histology.umsa.edu.ua · 2020. 12. 31. · 1 subject histology, cytology and embryology modul №1...
TRANSCRIPT
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Subject Histology, cytology and embryology
Modul №1 Cytology, embryology and basic histology
Submodul №1 Cytology
Topic 1 THE SUBJECT OF HISTOLOGY METHODS AND
MICROSCOPY Course 1
Faculty Dental
Hours: 2
1. The topic basis: the topic “The subject of histology” is very important for future
doctors in their professional activity, positively influences the students in their
attitude to the future profession, forms professional skills and experience as well as
taking as a principle the knowledge of the subject learnt.
2. The aims of the training course:
1) To have general knowledge of the topic studied.
2) To understand, to remember and to use the knowledge received.
3) To learn the classification, structure and functions of the different
histological methods.
4) To form the professional experience by reviewing, training and
authorizing it.
5) To be able to carry out laboratory and experimental work.
3. Materials for the before – class work self – preparation work:
3.1 Basic knowledge, experience, skills necessary for studying the topic in
connection with other subjects:
To know To be able to
Med. Biology the structure of the cells and
tissues
work with a light
microscope
Med. Physics the structure of the light and
electron microscopes
work with a light
microscope
Organic Chemistry the chemical content of the cell Speak of the topic
The contents of the topic:
Medical histology applies microscopy to the human body, seeking to discover the
nature of its smaller structures, how they relate to each other, and what they do.
Thinking in histology runs along these lines.
Histology is colourful. Almost everything seen is actually there; which is not to say
that what is not seen is absent. One handles and views actual slides - the source
material for most of histology, not just someone else's selected images. The
structures can be interpreted as parts in developmental and functional sequences,
and be fitted together by satisfying accounts, for example, of how cells defend the
body. So much is now known of the roles of cells and structures that histology is
both descriptive microanatomy, and an introduction to function for the whole body.
Preparation of the Material
1. A major distinction can be drawn between dead and living preparations Dead
Section - a thin slice of tissue or organ - on a glass slide or metal grid.
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Smear on a glass slide - suitable for suspensions, e.g., blood, urine, mucus, cyst
fluid, bone marrow, etc.
Spread sheet of tissue stretched thin, e.g., areolar connective tissue.
Teased apart fibrous elements, e.g., muscle.
Living
Such preparations may be out of the body in a tissue culture system, or
living within the body but in an observable situation, e.g., a transparent
chamber inserted into the ear or skin.
The first need is to keep the preparation alive. This seriously limits the
facilities for observation. For example, staining is usually impracticable.
Thus, phase-contrast or dark-field microscopy has to be used in order to
overcome the poor contrast between natural structures.
2. Steps needed to make and study a histological section
1. Fixation to prevent post-mortem decomposition, preserve structure, and intensify subsequent staining.
2. (a) Steps involved in imbedding the tissue in a block of wax or plastic, or (b) freezing of the material to a firm mass, for cutting into thin sections on a
microtome; 1-150 microns (µm) thick for light microscopy (LM); 30-60
nanometres (nm) for electron microscopy (EM).
3. Units: based on the metre (m): micron/micrometre (µm) = 10-6m; nanometre (nm)/ millimicron (mµ) = 10-9m; Ångström (Å) = 10-10m; 10Å=1nm.
4. Mounting of the section on a glass slide or metal grid. Staining of the section with one or more reagents, e.g., solutions of metallic salts, in one or more
stages.
5. For light microscopy, the removal of surplus stain and water, and steps involved in holding a thin glass coverslip to the section with a mounting
medium having a refractive index close to that of glass.
6. Observation and recording by means of the microscope, and notes, photomicrography, projection drawing, labelled sketches, counting and
reconstructions, digital and videorecording. A drawback to using our eyes as
part of the observing instrument is that the visual system does not record
accurately.
Microscopy
1. Microscopy in general
The main distinction is between light microscopy and electron microscopy. The
usual light microscope uses a visible light source with a system of condenser lenses
to send the light through the object to be examined. The image of this object is then
magnified by two sets of lenses, the objective and the eyepiece. Total
magnification is then the product of these two lens systems, e.g., 40 X 10 = 400.
The resolution or resolving power - how close two structures can be and still be
seen as separate - is a measure of the detail that can be seen, and for the light
microscope is about 0.25 µm. This limit to resolution is determined mostly by the
wavelength of the light; and, however powerful the lens, 0.25 µm cannot be
improved upon.
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The only way to improve resolving power is to reduce substantially the wavelength
of the light. This is achieved by the electromagnetic beam of the electron
microscope. The beam is focused through the object suspended on its metal grid,
and is magnified before striking a fluorescent screen to be transformed into a
visible image.
The resolutions so far achieved in biology with transmission electron microscopy
(TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. The other
forms of microscopy - phase-contrast, interference, fluorescence, confocal
scanning, atomic-force (and X-ray diffraction) - will be discussed in , in relation to
the problems for which they are suited.
2. Microscopy for the student
1. The usual class microscope has eyepieces/oculars magnifying X 7, and an objective nosepiece carrying X 8, X 20, X 40, and X 90 (oil immersion)
lenses. Normally the three lower-power lenses are kept mounted on the
nosepiece, whilst the oil immersion objective may be mounted or kept
separately.
2. Every time it is used, the microscope should be set up to the best optical advantage. How to do this is described briefly below.
3. Keep in mind the limit to resolution. In practical terms, make special note of those structures that need an oil immersion lens to be seen or are visible only
in electron microscopy.
4. The section has some thickness, so that the fine-focusing adjustment should be used continually during observation to bring out fine detail, e.g., cilia on
cells. Essentially, though, we are getting a two-dimensional picture from an
originally three-dimensional piece of material. For what the structure looked
like in the third dimension, the student can try to reconstruct mentally what
is going on in the missing dimension, and look up views of the structure in
scanning electron microscopy.
5. Artefacts (appearances not due to the original nature of the material as
obtained from the body) can arise at all stages in the treatment of the section.
Gross examples arise from: (1) clumsy excision from the body; (2) poor or
inappropriate fixation; (3) shrinkage and, worse, uneven shrinkage, leading to
artificial spaces and distorted relations; (4) cutting scores from a bad microtome
knife; (5) the section not flat on the slide; (6) water, dirt or bubbles on or in the
section; (7) dirt on the microscope lenses; (8) patchy or faded staining;
unbalanced staining when more than one stain has been applied; (9) precipitate
from fixative or stain; (10) tears and folds in the section.
6. Setting up the microscope – see in the album. 3. Differences between light and electron microscopy
1. Table 1 gives some differences between the two approaches. The detailed morphology revealed by EM may be called fine or submicroscopic
structure/ultrastructure.
2. The direct comparison of LM and EM images of a structure requires that the magnifications be of the same order. Noting the magnification, on the 'scope
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or in the figure legend, allows one to adjust one's expectations of what may
be seen, and should always be done.
3. A growing practice in histology and pathology is to fix and prepare the tissue by EM standards, imbed in plastic and cut semi-thin (1 µm) sections
for staining by modified LM methods. LM then reveals good cellular detail
and fewer artefacts.
4. Two other techniques yield anatomical images - fibre-optic endoscopy and scanning EM - are being digested by the anatomical texts. Endoscopy from
its low magnification is marginal to histology, but related in that endoscopy
is used to obtain biopsy specimens for histopathology.
SEM strengthens one's conception of microscopic structures, e.g., cilia, renal
podocytes, bone under resorption, and effectively counters the unavoidable
impression of structures existing only in two dimensions.
Table 1. Some differences between light and electron microscopy.
Light microscopy Electron microscopy
Image is presented directly to the eye.
Image keeps the colours given the
specimen by staining.
Image is in shades of green on the screen;
photographically, only in black and
white.
Modest magnification to X 1500; but a
wider field of view and easier
orientation.
High magnification, up to X 2,000,000
thus the range of magnification is greater.
Resolving power to 0.25 µm. Resolving power to 1 nm (0.001µm.)
Frozen sections can yield an image
within 20 minutes. Processing of tissue takes a day at least.
Crude techniques of preparation
introduce many artefacts.
(Histochemical methods are better.)
High resolution and magnification
demand good fixation (e.g. by vascular
perfusion), cleanliness and careful
cutting, adding up to fewer artefacts.
Section thickness (1-30 µm) gives a
little depth for focus for appreciation of
the third dimension. Serial sections can
be cut, viewed and used to build a
composite image or representation.
Very thin sections provide no depth of
focus, but 3-D information can be had
from: (a) thicker sections by high voltage
EM; (b) shadowed replicas of fractured
surfaces; (c) scanning electron
microscopy (SEM).
Most materials and structures cannot be
stained and viewed at the same time;
stains are used selectively to give a
partial picture, e.g. a stain for mucus
counterstained to show cell nuclei.
Heavy metal staining gives a more
comprehensive picture of membranes,
granules, filaments, crystals, etc.; but this
view is incomplete and even visible
bodies can be improved by varying the
technique.
Specimen can be large and even alive.
Specimen is in vacuo. Its small size
creates more problems with sampling and
orientation.
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Dynamic nature of the cell.
The cell is not a static entity in life. Its chemical constitution and morphology are
in continuous flux. Its complement of organelles is altering, with wearing-out and
replacement, i.e., the cell is having to synthesize its own material. The cell itself
represents a system of activities isolated to partial extent from an extracellular
environment. Within the cell things are constantly being altered, moved around and
joined up within the membranes. The membranes define temporary compartments
separated from the cytoplasm, where particular activities can be confined and
controlled by enzymes attached to the extensive membrane surfaces. Dynamic
aspects of the cell's existence are partly deduced from a study of its morphology in
specimens fixed in various states, partly from microscopical observations of living
cells, and from histophysiological experiments outlined in.
➢ Biological tissues must undergo a series of treatments to be observed with light and electron microscopes. The process begins by stabilization of the tissue with
chemical fixatives. Next, the tissue is made rigid to allow sectioning. Finally, it is
stained to provide contrast for visualization in the microscope.
➢ Steps in tissue preparation 1. Fixation 2. Dehydration 3. Infiltration and embedding 4. Sectioning 5. Staining 6. Chemical Fixation
➢ Preserves cellular structure and maintains the distribution of organelles.
➢ Formaldehyde and glutaraldehyde are the most commonly used chemical fixatives. They stabilize protein by forming cross-links between primary amino
groups. Formaldehyde in solution is referred to as formalin.
➢ Osmium tetraoxide is a fixative used to preserve lipids, which aldehydes cannot do. Osmium combines with and stabilizes lipid and, in addition, also adds a brown
color (light microscopy) or electron density (electron microscopy) at the site of the
lipid. Osmium fixation is required for electron microscopy, especially to preserve
the lipid in membranes.
DEHYDRATION, INFILTRATION, AND EMBEDDING
➢ Tissue water is not miscible with the embedding solutions and must be replaced using a series of alcohols at increasingly higher concentrations. This step is
followed by alcohol replacement with an intermediate solvent that is miscible with
both alcohol and the embedding solutions.
➢ Infiltration and embedding. The liquid form of the embedding compound, for example, paraffin wax or epoxy plastic, replaces the intermediate solvent. The
liquid embedding medium is allowed to solidify, thereby providing rigidity to the
tissue for sectioning.
Sectioning
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➢ The embedded tissue is cut thin enough to allow a beam of light or electrons to pass through.
➢ Section thickness
Light microscopy. 1–20 microns
Electron microscopy. 60–100 nanometers
➢ Section planes 1. Cross-section (cs) or tranverse section (ts) is a section that passed
perpendicular to the long axis of a structure.
2. Longitudinal section (ls) is a section that passed parallel to the long axis of a structure.
3. Oblique (tangential) section is any section other than a cross- or longitudinal section.
STAINING
➢ Most tissues have no inherent contrast; thus, stains must be applied to visualize structures.
➢ Conventional staining. Relies mostly on charge interactions. 1.Light microscopy
Hematoxylin and eosin (H&E). These two dyes are the most commonly used stains in routine histology and pathology slides. Most conventional stains
bind to tissue elements based on charge interactions, that is, positive charge
attraction for a negatively charged structure. Hematoxylin binds to
negatively charged components of tissue, the most prominent being nucleic
acids. Hematoxylin imparts a purple/blue color to structures and, therefore,
the nucleus and accumulations of rough endoplasmic reticulum in the
cytoplasm, which contains large amounts of nucleic acid, appear blue or
purple in sections.
Structures, like the nucleus and rough endoplasmic reticulum that stain with hematoxylin, are referred to as basophilic or “base loving.” The term
basophilia, refers to the property of a structure or region that stains with a
basic dye, such as hematoxylin. Structures that stain with eosin, for example,
the cytoplasm of most cells and collagen fibers, appear pink or orange and
are referred to as eosinophilic.
2.Electron microscopy
Images in the electron microscope are produced by passing a beam of
electrons though the tissue that has been “stained” with salts of heavy
metals, usually lead (lead citrate) and uranium (uranyl acetate). These metals
bind to areas of negative charge and block the passage of the electrons
through the section, resulting in a dark area in the electron micrograph.
Electron density is also achieved using osmium tetroxide, which also serves
as a lipid fixative.
Areas or structures in tissue that bind the metals are referred to as electron dense. Areas where the metals do not bind appear light and are referred to
electron lucent.
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➢ Histochemical staining. Localizes chemical groups
Osmium tetroxide. Stains lipids
Periodic acid–Schiff stain (PAS). Stains carbohydrates
Staining Reactions
Staining reactions have both physical and chemical characteristics. The
mechanisms involved in staining include the following:
The dye may actually be dissolved in the stained substance. Most fat staining is
accomplished in this fashion. A dye may be absorbed on the surface of a structure,
or dyes may be precipitated within the structure, simply because environmental
factors (pH, ionic strength, temperature, etc.) favor absorption or precipitation.
Most staining reactions involve a chemical union between dye and stained
substance through salt linkages, hydrogen bonds, or others. Staining with these
dyes results in a predictable color pattern based in part on the acid base
characteristics of the tissue. However, color and color distribution are not
absolutely reliable for discrimination between tissue components. Color will vary
not only with specific stains used, but also with the conditions that exist during
preparation of the slide. These include everything from the initial fixing solution to
the ionic strength of the staining solution and the differentiating solvents utilized
after staining.
Acid and Basic Dyes
Most histologic dyes are classified either as acid or as basic dyes. An acid dye
exists as an anion (negatively charged) in solution, while a basic dye exists as a
cation (positive charge). For instance, in the hematoxylin-eosin stain (H&E), the
hematoxylin-metal complex acts as a basic dye. The eosin acts as an acid dye.
Any substance that is stained by the basic dye is considered to be basophilic; it
carries acid groups which bind the basic dye through salt linkages. When using
hematoxylin, basophilic structures in the tissue appear blue (or purple or brown;
this varies according to the stain that is being used). A substance that is stained by
an acid dye is referred to as acidophilic; it carries basic groups which bind the acid
dye. With eosin, acidophilic structures appear in various shades of pink. Since
eosin is a widely used acid dye, acidophilic substances are frequently referred to as
eosinophilic.
Trichrome Stains
In the trichrome stains, which commonly employ more than one acid dye, use is
made of dye competition. For instance, acid fuchsin and picric acid are used in Van
Gieson's trichrome stain. In the picric acid-fuchsin mixture, the small picric acid
molecule reaches and stains the available sites in muscle before the larger fuchsin
molecules can enter. Used by itself, acid fuchsin has no difficulty in staining
muscle.
Neutral Stains
These are compounds of an acid dye and basic dye. For instance, aqueous
solutions of acid fuchsin may be neutralized by addition of aqueous methyl green.
The resulting neutral product is water insoluble, but may be kept in solution by the
presence of excess amounts of either component. The tissue stained with such a
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solution may show affinity for the acid dye, the basic dye, and for the whole
compound. Some blood stains are "neutral stains." Wright's Stain, for instance, is
formed by the combination of partially oxidized and demethylated methylene blue
and eosin. Such a stain can be used to differentiate between blood cells that contain
acidic, basic, and neutral granules.
Hematoxyl and Eosin (H&E)
This is a good general stain and is widely used. Most of your slides are stained
with H&E. A hematoxylin-metal complex acts a as a basic dye, staining nucleic
acids in the nucleus and the cytoplasm blue, brown, or black. Eosin is an acid
aniline dye which stains the more basic proteins within cells (cytoplasm) and in
extracellular spaces (collagen) pink to red. Cartilage and mucus may stain light
blue.
Masson Trichrome Stain
A staining sequence involving iron hematoxylin, acid fuchsin, and light green. It is
a good stain for distinguishing cellular from extracellular components. Collagen
fibers stain an intense green. Black or brown nuclei; mucus and ground substances
take on varying shades of green. Cytoplasm stains red. Elastic fibrils, red blood
cells and nucleoli stain pink.
Aldehyde Fuchsin
Stains elastic fibers purple to black. Can be counter-stained with a dye of
contrasting color, such as metanil yellow.
Verhoeff's Hematoxylin
Another variant of the versatile hematoxylin stains. This method stains elastic
fibers black in addition to nuclei.
Reticular Fiber Stain – Weigert
Reticular fibers are impregnated with a silver salt and appear as sharp black.
Collagenous fibers usually stain purple. This stain can be used with a counterstain
or without, if the silver stain turned out very dark.
Wright's/Giemsa Stain
This and similar stains for blood and bone marrow smears are mixtures of basic
(methylene blue derivatives) and acid dyes (usually eosin). According to the
number of acid and basic groups present, cell components take up the dyes from
the mixture in various proportions. Some blood stains use acid and basic dyes in
separate dye baths.
Metachromatic Stain
Certain basic dyes, such as toluidine blue, stain nucleic acids blue (the
orthochromatic color), but sulfated polysaccharides purple (the metachromatic
color). When dye molecules bound to sulfate groups are stacked closely together,
the dye experiences a color shift from blue to purple. Thus, a metachromatic
reaction often indicates the presence of numerous closely packed sulfate groups.
Plastic Sections Stained with Toluidine Blue or with H&E
Plastic embedded tissues can be cut as thin as 0.1 um with a glass knife. These
sections are then stained with toluidine blue in an alkaline solution. Almost all
tissue components are stained more or less deeply (usually a bluish-purple) and
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structural detail is very sharp. For the knowledgeable observer, this type of
preparation may be very informative.
Periodic Acid Schiff (PAS)
Adjacent hydroxyl groups (1, 2 glycols) or amino and hydroxyl groups are
oxidized to aldehyde groups with periodic acid. Schiff's Reagent then produces a
red or magenta addition product with the aldehyde groups and this technique
identifies a number of polysaccharides and carbohydrate-containing compounds.
The slide may also be counter stained with hematoxylin. Feulgen Reaction: Mild
hydrolysis with HCl frees the aldehyde group of deoxyribose, which is then reacted
with the Schiff's reagent. This reaction is highly specific for DNA and may also be
used with a counter stain for the cytoplasm.
➢ Immunocytochemistry. Localization of specific antigens in cells using labeled antibodies
➢ In situ hybridization. Detection of messenger RNA or genomic DNA sequences using labeled nucleotide probes
ARTIFACT
➢ The term artifact is used to refer to any feature of a tissue section that is present as a result of the tissue processing. These include tears and folds, shrinkage, spaces
resulting from extracted cellular contents (e.g., lipid, precipitates), and
redistributed organelles.
MICROSCOPY
➢ Properties 1.Resolution is the smallest degree of separation at which two objects can still be
distinguished as separate objects and is based on the wavelength of the
illumination.
Light microscopy. Approximately 200nm
Electron microscopy. Approximately 1nm 2.Magnification. Enlargement of the image
➢ Bright field microscope
An image is formed by passing a beam of light through the specimen and then focusing the beam using glass lenses.
The bright field microscope is called a compound microscope because it uses two lenses, objective and ocular, to form and magnify the image. The
compound microscope typically has a total magnification range of 40–1000
times.
➢ Electron microscope 1.Transmission electron microscope (TEM)
An image is formed by passing a beam of electrons through the specimen and focusing the beam using electromagnetic lenses.
Similar arrangement of lenses is used as with optical microscopy; magnification is up to 400,000 times, which is sufficient to visualize
macromolecules (e.g., antibodies and DNA).
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2.Scanning electron microscope (SEM). The image is formed by electrons that are
reflected off the surface of a specimen, providing a three-dimensional image;
magnification ranges from 1–1000 times.
➢ Freeze fracture technique
This technique is used to examine the number, size, and distribution of membrane proteins.
A tissue is frozen and mechanically fractured; the exposed membrane surface is coated with a thin metal film called a “replica.”
The replica is viewed by TEM. Membrane proteins appear either as bumps or pits in the replica.
SECTION INTERPRETATION
➢ In histology, three-dimensional tissues are viewed in two dimensions; therefore, it is extremely important to learn to visualize the threedimensional structure from
the two-dimensional image. For example, a cross-section through a tubular
structure appears as a ring, whereas a longitudinal section appears as two parallel
bands. As an added challenge, most sections pass obliquely to these perpendicular
axes and, thus, require further “mental gymnastics.”
UNITS OF MEASURE
➢ Millimeter (mm) = 1/1000 meter, 10-3M
➢ Micron, micrometer (mm) = 1/1000mm, 10-3mm, 10-6M
➢ Nanometer (nm) = 1/1000mm, 10-3mm, 10-9M
➢ Еngstrцm unit (Е) = 1/10nm, 10-10M Cytological description of an individual cell.
In light microscopy involves: (1) relative and absolute size; (2) shape; (3) number
of nuclei; (4) shape and size of nucleus/nuclei; (5) intensity of nuclear staining; (6)
amount of cytoplasm; (7) staining affinity of cytoplasm, e.g., basophilic,
acidophilic (eosin), argentophilic (silver stains), or chromophobe (liking no stain);
(8) granular cytoplasm; (9) nature of any inclusions, for instance, melanin pigment,
fat, carbon, bacteria, zymogen granules, glycogen, mucus; (10) specializations of
the cell membrane, e.g., cilia, a brush/striated border (many microvilli); (11)
distinctive organelles in cytoplasm and their position, e.g., prominent Golgi
complex, many fibrils, numerous orderly mitochondria giving another striated
effect, Nissl substance (GER) in nerve cells; (12) whether the cell is in some phase
of mitosis or meiosis; (13) the cell's surroundings; (14) manifest properties of the
living cell, e.g., motility, phagocytosis, contractility.
3.3. Literature recommended
Main Sources:
1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004. 3. “Color Atlas of basic histology” – R.Berns – 2006. 4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997.
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7. Ten Cate A.R. Oral Histology.- St.Louis, Baltimore, Toronto.- 1995. 8. William A., Beresford M.A. Cytology and Histology.- Anatomy department,
West Virginia University, USA.- 1992.
9. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington –
1991.
Additional ones:
1. Methodical Instructions.
3.4 How to work with the literature recommended:
Main tasks Recommendations
To review the material
To learn the material
To read and compose the plan
To answer the questions
To do the test on the material
To be ready to answer the topic
To use the material studied
To use the material on pages
To learn the new material and be
ready to write a summary
To be ready to give an answer to
the following:
3.5. Self-control material:
A. Questions to be answered:
1. Technique of light microscopy. 2. Special methods of light microscopy. 3. Trasmission and sweepable electronic microscopy. 4. Methods of research of living cages and fabrics Intravital painting. 5. Research of living cells and fabrics is in a culture (in vitro). 6. Research of separate cells and their cultivation. 7. Іmmunofluorescent, methods of radioautographic research. 8. Modern methods of study of cellular content. 9. Quantitative methods of research. 10. Basic principles of making of preparations for a light microscopy. 11. Fixing. Types of fixings. Method of making of cuts. 12. Dehydration, compression and inundation. 13. Colouring and contrasting. 14. Classification of histological dyes to on by chemical properties 15. Conclusion of histological preparations 16. Types of microslides are a cut, stroke, imprint, tapes, microsection. 17. Methods of analysis of image of cellular and tissue structures 18. Prizhiznennye methods of research. 19. Intravital and supravital painting. B. Test tasks to be done: Tests are applied
4. Self-preparation in the classroom.
1) Listen to the information.
2) Work with the tables and a Light microscope.
3) Ask about the problems that haven’t been found in the information given.
4) To sketch in the album the investigated preparations.
5. Self-preparation work at home.
1) Review the material learnt in the classroom.
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2) Compose the plan of your answer.
3) Answer the questions to this topic.
4) Do the test given above.
6. The subject of the research work.
- “The History of Histology”. - “New methods in histological research”.
Subject Histology, cytology and embryology
Modul №1 Cytology, embryology and basic histology
Submodul №1 Cytology
Topic 2 CELLS. PLASMALEMMA. CELL JUNCTIONS
Course 1
Faculty Dental
Hours: 2
1. The topic basis: the topic “Plasmalemma. Cell junctions.” is very important for
future doctors in their professional activity, positively influences the students in
their attitude to the future profession, forms professional skills and experience as
well as taking as a principle the knowledge of the subject learnt.
2. The aims of the training course:
1) To have general knowledge of the topic studied.
2) To understand, to remember and to use the knowledge received.
3) To learn the classification, structure, functions of cell’s surface.
4) To form the professional experience by reviewing, training and
authorizing it.
5) To be able to carry out laboratory and experimental work.
3. Materials for the before – class work self – preparation work:
3.1 Basic knowledge, experience, skills necessary for studying the topic in
connection with other subjects:
To know To be able to
Anatomy - -
Histology - -
Med. Biology the structure and functions
of cell’s surface
work with the light
microscope
Med. Physics the structure of the light and
electron microscopes
work with the light
microscope
Organic Chemistry Chemical content of the cell Speak on the subject
3.2.The contents of the topic:
1. Body components
The human body content of the cells, tissues, organs and organ’s systems. These
are cells, extracellular substances, and body fluids. Fluids can have their suspended
solid constituents viewed microscopically as smear preparations (see Blood), but
are otherwise of limited histological interest. Extracellular substances are
important for the cells whose environment they form: they reflect and help control
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cellular activities, aside from their critical structural mechanical properties.
Individual materials can be seen and localised by histochemistry.
2. Cells: chemical constitution and fixation
1. Composition: much water; proteins, nucleic acids, lipids, carbohydrates,
amino acids, minerals, hormones, vitamins, etc.
2. Fixation stabilizes mainly proteins, and protein conjugates. These substances are used as building materials for the firmer structures of the cell. Lipids,
minerals, glucose, and smaller molecules are usually lost from the section
during processing. What is left is a skeleton of structures - membranes,
granules, filaments - made up of proteins, polypeptides, polysaccharides and
some other macromolecular materials. The special steps of histo- and
cytochemistry serve and reveal some smaller molecules.
3. Cells: living properties and specialization
1. Properties of cells: (a) general - communication, respiration and energy storage and release, synthesis, excretion, growth, differentiation,
reproduction; (b) specialized - irritability to stimuli (excitability), motility,
contractility, conductivity, absorption, phagocytosis, secretion.
2. During development from the fertilized oocyte, a great variety of cells is formed in the mammal, each kind specializing in a certain function, e.g.,
secretion, but many activities, such as energy production, are common to all
cells. The cells of the four primary tissues - epithelial, connective, nervous
and muscular - are divided along lines of specialized function, e.g., muscle
for contractility and excitability.
4. Cell morphology
1. Cells performing a given function have a characteristic size, form and fine structure adapted to that task. However it may help at this stage to think in
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terms of a composite cell having all the features the various cells of the body
display.
2. The cell is defined as a distinct entity by having a thin skin or plasmalemma/cell membrane separating off from the outside a soft, viscous,
almost fluid cytoplasm, in which are suspended a number of firmer,
recognizable structures - organelles and inclusions - and one or more nuclei.
The nucleus, likewise, is a mass of material enclosed in nuclear membranes.
5. Cell components – cytoplasm, plasmalemma and nucleus.
1.Cytoplasm. It consist of organelles, includings and hyaloplasm - the so-called
soluble phase of the cell, consisting mostly of water, dissolved solutes, and
larger molecules in suspension tending to link repetitively with covalent bonds
giving the cytoplasm a dense, viscous colloidal sol or gel consistency.
2.Cell or plasma membrane/plasmalemma
o Made of unit membrane roughly 7.5 nm wide; in the EM picture with
appropriate fixation it has a trilaminar appearance of two dark bands with a
light layer betwen them; the light layer is mostly non-polar lipid, the dark
layers mostly the charged ends of the lipid molecules with attached protein.
o A coat of glycoproteins (sugars+protein) - glycocalyx - adheres to the
outside of most cells – undermembranous complex.
o The plasma membrane is flexible, semi-permeable, and experiences active
transport and potential differences across it.
o By the cell membrane's fusing with the intracellular membrane around
stored material, then breaking open at a point along the line of fusion the
material can be released to outside the cell - exocytosis/emiocytosis. By
bringing extracellular things into an invagination, followed by selective
membrane fusion and separation, materials are brought into the cell -
endocytosis: phagocytosis for solids, pinocytosis for fluids; endocytosis
includes these bulk movements, but also signifies the internalization of
membrane receptors and bound ligands on a smaller scale.
o Specializations of form shown by the plasma membrane include: microvilli,
cilia with basal bodies, (iii) pinocytotic vesicles/pinosomes/caveolae
intracellulares, infoldings or plications, desmosomes, gap junctions/nexuses,
occluding junctions, stereocilia (long microvilli).
o Some vesicles involved in transport into the cell have prominent coats of
clathrin attached to the membrane.
o Proteins of internal surface of the plasmalemma form the
cytoskeleton(submembranous complex)
3.Cell nucleus consist of Nuclear membrane, Chromatin, Nucleoplasma and
Nucleola.
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15
6. Cell Membranes
I. Functions of the membrane are:
1. Separation the cell from the external enviropment. 2. Firm attachment to other cells or a basal lamina; membrane specializations
for this are: junctional complexes, gap junctions/ nexuses, desmosomes,
hemidesmosomes, intercalated discs, and membrane interdigitations.
3. Transport of materials in and out of the cell served by: permeability (selective) of the membrane, active transport through the membrane,
endocytosis, and its more scaled up forms - pinocytosis and phagocytosis,
exocytosis; and increased exchange surface area by microvilli (thousands on
a cell), and infoldings of membrane.
4. Movement of the cell itself by pseudopodial, filipodial, or lamellipodial extensions (think karate: fist, finger, or side of the hand) and the release of
any firm attachments, or by flagellate activity, e.g., by sperm. (Microspikes
and ruffles are alternative names for filopodia and lamellipodia,
respectively.)
5. Movement of materials outside the cell by the activity of cilia, e.g., ciliated epithelia of the respiratory tract and uterine tube. The wide-spread
occurrence of solitary cilia (flagella), e.g., on neurons, adrenal cells, smooth
muscle, may involve a vestigial body or one still functional. The stereocilia
of the male reproductive tract are non-motile, clumped, long microvilli,
probably absorptive.
6. Communication and transduction. Each cell collaborates with both adjacent cells, and those of the whole body, for development, growth,
homeostasis, regeneration, and its own particular task. The importance of the
cell membrane in receiving and sending the necessary signals is stressed by
the number of examples given:
The binding of hormones to receptors on the membrane.
The binding of the lymphocyte's membrane receptor to an antigen.
Transmitter chemicals depolarize neurons and muscle cells.
Excitable tissues propagate action potentials along the membranes.
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16
Schwann cells wrap their membranes many times round an axon's to make
myelin sheath segments for faster signalling.
Chemical stimuli are transduced into nerve impulses in chemoreceptors;
mechanical stimuli in mechanoreceptors.
Gap junctions permit ions and excitation to spread from cell to cell, and
unify and synchronise actions of many cells/cell assemblies.
In development, epithelial and mesenchymal cells interact in sequence to
induce cell differentiations, e.g., in tooth and glands.
Cells attract and fuse with one another to form multinucleated cells, e.g.,
skeletal muscle and osteoclasts.
Chemotactic agents act on phagocytic cells to attract them to their targets.
Keratinocytes of the skin phagocytose melanin pigment offered to them in
the processes of melanocytes.
Macrophages detect spent or abnormal red blood cells, and hold and engulf
either the whole cell, or the part holding an unwanted body.
II. Molecules Wherever such actions are described, special molecules are acting,
by binding to each other, changing their conformation, or some other means.
o Spectrin/fodrin provides a subplasmalemmal skeleton attached to the cell
membrane by ankyrin, and to actin of the cytoskeleton, to permit control of the
membrane's shape and movement.
o Cell adhesion molecules (CAMs) allow cells to attach to only certain cell
types.
o Integrins are cell-surface-membrane dimeric molecules (an alpha with a
beta), by which cells choose to which extracellular matrix (ECM) components
they wish to fasten, e.g., laminin.
o Connexins are proteins that combine as hexamers to form connexons - the
gap-junction channels, allowing ions and small molecules to pass between
cells. Connexins and the transports allowed vary among liver cells, neurons.
o Occludins are responsible for the seal preventing passage of materials past
inter-epithelial tight junctions.
7. Basic Membrane structure
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17
The proteins of the membrane are: integrals, semi-integrals (external and internal)
and peripheral (external and internal). o They may form are esssential part of the structure of the membrane
i.e., they may be structural proteins. o Some proteins play a vital role in transport across the membrane and
act as pumps. Ions get attached to the protein on one surface and
move with the protein to the other surface. o Some proteins are so shaped that they form passive channels through
which substances can diffuse through the membrane. However, these
channels can be closed by a change in the shape of the protein. o Other proteins act as receptors for specific hormones or
neurotransmitters. o Some proteins act as enzymes.
8. Contacts between adjoining cells In tissues in which cells are closely packed the cell membranes of adjoining cells
are separated, over most of their extent by a narrow space (about 20 nm). This
contact is sufficient to bind cells loosely together, and also allows some degree of
movement of individual cells. In some regions the cell membranes of adjoining
cells come into more intimate contact: these areas are marked by structural
specializations as described below.
Zonula Occludens At such a junction the two plasma membranes are in actual contact. These
junctions act as barriers that prevent the movement of molecules into the
intercellular spaces. For example, intestinal contents are prevented by them from
permeating into the intercellular spaces between the lining cells. Zonulae
occludens are, therefore, also called tight junctions. Apart from epithelial cells,
zonulae occludens are also present between endothelial cells. In some situations
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18
occlusion of the gaps between the adjoining cells maybe incomplete and the junc-
tion may allow slow diffusion of molecules across it. These are referred to as leaky
tight junctions.
Desmosomes (Maculae Adherens)
This is the most common type of junction between adjoining cells. A desmosome
is a small circumscribed area of attachment. At the site of a desmosome the plasma
membrane (of the cell) is thickened because of the presence of a dense layer of
protein on its inner surface (i.e., the surface towards the cytoplasm). The thickened
areas of the two sides are separated by a gap of 20 nm or more. The region of the
gap is rich in a glycoprotein called desmoglea. The thickened areas of the two
membranes are held together by fibrils that appear to pass from one membrane to
the other across the gap. Closer examination shows, however, that the fibrils do not
pass from one cell to the other. Instead the fibrils of each side are in the form of
loops: the loops of the opposing membranes interlock. The cytoplasmic aspect of
the thickened areas of the cell membrane also gives attachment to numerous fibrils
that pass into the cytoplasm. Desmosomes are present where strong anchorage
between cells is needed. Zonula Adherens In some situations, most typically near
the apices of epithelial cells, we see a kind of junction called the zonula adherens.
This is similar to a desmosome in being marked by thickenings of the two plasma
membranes, to the cytoplasmic aspects of which fibrils are attached. However, the
junction differs from a desmosome in that instead of being a small circumscribed
area of attachment the junction is in the form of a continuous band around the
apical part of the epithelial cell; and in that the gap between the thickenings of the
plasma membranes of the two cells is not traversed by filaments. An adhesive
material is probably present in this situation. Apart from epithelial cells zonulae
adherens are also seen between smooth muscle cells, and between myocytes of
cardiac muscle in the region of intercalated discs. Gap Junctions (Nexuses) At
these junctions the plasma membranes are not in actual contact (as in a tight
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19
junction), but lie very close to each other, the gap being reduced (from the normal
20 nm) to 3 nm. Placed in this gap there are bead-like structures arranged in the
form of hexagons. A minute canaliculus passing through each 'bead' connects the
cytoplasm of the two cells thus allowing the free passage of substances from one
cell to the other. Gap junctions are, therefore, also called maculae comnumicantes.
They are widely distributed in the body. Junctional Complex Near the apices of
epithelial cells the three types of junctions described above, namely zonula oc-
cludens, zonula adherens and macula adherens are often seen arranged in that
order. They collectively form a junctional complex.
Stem cells.
For a stable population, the corollary to cell death is cell renewal. This requires:
o the proliferation of cells;
o an enduring population of stem cells;
o controls (+ & -) that promote division of stem cells to maintain their
numbers - self-replication;
o controls that cause differentiation of certain of the stem cells to
become the determined/committed precursors of the mature cells of
the tissue;
o factors to promote division of the precursors/progenitors and their
further differentiation. The controlling factors include cytokines.
More is known about the ensuing progenitor cells than about the stem cells.
Although not essential to the concept of stem cells, at step (iv) above, stem
cells usually give rise to more than one lineage of differentiated cells, in
order to furnish the needed diversity of cell types in blood and most
epithelia. 3.3. Literature recommended
Main Sources:
1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004.
3. “Color Atlas of basic histology” – R.Berns – 2006. 4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997. 7. William A., Beresford M.A. Cytology and Histology.- Anatomy department,
West Virginia University, USA.- 1992.
8. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington – 1991.
Additional ones:
1. Methodical Instructions.
3.4. How to work with the literature recommended:
Main tasks Recommendations
To review the material
To learn the material
To read and compose the plan
To use the material studied
To use the material on pages
To learn the new material and be
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20
To answer the questions
To do the test on the material
To be ready to answer the topic
ready to write a summary
To be ready to give an answer to
the following:
3.5. Self-control material:
A .Questions to be answered:
1.What is the cell?
2. What basic functions of cells do you know?
3. Name the basic components of a cell.
4. What are the basic functions of plasmalemma?
5. What specific membrane proteins distinguish?
6. How is the elementary biological membrane constructed?
7. What are the contacts between adjoining cells?
8. Describe a structure of zonula occludense?
9. Describe a structure of desmosomes (maculae adherens) and zonula adherens.
10. What is nexus?
11. What is the junctional complex?
B. Test tasks to be done: See enclosure.
4. Self-preparation in the classroom.
1) Listen to the information.
2) Work with the tables and microscope.
3) Ask about the problems that haven’t been found in the information given.
4) To sketch in the album the investigated preparations.
5. Self-preparation work at home.
1) Review the material learnt in the classroom.
2) Compose the plan of your answer.
3) Answer the questions to this topic.
4) Do the test given above.
6. The subject of the research work.
- “The active and passive transport through a cellular wall”. - “The role of intercellular contacts in the preservation of homeostasis”.
Subject Histology, cytology and embryology
Modul №1 Cytology, embryology and basic histology
Submodul №1 Cytology
Topic 3 ORGANELLES AND INCLUSION
Course 1
Faculty Dental
Hours: 2
1. The topic basis: the topic “Organelles and Inclusion.” is very important for
future doctors in their professional activity, positively influences the students in
their attitude to the future profession, forms professional skills and experience as
well as taking as a principle the knowledge of the subject learnt.
2. The aims of the training course:
1) To have general knowledge of the topic studied.
2) To understand, to remember and to use the knowledge received.
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21
3) To learn the classification, structure and functions of organelles
and inclusion.
4) To form the professional experience by reviewing, training and
authorizing it.
5) To be able to carry out laboratory and experimental work.
3. Materials for the before – class work self – preparation work:
3.1 Basic knowledge, experience, skills necessary for studying the topic in
connection with other subjects:
To know To be able to
Med. Biology the structure of the cells and tissues work with a light
microscope
Med. Physics the structure of the light and electron
microscopes
work with a light
microscope
Organic Chemistry the chemical content of the cell Speak of the topic
3.2.The contents of the topic:
The cytoplasm of a typical eukaryotic cell contains various structures that
are referred to as organelles. They include the membrane-bound and the non-
membranous organelles. Other components of cytoplasm are gialoplasm and
inclusions.
Mitochondria Mitochondria can be seen with the light microscope in specially stained
preparations. They are so called because they appear either as granules or as rods
(mitos = granule; chondrium = rod). The number of mitochondria varies from cell
to cell being greatest in cells with high metabolic activity (e.g., in secretory cells).
Mitochondria vary in size, most of them being 0.5 to 2μm in length.
The mitochondrion is bounded by a smooth outer membrane within which
there is an inner membrane. The inner membrane is highly folded on itself forming
incomplete partitions called cristae. The space bounded by the inner membrane is
filled by a granular material called the matrix. This matrix contains numerous
enzymes. It also contains some RNA and DNA: these are believed to carry
information that enables mitochondria to duplicate themselves during cell division.
An interesting fact, discovered recently, is that all mitochondria are derived from
those in the fertilized ovum, and are entirely of maternal origin.
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22
Mitochondria are of great functional importance. They contain many
enzymes including some that play an important part in Kreb's cycle. ATP and GTP
are formed in mitochondria from where they pass to other parts of the cell and
provide energy for various cellular functions. These facts can be correlated with
the observation that within cell mitochondria tend to concentrate in regions where
energy requirements are greatest. The enzymes of Kreb's cycle are located in the
matrix, while enzymes associated with the cytochrome system are present on the
inner mitochondrial membrane. Mitochondria are also concerned with fatty acid
metabolism, and various other chemical reactions. Endoplasmic Reticulum
The cytoplasm of most cells content with com of membranes that
constitute the endoplasmic reticulum. The membranes form the boundaries of
channels that may be arranged in the form of flattened sacs (or cistern) or of
tubules. Because of the presence of the endoplasmic reticulum the cytoplasm is
divided into two components, one within the channels and one outside them.
In most places the membranes forming the endoplasmic reticulum are studded with
minute particles of RNA called ribosomes. The presence of these ribosomes gives
the membrane a rough appearance. Membranes of this type form what is called the
rough (or granular) endoplasmic reticulum. In contrast some membranes are
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23
devoid of ribosomes and constitute the smooth or agranular endoplasmic reticulum. Rough endoplasmic reticulum represents the site at which proteins are
synthesized. The attached ribosomes play an important role in this process. Smooth
endoplasmic reliculum is associated with numerous biochemical processes in-
cluding carbohydrate metabolism. Products synthesized by the endoplasmic
reticulum are stored in the channels within the reticulum. Ribosomes, and
enzymes, are present on the 'outer' surfaces of the membranes of the reticulum.
Golgi Complex The Golgi complex (or Golgi apparatus) can be seen as a small structure of
irregular shape, usually present near the nucleus. When examined with the EM the
complex is seen to be made up of membranes similar to those of smooth
endoplasmic reticulum. The membranes form the walls of a number of flattened
sacs that are stacked over one another. Towards their margins the sacs are
continuous with small rounded vesicles. The Golgi complex is intimately
connected with the formation of several secretory products, specially those
containing carbohydrates.
The protein component of these products is synthesized in rough
endoplasmic reticulum. As the proteins pass through successive sacs of the Golgi
complex they undergo a process of purification. In the Golgi complex
carbohydrates are added to the proteins to form protein-carbohydrate complexes.
These complexes are formed within the cisternae of the Golgi apparatus. They pass
to the margins of the cisternae where they separate from the Golgi complex
forming membrane bound secretory vacuoles.The membranes of the Golgi
complex give attachment to enzymes associated with carbohydrate synthesis.
Lysosomes may also be produced in the Golgi complex. Lysosomes
These vesicles (primary, secondary, rest bodies and autophagosomes)
contain enzymes that can destroy unwanted material present within a cell. Such
material may have been taken into the cell from outside (e.g., bacteria); or may
represent organelles that are no longer of use to the cell. The enzymes present in
lysosomcs include proteases, Lipases, curbohydrases, and acid phosphatase. (As
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24
many as 40 different lysosomal enzymes have been identify. Passing along the
channel of the reticulum they reach the Golgi complex. Here the enzymes come to
be surrounded by membranes and are set free into the cyloplasm in the form of
vesicles that bud off from marginal areas of the Golgi complex.
Lysosomes help in 'digesting' the material within phagosomes (described
above) as follows. A lysosome, containing appropriate enzymes, fasts with the
phagosome so that the enzymes of the former can act on the material within the
phagosome. These bodies consisting of fused phagosomes and lysosomes arc
refried to a secondary lysosomes or phagolysosomes. In a similar manner lysosomes may also fuse with pinocytotic vesicles. The
structures formed by such fusion often appear to have numerous small vesicles
within them and called multivesicular bodies. After the material in phagosomes or pinocytotic vesicles has been 'digested'
by lysosomes, some waste material may be left. Some of it is thrown out of the cell
by exocytosis. However, some material may remain within the cell in the form of
membrane bound residual bodies. Lysosomal enzymes play an important role in
the destruction of bacteria phagocytosed by the cell. Lysosomal enzymes may also
be discharged out of the cell and may influence adjoining structures. Lysosomes
are present in all cells except mature erythrocytes. They are a prominent feature in
neutrophil leucocytes.
Ribosomes We have seen above that ribosomes are present in relation to rough
endoplasmic reticulum. They may also lie free in the cytoplasm. They may be
present singly in which case they are called monosomes; or in groups which are
referred to as potyribosomes (or polysomes). Each ribosome consists of proteins
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25
and RNA (ribonucleic acid) and is about 15 nm in diameter. The ribosome is made
up of two subunits one of which is larger than the other. Ribosomes play an
essential role in protein synthesis.
Microfilaments & Microtubules
The cytoplasm of many varieties of cells contains thin elongated elements.
Some of these are tubular, and are circular in cross section: they are called
microtubules. Others, called micro filaments, are solid fibres. These elements can
be made out in light microscopic preparations of dividing cells in which they form
the mitotic spindle. With the EM they can be identified in many other cells and the
distinction between tubule and filaments can also be made out. Microtubules and
microfilaments (along with some other filaments present in the cytoplasm)
constitute the cytoskeleton.
Both microtubules and microfilaments are made up of proteins. The proteins
forming microtubules are called tubulins. Microfilaments are usually composed of
a protein called actin, but in some situations (e.g., in neurons) they may be
composed of other proteins. A microtubule is about 24 nm in diameter. A microfilament is 6-8 nm in
diameter. Intermediate filaments about 10 nm in diameter are found in many cells
where structural strength is required: these include nerve cells (in which they are
seen as neurofilaments), epithelial cells and neuroglial cells.
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26
Centrioles
All cells capable of division (and even some which do not divide) contain a
pair of structures called centrioles. With the light microscope the two centrioles are
seen as dots embedded in a region of dense cytoplasm which is called the
centrosome. With the EM the centrioles are seen to be short cylinders that lie at
right angles to each other. When we examine a transverse section across a
centrioles (by EM) it is seen to consist essentially of a series of microtubules
arranged in a circle. There are nine groups of tubules each group consisting of
three tubules. Centrioles play an important role in the formation of various cellular
structures that are made up of microtubules. These include the mitotic spindles of
dividing cells, cilia, flagella, and some projections of specialized cells (e.g., the
axial filaments of spermatozoa). It is of interest to note that cilia, flagella and the
tails of spermatozoa all have the 9+2 configuration of microtubules that are seen in
centrioles.
SPETIAL ORGANELLES Many cells show projections from the cell surface. The various types of
projections are described below. Cilia These can be seen, with the light
microscope, as minute hair-like projections from the free surfaces of some
epithelial cells. In the living animal cilia can be seen to be motile. Details of their
structure, described below, can be made out only by EM. The free part of each
cilium is called the shaft. The region of attachment of the shaft to the cell surface is
called the base (also called the basal body, basal granule, or kinetosome). The free
end of the shaft tapers to a tip. In structure the cilium consists of an outer covering
which is formed by an extension of the cell membrane; and an inner core that is
formed by microtubules arranged in a definite manner. It has a striking similarity
to the structure of a centriole (described above). There is a central pair of tubules
which is surrounded by nine pairs of tubules. The outer tubules are connected to
the inner pair by radial structures (which are like the spokes of a wheel). Other
projections pass outwards from the outer tubules. As the tubules of the shaft are
traced towards the tip of the cilium it is seen that one tubule of each outer pair ends
short of the tip so that near the tip each outer pair is represented by one tubule
only. Just near the tip, only the central pair of tubules is seen. At the base of the
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27
cilium one additional tubule is added to each outer pair so that here the nine outer
groups of tubules have three tubules each, exactly as in the centriole. Functional
significance of cilia The cilia lining an epithelial surface move in coordination
with one another the total effect being that like a wave. As a result fluid, mucous,
or small solid objects lying on the epithelium can be caused to move in a specific
direction. Movements of cilia lining the respiratory epithelium help to move
secretions in the trachea and bronchi towards the pharynx. Ciliary action helps in
the movement of ova through the uterine tube, and of spermatozoa through the
male genital tract. In some situations there are cilia-like structures that perform a
sensory function. They may be non-motile, but can be bent by external influences.
Such 'cilia' present on the cells in the olfactory mucosa of the nose are called
olfactory cilia: they are receptors for smell. Similar structures called kinocilia are
present in some parts of the internal ear. In some regions there are hair-like
projections called stereocilia: these are not cilia at ah1, but are large microvilli.
Flagella These are somewhat larger processes having the same basic structure as
cilia. In the human body the best example of a flagellum is the tail of the sper-
matozoon. The movements of flagella are different from those of cilia. In a
flagellum movement starts at its base. The segment nearest the base bends in one
direction. This is followed by bending of succeeding segments in opposite
directions so that a wave like motion passes down the flagellum. When a
spermatozoon is suspended in a fluid medium this wave of movement propels the
spermatozoon forwards (exactly in the way a snake moves forwards by a wavy
movement of its body). Microvilli These are finger-like projections from the cell
surface that can be seen only with the EM. Each microvillus consists of an outer
covering of plasma membrane and a cytoplasmic core in which there are numerous
microfilaments. Numerous enzymes have been located in microvilli. With the light
microscope the free borders of epithelial cells lining the small intestine appear to
be thickened: the thickening has striations perpendicular to the surface. This
striated border of light microscopy has bcc n shown by EM to be made up of long
microvilli arranged parallel to one another. In some cells the microvilli are not arranged so regularly. With the light
microscope the microvilli of such cells give the appearance of a brush border.
Microvilli greatly increase the surface area of the cell and are, therefore,
seen most typically at sites of active absorption e.g., the intestine, and the proximal
and distal convoluted tubules of the kidneys. Modified microvilli called stereocilia
are seen on receptor cells in the internal ear, and on the epithelium of the
epididymis. Cell inclusions Non-living, non-participating, poorly structured cell
elements, very rarely seen in an intra-nuclear position; usually cytoplasmic.
3.3. Literature recommended
Main Sources:
1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004. 3. “Color Atlas of basic histology” – R.Berns – 2006.
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28
4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997. 7. Ten Cate A.R. Oral Histology.- St.Louis, Baltimore, Toronto.- 1995.
8. William A., Beresford M.A. Cytology and Histology.- Anatomy department, West Virginia University, USA.- 1992.
9. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington – 1991.
Additional ones:
1. Methodical Instructions.
3.4 How to work with the literature recommended:
Main tasks Recommendations
To review the material
To learn the material
To read and compose the plan
To answer the questions
To do the test on the material
To be ready to answer the topic
To use the material studied
To use the material on pages
To learn the new material and be
ready to write a summary
To be ready to give an answer to the
following:
3.5. Self-control material:
A. Questions to be answered:
1. What is the cytoplasm?
2. Give the classification of the organelles?
3. Describe a structure of the mitochondria?
4. Give the characteristics of the endoplasmic reticulum?
5. What is the Golgi complex? 6. Describe the structure of lysosomes?
7. Give the characteristics of the ribosomes? 8. Describe the structure of microfilaments and microtubules? 9. Give the characteristics of centrioles? 10. Describe the structure of the cilia? 11. Give the characteristics of flagella? 12. Describe the structure of the microvilli? 13. Give the characteristics of cell inclusions?
B. Test tasks to be done: See enclosure.
4. Self-preparation in the classroom.
1) Listen to the information.
2) Work with the tables and microscope.
3) Ask about the problems that haven’t been found in the information given.
4) To sketch in an album the investigated preparations.
5. Self-preparation work at home.
1) Review the material learnt in the classroom.
2) Compose the plan of your answer.
3) Answer the questions to this topic.
4) Do the test given above.
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29
6. The subject of the research work.
- “Principal causes of lysosomal diseases development”. Subject Histology, cytology and embryology
Modul №1 Cytology, embryology and basic histology
Submodul №1 Cytology
Topic 4 NUCLEUS. DIVISION OF CELLS. CELL’S CYCLE Course 1
Faculty Dental
Hours: 2
1. The topic basis: the topic “Cell nucleus. Division of cells” is very important for
future doctors in their professional activity, positively influences the students in
their attitude to the future profession, forms professional skills and experience as
well as taking as a principle the knowledge of the subject learnt.
2. The aims of the training course:
1) To have general knowledge of the topic studied;
2) To understand, to remember and to use the knowledge received;
3) To learn the classification, structure, functions of the Nucleus;
4) To form the professional experience by reviewing, training and
authorizing it;
5) To be able to carry out laboratory and experimental work.
3. Materials for the before – class work self – preparation work:
3.1 Basic knowledge, experience, skills necessary for studying the topic in
connection with other subjects:
To know To be able to
Med. Biology the structure of the cells and
tissues
work with a light
microscope
Med. Physics the structure of the light and
electron microscopes
work with a light
microscope
Organic Chemistry the chemical content of the
cell
Speak of the topic
3.2. The contents of the topic:
The nucleus constitutes the central, more dense part of the cell. It is usually
rounded or ellipsoid. Occasionally it may be elongated, indented or lobed. It is
usually 4-10 um in diameter. The nucleus contains inherited information which is
necessary for directing the activities of the cell as we shall see below.
Nuclear constituents
1. Chromatin - mainly DNA and dispersed at interphase (except for one female X chromosome); has a fine granular appearance in routine EM, but is
fibrillar; some RNA is present.
2. Nucleolus/nucleoli - dense clumped granules; nucleic acid is mainly RNA, but some DNA is there. Perinucleolar/nucleolus-associated chromatin has a
special relation to the nucleolus. A range in the number of nucleoli present
usually exists, e.g., in the liver cells with one nucleus, 1 to 6, with 2 the
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modal value. Removal of DNA by DNase allows one to see nucleoli in the
very dense nuclei of lymphocytes and some other cells.
3. Nuclearmembrane – a double layered membrane that surrounded a nucleus. It content the nuclear pores.
4. Nucleoplasma – the fluid spaces between the various constituents of the nucleus.
Histological methods for the nucleus:
o Nuclear structure is seen in TEM.
o Radioautography for rates of cell division uses tritium-labelled
precursors of nucleotides, e.g., thymidine and cytidine.
Bromodeoxyuridine (BrdU) is incorporated in place of thymidine, so
that cells synthesizing DNA prior to division can be revealed by a
labelled antibody to the BrdU.
o Chromosomes and their banding patterns are studied in cytogenetics.
o Fluorescent in-situ hybridisation uses 'coloured' nucleotide probes to
identify a particular chromosome and, on it, the gene of clinical
interest - normal, mutated, translocated, deleted, duplicated, truncated,
etc.
o Silver staining reveals the nucleolar organizer regions (NORs) as
intranuclear black dots, because it marks the acidic proteins binding to
the genes coding for ribosomal RNA located on certain chromosomes.
An increase in the number of NORs so shown (AgNORs) correlates
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with dysplastic (abnormal) growth, and may indicate malignant
tendencies in epithelial cells.
Chromatin
In recent years there has been a considerable advance in our knowledge of the
structure and significance of chromatin. It is made up of a substance called
deoxyribonucleic acid (usually abbreviated to DNA); and of proteins. Most of the
proteins in chromatin are histones. During cell division the entire chromatin within
the nucleus becomes very tightly coiled and takes on the appearance of a number
of short, thick, rodlike structures called chromosomes. Chromosomes are made up
of DNA and proteins. Proteins stabilize the structure of chromosomes.
In usual classroom slides stained with haematoxylin and eosin, the nucleus stains
dark purple or blue while the cytoplasm is usually stained pink. In some cells the
nuclei are relatively large and light staining. Such nuclei appear to be made up of a
delicate network of fibres: the material making up the fibres of the network is
called chromatin (because of its affinity for dyes). At some places (in the nucleus)
the chromatin is seen in the form of irregular dark masses that are called
heterochromalin (condensering). At other places the network is loose and stains
lightly: the chromatin of such areas is referred to as euchromatin
(decondensering). Nuclei which are large and hi which relatively large areas of
euchrouiatin can be seen are referred to as open-faced nuclei. Nuclei which are
made up mainly of heterochiomatin are referred to as closed-face nuclei.
In addition to the masses of heterochromatin (which are irregular in outline), the
nucleus shows one or more rounded, dark staining bodies called nucleoli. The
nucleus also contains various small granules, fibres and vesicles (of obscure
function). The spaces between the various constituents of the nucleus described
above are filled by a base called the nucleoplasm.
Nuclear membrane With the EM the nucleus is seen to be surrounded by a double layered nuclear
membrane. The outer layer of the nuclear membrane is continuous with
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endoplasmic reticulum. The inner layer provides attachment to the ends of
chromosomes. Deep to the inner membrane there is a layer containing proteins and
a network of filaments: this layer is called the nuclear lamina. At several points the
inner and outer layers of the nuclear membrane fase leaving gaps called nuclear
pores. Each pore is surrounded by dense protein: the region of dense protein and
the pore together - form the pore complex.
Nuclear pores represent sites at which substances can pass from the nucleus to the
cytoplasm and vice versa. The nuclear pore is about 80 nm across. It is partly
covered by a diaphragm which allows passage only to particles less than 9 nm in
diameter. A typical nucleus has 3000 to 4000 pores.
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Nucleoli We have seen that nuclei contain one or more nucleoli. They stain intensely with
basic dyes like haematoxylin. In ordinary preparations they can be distinguished
from heterochromatin by their rounded shape. (In contrast masses of
heterochromatin are very irregular).
Using histochemical procedures that distinguish between DNA and RNA it is seen
that the nucleoli have a high RNA content. With the EM nucleoli are seen to have a
central filamentous zone (pars filamentosa) and an outer granular zone (pars
granulosa) both of which are embedded in an amorphous material (pars
amorphosa). Nucleoli are formed in relationship to the secondary constrictions of specific
chromosomes. These regions are considered to be nucleolar organizing centres.
Parts of the chromosomes located within nucleoli constitute the pars chromosoma
of nucleoli.
Nucleoli are sites where ribosomal RNA is synthesized. The templates for this
synthesis are located on the related chromosomes. Ribosomal RNA is at first in the
form of long fibres that constitute the fibrous zone of nucleoli. It is then broken up
into smaller pieces that constitute the granular zone. Finally, this RNA leaves the
nucleolus, passes through a nuclear pore, and enters the cytoplasm where it takes
part in protein synthesis.
Multiplication of cells takes place by division of pre-existing cells. Such
multiplication constitutes an essential feature of embryonic development. Cell
multiplication is equally necessary after birth of the individual for growth and for
replacement of dead cells. We have seen that the chromosomes within of nuclei of cells carry genetic
information that call trolls the development and functioning of vat rows cells and
tissues — and, therefore, of the body as a whole. When a cell divides it is essential
that the whole of the genetic information within it be passed on to both the
daughter cells resulting from the division. In other words the daughter cells must
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have chromosomes identical in number and in genetic content to those in the
mother cell. This type of cell division is called mitosis. A different kind of cell division called meiosis occurs during the formation of
gametes. This consists of two successive divisions called the first and second
meiotic divisions. The cells resulting from these divisions (i.e., the gametes) differ
from other cells in the body in that the number of chromosomes is reduced to half
the normal number, and the genetic information in the various gametes produced is
not identical. Mitosis
Many cells of the body have a limited span of functional activity at the end
of which they undergo division into two daughter cells. The daughters cells hi turn
have their own span of activity followed by another division. The period during
which the cell is actively dividing is the phase of mitosis. The period between two
successive divisions is called the interphase.
The greater part of interphase is called the G1 stage, which may last from a
few hours to many years. During this period the cell carries out its 'normal'
functions. Protein synthesis takes place mainly in this phase. About 12 hours
before the onset of mitosis the synthesis of DNA takes place and is completed in
about 7 hours: this period is called the S stage (S for synthesis). The last five hours
before mitosis are utilized for synthesis of proteins required for cell division. This
is called the G2 stage of interphase.
Mitosis is conventionally divided into a number telophase. At this stage each
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chromosome consists of stages called prophase, metaphase, anaphase and
telophase. The sequence of events of the mitotic cycle is best understood by
starting with a cell in thelophase. With the progress of telophase the chromatin of
the chromosome uncoils and elongates and the chromosome can no longer be
identified as such.
However, it is believed to retain its identity during the interphase (which
follows telophase). During the S stage of interphase the DNA content of the
chromosome is duplicated so that another chromatid identical to the original one is
formed: the chromosome is now made up of two chromatids. When mitosis begins
(i.e., during the prophast) the chromatin of the chromosome becomes gradually
more and more coiled so that the chromosome become recognizable as a thread-
like structure that gradually acquires a rod-like appearance.
While the changes described above are occurring in the chromosomes a
number of other events are taking place. The two centrioles separate and move to
opposite poles of the cell. They produce a number of microtubules that pass from
one centriole to the other and form a spindle. Tubules radiating from each centriole
create a star like appearance or aster.
The spindle and the two asters collectively form the diasler (also called
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amphiaster or achromatic spindle). Meanwhile the nuclear membrane breaks down
and the nucleoli disappear. With the formation of the spindle the chiomosomes
move to a position midway between the two centrums u c., at the equator of the
cell) where each chromosome becomes attached to microtubules of the spindle by
its centromere. This stage is referred to as metaphase. The plane along which the
chromosomes lie during metaphase is the equatorial plate.
In the anaphase the centromere of each chromosome splits longitudinally
into two so that the chromatids now become independent chromosomes. At this
stage the cell can be said to contain 46 pairs of chromosomes. One chromosome of
each such pair now moves along the spindle to either pole of the cell. This is
followed by telophase in which two daughter nuclei are formed by appearance of
nuclear membranes around them. The chromosomes gradually elongate and
become indistinct. Nucleoli reappear. The centriole is duplicated at this stage or in
early interphase.
The division of the nucleus is accompanied by the division of the cytoplasm.
In this process the organelles are presumably duplicated and each daughter cell
comes to have a full complement of them. The rate of cell division varies from tissue to tissue being greatest fit those
epithelia which lose cells because of friction (e.g., the epidermis and the lining
cells of the intestine). The rate varies with demand becoming much greater during
repair after injury. The rate is precisely controlled to correlate with demand.
Failure of such control results in uncontrolled growth leading to formation of
tumours. Various abnormalities in mitosis may be produced by exposure to various
radiations, the most important being nuclear radiation. Mitosis can be arrested by
chemicals. One of them — colchicin (or colcemide) — stops mitosis at metaphase
and allows us to study chromosomes at this stage. Meiosis
As already stated meiosis consists of two successive divisions called the first
and second meiotic divisions. During the interphase preceding the first division
duplication of the DNA content of the chromosomes takes place as in mitosis.
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First Meiotic Division The prophase of the first meiotic division is prolonged and is usually divided
into a number of stages as follows. (a) Leptotene: The chromosomes become
visible (as in mitosis). Although each chromosome consists of two chromatids
these cannot be distinguished at this stage. At first the chromosomes are seen as
threads bearing bead-like thickenings (cliromomeres) along their length. One end
of the thread is attached to the nuclear membrane. During leptotene the
chromosomes gradually become thicker and shorter. (b) Zygotenc: We have seen
that the 46 chromosomes in each cell consist of 23 pairs (the X and Y
chromosomes of the male being taken as a pair). The two chromosomes of each
pair come to lie parallel to each other, and are closfcly ap-posed. This pairing of
chromosomes is also referred to as synopsis or conjugation The two
chromosomes together constitute a bivalent. (c) Pachytene: The two chromatids
of each chromosome become distinct. The bivalent now has four chromatids in it
and is called a tetrad. There are two central and two peripheral chromatids,
one from each chromosome. An important event now takes place. The two central
chromatids (one belonging to each chromosome of the bivalent) become coiled
over each other so that they cross at a number of points. This is called crossing
over. At the site where the chromatids cross they become adherent: the points of
adhesion are called chiasmata. (d) Diplotene: The two chromosomes of a bivalent
now try to move apart. As they do so the chromatids 'break' at the points of
crossing and the 'loose' pieces become attached to the opposite chromatid. This
results in exchange of genetic material between these chromatids. A study of that
as a result of this crossing over of genetic material each of the four chromatids of
the tetrad now has a distinctive genetic content.The metaphase follows. As in
mitosis the 46 chromosomes become attached to the spindle at the equator, the two
chromosomes of a pair being close to each other.The anaphase differs from that in
mitosis in that there is no splitting of the centromeres. One entire chromosome of
each pair moves to each pole of the spindle. The resulting daughter cells, therefore,
have 23 chromosomes, each made up of two chromatids.The telophase is similar to
that in mitosis.The first meiotic division is followed by a short interphase. This
differs from the usual interphase in that there is no duplication of DNA. Such
duplication is unnecessary as the chromosomes of the cells resulting from the first
meiotic division already possess two chromatids each.
Second Meiotic Division The second meiotic division is similar to mitosis. However, because of the
crossing over that has occurred during the first division, the daughter cells are not
identical in genetic content. This is the reason for regarding it as a meiotic division. At this stage it may be repeated that the 46 chromosomes of a cell consist of
23 pairs, one chromosome of each pair being derived from the mother and one
from the father. During the first meiotic division the chromosomes derived from
the father and those derived from the mother are distributed between the daughter
cells entirely at random. This, along with the phenomenon of crossing over, results
in thorough shuffling of the genetic material so that the cells produced as a result
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of various meiolic divisions (i.e., ova and spermatozoa) all have a distinct genetic
content. A third step in this process of genetic shuffling takes place at fertilization