Succinate dehydrogenase. Myocardial cells. Nitro blue
tertrazolium
Slide 51. Mitochondria. Nephrocytes. Kidney. Ferrical hematoxylin Stain
Slide 49. Mitochondria. Succinate dehydrogenase. Hepatocytes. Liver. Nitro blue

tertrazolium
On this slide the specific structure of the liver can be recognized. The hepatocytes are
organized in cords having an arrangement like the sun rays in relation to central vein.
The blue granules suggest the presence of the mitochondria in the cytoplasm of the
cells. The nuclei have an empty appearance.
Slide 49 a, 10x
145
Slide 49 b, 20x
Slide 49 c, 40x
146
Slide 50. Mitochondria. Succinate dehydrogenase. Myocardial cells. Nitro blue

tertrazolium
The cells that comprise cardiac muscle are called myocardiocytes and are
multinuclear. In contrast to skeletal muscle fibers, myocardiocytes may be branched instead
of linear and longitudinal. Cardiac muscle cells exhibit cross striations formed by alternating
segments of actin and myosin protein filaments. The nuclei are not visible. The blue granules
show the places where the mitochondia are located.
The myocard muscle is adapted to be highly resistant to fatigue. It has a large number
of mitochondria, enabling continuous aerobic respiration via oxidative phosphorylation,
numerous myoglobins and a good blood supply, which provides nutrients and oxygen.
Slide 50 a, 20x
147
Slide 50 b, 40x
Slide 51. Mitochondria. Nephrocytes. Kidney. Ferrical hematoxylin Stain

The mitochondria as energy providers, and numerous in active cells that need high
quantity of energy for their activity, are present also in nephrocytes, the cells of the proximal
convoluted tubules of the kidney. On this slide the mitochondria are located in the cytoplasm
of the nephrocytes. They have different shapes: rods, spheres, small filaments that may be
viewed by ferrical hematoxylin stain in black colour. The nuclei of the cells remain unstained.
148
Slide 51 a, 10x
Slide 51 b, 20x
149
Slide 51 c, 40x
150
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MARIANA CORNELIA TILINCA

MARIANA CORNELIA TILINCA

CIP nr.00703 / 10.01.2011

University Press Publisher - Trgu Mure

Mariana Cornelia Tilinca

Cell Biology Practical Works Guide

1.1. LIGHT MICROSCOPY

The optical microscope, often referred to as the "light microscope", is a type of

Cell Biology Practical Works Guide

12. condenser focus knob

Figure 1. The optical microscope

All optical microscopes share the same basic components:

Cell Biology Practical Works Guide

Figure 2. The eyepiece

Cell Biology Practical Works Guide

Figure 4. The immersion lens

Cell Biology Practical Works Guide

The tube connects the eyepiece to the objective lenses.

Figure 5. The stage with the stage clips

Cell Biology Practical Works Guide

Cell Biology Practical Works Guide

Figure 7. The light source

How to Focus Your Microscope

Cell Biology Practical Works Guide

Figure 8. The position of the slide for examination

Cell Biology Practical Works Guide

The essence of a microscope is its ability to magnify a specimen. Total magnification

Main rules regarding the examination with an optical microscope

1. Plug in the microscope

Cell Biology Practical Works Guide

1.2. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY

One of the oldest methods of preparing tissue samples is by making sections on a

Figure 9. The slide and the cover slide

Cell Biology Practical Works Guide

The Steps of the Specimen Preparation for Light Microscopy

Cell Biology Practical Works Guide

Figure 10. Fixation in formalin

Cell Biology Practical Works Guide

Figure 11. The paraffin block with the tissue sample

Cell Biology Practical Works Guide

Figure 12. A microtome for cutting wax embedded biological specimens

Figure 13. Collecting paraffin embedded slices in water bath

Cell Biology Practical Works Guide

Figure 14. The section mounted on glass slide

Figure 15. The staining battery

Cell Biology Practical Works Guide

Figure 16. Microscopic specimen

Cell Biology Practical Works Guide

1.3. ELECTRON MICROSCOPY

Cell Biology Practical Works Guide

Image 1. Golgi apparatus, secreting cell, adrenal medulla, TEM

Image 2. Rough endoplasmic reticulum, hepatocyte, liver, TEM

Cell Biology Practical Works Guide

Image 3. Mitochondrion, neuron, TEM

Image 4. Mitochondria, secreting cell, adrenal cortex, TEM

Cell Biology Practical Works Guide

Image 5. Mitochondria, hepatocyte, liver, TEM

Image 6. Mitochondria, striated muscle fiber, TEM

Cell Biology Practical Works Guide

Image 7. Primary lysosomes, nephrocyte, kidney, TEM

Image 8. Secondary lysosome, secreting cell, adrenal cortex, TEM

Cell Biology Practical Works Guide

Image 9. Peroxysome, hepatocyte, liver, TEM

Cell Biology Practical Works Guide