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Lab 1. Cells, Epithilia, Glands & Mitosis

A. The Cell

Throughout the year you will be examining slides showing the various tissues and organs of the body. Tissues and organs are made up of differentiated cells, usually organized in a particular manner, and extra-cellular matrix components. The study of Histology requires you to understand the structure of cells and recognize specific cell types based on differences in their structure. The stains used in Histology allow visualization of cellular components and give distinct colors to organelles. The stain most commonly used is Hematoxylin and Eosin blue (H & E stain). Hematoxylin is basic and therefore binds to acids such as RNA and DNA. Eosin is acidic and binds to proteins in general. A variety of other stains are available for specialized purposes but H & E is the stain routinely used in Pathology. Most of your slides are H & E but a few use more specialized techniques.

To get a feel for the appearance of cells, take slide 62 (liver) from your slide box and visually inspect it against a white background or the overhead lights. You will see a squared-off piece of tissue, red in color with clear holes in it and very pale blue or purple shading honeycombed throughout the red square.

The slide was stained with hematoxylin (blue chromophore) and eosin (red chromophore). The redness of the tissue indicates that the tissue is eosinophilic (or acidophilic). See explanation 1 for discussion of staining.

Now, remove an ocular (eyepiece) from the microscope and examine the tissue by reversed ocular (R.O.). (Take the ocular from the microscope, place the lens near the slide and look at the tissue through the tube. Be sure the background is a white sheet of paper or a light). The uniform red color will now appear to have tiny blue granules within it, and the blue honeycomb previously mentioned will be clearer, and may have a small red space at the center of the zone it delimits.

NOW put the slide on the microscope. With the 4x objective you can see the small blue dots that made the generalized granularity seen with R.O. You can see that most of the blue dots are round or oval, and some are flat. These are nuclei. Turn the 10x objective lens into position and note that the blue color of the nuclei is due to blue blocks of dark and light material (chromatin). With the 40x objective, observe that the dark blue blocks of DNA-containing chromatin are concentrated at the edge of the nucleus, against the nuclear envelope. This darker material is heterochromatin. You may also see one or more large, dark blobs of chromatin. These are the nucleoli and are present near the center of the nucleus. You can also see that the cytoplasm of the cell is granular in texture and eosinophilic (pink) in color and may contain blue staining blocks if recently stained (dye colors fade with age like histology teachers).

What might the cytoplasmic granules be? The blue masses? Attempt to discern the site of the cell membrane. Between the plates of hepatic parenchymal cells are blood vessels that appear as spaces and may contain blood cells.

B. Epithelium

Epithelium is one of the 4 primary tissues of the body. It consists of cells usually arranged in sheets or tubules that are attached to the underlying basement membrane. The basement membrane, a structure seen with the light microscope, has been subdivided into a basal lamina (thought to be produced by the epithelium) and a reticular lamina (produced by connective tissue cells). You will usually see the basal lamina only with the electron microscope.

Epithelium is found covering the numerous internal and external surfaces of the body and may also be modified to form glandular structures. As a physician, it is very important to be able to recognize and identify the various types of epithelia since they are important in organ identification and essential in the detection of pathological processes.

In general, the various types of epithelia are classified according to the shape of the cells and whether or not they are layered. In a simple epithelium the cells form a single layer and are all attached to the basement membrane. A stratified epithelium consists of multiple layers of cells in which only the basal layer is attached to the basement membrane. A simple epithelium which appears to be stratified is classified as "pseudostratified" (the nuclei are in different layers, but all the cells are in contact with basement membrane). A stratified epithelium in which the number of cell layers varies upon physiological stretching is called "transitional". Epithelial cells are classified by shape into squamous (flat), cuboidal and columnar varieties. The name of a stratified epithelium is determined from the shape of the apical layer of cells.

Objectives

You should aim to:

  1. Recognize the presence of an epithelium and understand its defining characteristics.
  2. Distinguish the different morphological types of epithelium.
  3. Relate morphology to function where possible.
  4. Identify ultrastructural features of epithelial cells and their functional significance.

1. Simple Squamous Epithelium

Squamous cells are flattened cells that are wider than they are tall (think of a fried egg). Look at slide # 74 (kidney). In one area of this slide you will find roughly circular structures containing a tangle of cells separated by a clear space from the outer wall. These are renal glomeruli which filter the blood and the space is where the filtrate enters the renal tubules. The outer wall is composed of a sheet of squamous epithelial cells. They are so thin that you can only really see the nucleus of the cell in the light microscope. Try to imagine this sheet of cells in 3-dimensions. If you look at an area outside the glomerulus you will see a mass of tubules formed by cuboidal epithelial cells. If you look carefully you will see that they are not identical but vary in morphology and coloring. Later on you will come to appreciate that the appearance of cells in different organs is characteristic.

The EM view of a simple squamous epithelium shows how flatttened these cells can be but allows you to see the cytoplasm and intracellular organelles.

 The coelomic cavities within the body are covered by a simple squamous epithelium which is called mesothelium. Slide #2 (Mesentery) is a piece of mesentery which has been spread flat on the glass and stained with silver nitrate. In this slide you are not viewing a cross-section but looking head-on at the surface. The mesentery is a sandwich composed of two sheets of simple squamous epithelium separated by a relatively thicker layer of connective tissue, which may contain blood vessels and fat cells. The edges of the squamous cells can be made out as uneven corrugated black lines due to precipitation of silver in the intercellular space. By changing focus, both epithelial layers may be brought into view separately in certain areas of the slide (see diagram).

Blood and lymphatic vessels are lined with a special type of simple squamous epithelium called endothelium. Examine slide #70 (nasal mucosa) for blood vessels and locate their endothelial lining. Blood vessels are usually recognized by their content of red blood cells (small red staining spheres). They vary in size and in this region of the body there are many large vessels that provide warmth and moisture to inhaled air. The nuclei of the squamous endothelial cells are prominent, but cell boundaries cannot be made out because the cells are so flattened. The cells are generally oriented with their long axis parallel to the axis of the vessel. Try to contrast longitudinal and cross-sectional views of blood vessels.

2. Simple Cuboidal Epithelium

This type of epithelium consists of a single layer of cuboidal cells. Cuboidal epithelial cells are approximately as wide as they are tall and can be found in the kidney tubules, glands and ducts. A good example is the thyroid gland, slide #67 which is composed of follicles (hollow spherical structures) that are discrete areas of colloidal material surrounded by a simple cuboidal epithelium. First locate a well-sized follicle. Note the central lumen containing pinkish material (colloid). The wall of the follicle consists of a sheet of roughly cuboidal cells. Each cell has a central, bluish-colored nucleus. The blue color is due to the hematoxylin component of the dye. Compare this to the pinkish (eosin) color of the surrounding cytoplasm. You will see later that staining of both nuclei and cytoplasm varies according to the packing of chromatin and the amount of ribosomes in the cytosol. Between the follicles there is connective tissue which will be studied in the next class.

3. Simple Columnar Epithelium

This epithelium is composed of a single layer of cells which are taller than they are wide and can be found lining the stomach, intestine, uterus and gall bladder. Locate the columnar epithelium on slide #3 (intestine) noting the presence of mucous-secreting goblet cells (they appear as clear, unstained areas due to mucous extraction) and the apical brush border (see below). The nuclei are not centrally located but lie nearer the basal plasma membrane. This epithelium provides an opportunity to think about planes of section. Since the epithelial cells are asymmetric, their appearance in the microscope depends on how the cells were sectioned. This time think of a hard-boiled egg with its yolk closer to one end than the other. If the plane of section passes through the long axis of the egg you will see an ovaloid structure with the yolk (nucleus) nearer one end than the other and this more white at one end. If the section passes through the short axis at the base you will see predominantly yolk surrounded by a thin rim of white. However, the section could also pass through the white at the other end and miss out the yolk altogether. Try to find areas on your slides corresponding to each of these possibilities.

4. Stratified Cuboidal and Columnar Epithelia

These epithelia can be found in the tubules of the testis, ducts of some glands, and in regions of the urinary and respiratory tracts. They will be examined later when you study organ systems.

5. Pseudostratified Columnar Ciliated Epithelium

This epithelium is found lining the respiratory system. Examine the epithelium on slide #70 (nasal mucosa) which is made up of three types of cells: basal, columnar and fusiform. The first two are easy to see, but fusiform cells are difficult to demonstrate due to their shape (see diagram). All cells rest on the basement membrane, but vary in height. As a result, the nuclei are at different levels giving the appearance of stratification (see diagram). You will not have to distinguish between the different cell types. Also examine apical membrane cilia on this slide. They appear like bristles extending from the apical surface (i.e. the surface furthest from the basement membrane)

6. Stratified Squamous Epithelium

Stratified squamous epithelium is found in the skin and passageways leading to the exterior such as the mouth, anus and vagina. Examine slide #50 (esophagus) and note the epithelium lining the lumen. In the odd-numbered boxes, it is cut in cross-section and the epithelium lines the luminal surface of the tube; in the even-numbered boxes it is a longitudinal piece of tissue with the epithelium on one surface. By visual inspection, most of the tissue is pink. The epithelium will be the most basophilic material in the slide. Inspection of the epithelial layer at low-power indicates that the basal surface of the epithelium is indented by connective tissue papillae. If these papillae are cut in cross-section, they appear as circular areas within the epithelium. Using the 10x objective, observe the many layers of cells. With this and the 40x objective, examine the changes in shape the cells undergo from the basal to the lumenal layers. New cells are formed in basal areas of the epithelium, are pushed towards the free surface eventually and are sloughed off.

If the epithelium is subject to a great deal of abrasion, such as in the skin, the outer cells undergo a special type of transformation. Keratin is formed in these cells as they migrate towards the surface. The nuclei disappear and the cells become dehydrated. This process will be discussed in the lecture on skin. Slide #43 (Thick skin), is a section through the plantar surface of a foot. The pink material is connective tissue and the epithelium is the blue layer next to it. The thick outer layer is composed of the keratinized cells mentioned above. It may be partially or completely detached in some slides. If so, examine your neighbor's slide.

7. Transitional Epithelium

This epithelium is found in urinary tract. Its appearance varies depending on whether it is in a stretched or relaxed state. Examine slide #76 (human bladder) and slide #9 (rat bladder) made from relaxed bladders. The cells in the outermost layer characteristically have a large amount of cytoplasm, which may give the cell a swollen, balloon shape appearance ("cap" cells). In slide #9, many cells in the outer layer appear to be covered by a dense-layer of material which is not part of the epithelium.

Specializations of Epithelial Cells

Epithelial cells have polarity in terms of their morphology and orientation. The plasma membrane nearest the basement membrane is the basal membrane; the apical membrane lines the free surface, and the lateral membrane is between adjacent cells. The various membranes may be modified to perform specialized functions. Projections from the cell-surface may be found on different cells depending on their function. These can be visualized in the light microscope but their structure is only apparent in electron micrographs (E.M.s). Examine the E.M.s in your textbook. Non-motile processes called microvilli may project from the surface of cells. Microvilli vary in size from extremely small processes which can only be seen with the electron microscope, to larger structures which can be seen with the light microscope. In the small intestine (slide #3), individual microvilli are not seen but the brush border they form is visible. Explain why. What is the function of microvilli?

Motile processes called cilia are present in the pseudostratified epithelium of the trachea (slide #72). Observe these and then study the E.M.s noting the characteristic 9 + 2 microtubule arrangement. Notice in the thin section EM of a simple columnar ciliated epithelium numerous cilia cut in both cross section and longitudinal section can be observed at the apical membrane. The cilia are attached to basal bodies in the apical cytoplasm

Cell Junctions

Thin section EM of a tight junction on the lateral membrane of adjacent epithelial cells. Using this procedure, the tight junction appears as punctate membrane fusions.

In freeze-fracture EM, these membrane fusions appear as intramembrane ridges. The function of tight junctions is to seal the intercellular space between epithelial cells and prevent transepithelial diffusion. Tight junctions also maintain epithelial cell surface polarity.

Desmosomes are observed on the lateral membrane between epithelial cells. They are involved in cell-cell adhesion and are abundant in organs that undergo severe mechanical stress such as the skin. Hemidesmosomes are found on the epithelial basal membrane and anchor the epithelium to the basal lamina.

Gap junctions appear as plaques of tightly packed intramembrane particles in freeze-fracture electron microscopy. For comparison, the intramembrane particles of the adjacent plasma membrane (labelled M in the picture) are less densly packed. In thin sections gap junctions are seen as close apposition of the adjacent membranes with a 2 nm gap between them. Electron dense stains can permeate this type of junction, unlike tight junctions. Gap junctions are involved in cell-cell communication.

More electron micrographs can be found by clicking on this link.

Students are expected to become familiar with the EM appearance of the structures pertinent to each laboratory session

C. Exocrine Glands

Exocrine glands are extensions of an epithelial surface. They arise by mitosis of the epithelial cells and their growth downward through the connective tissue to form a tubule. The gland differentiates into two regions: the duct and the secretory end-piece. The duct forms a conduit through which secretions are delivered to the surface while the cells of the secretory acini produce a secretory product and release it into the lumen. All along the duct and in the secretory end-piece the cells sit on a basement membrane and are polarized with basal and apical surfaces. Examples are the pancreas and salivary glands and you will meet many of these glands in later classes.

To get an appreciation of their structure look again at slide # 70 (nasal mucosa) where you will find glands in the connective tissue just beneath the epithelium. They are roughly rounded structures composed of a cuboidal epithelium and most have a deep coloration. The epithelial cells surround a central lumen. Don’t confuse these glandular structures with blood vessels – in what way are they different? Mostly you will observe secretory end-pieces but you may also find groups of cells coursing up towards the epithelial surface. These are ducts and in some cases you can see one penetrating up to and joining the surface.

D. Cell Division

OBJECTIVES

  1. recognize and distinguish the various stages of mitosis
  2. understand the changes in DNA content and chromosome number as cells progress through the cell-cycle.

Mitosis is preceded by an exact duplication of the DNA and associated proteins in the nucleus. This occurs during interphase - the period between actual mitotic divisions. After this doubling, the chromosomes are organized, complete division and are then equally distributed to the two daughter cells during mitosis.

All somatic cells pass through the mitotic and interphase periods at one time or another. The duration of the mitotic period is usually about 1-2 hours, while the intermitotic period can vary from a few hours to many years. The mitotic index (proportion of cells undergoing division at any one time) varies greatly depending upon the type of tissue and the physiological needs of the organism. A high mitotic index characterizes all growing embryonic tissues, and certain adult tissues such as bone marrow and intestinal crypts as well as cancer cells.

Examine the section of onion root tips (slide #1). Using the reversed 10x ocular, note that it consists of several sections mounted on the slide. Locate the growing tip (smaller, tapering, end). Under high magnification it can be seen that many of the cells are undergoing mitosis. With the aid of the textbook, pick out the various stages of mitosis and identify the components of the mitotic figures. It is important to realize that the stages of mitosis are defined only with reference to the nucleus and chromosomes. Cytokinesis, the division of the cytoplasm, may fail to occur giving rise to binucleated and/or polyploid cells. Although the actual process of mitosis is continuous, it is usually arbitrarily divided into 4 stages.

Interphase (not a stage of mitosis): The chromosomes cannot be distinguished and appear as scattered granules connected by a network of pale-staining strands within a distinct nuclear membrane.

1. In PROPHASE, morphologically distinct chromatin threads appear and these shorten and thicken, forming distinct chromosomes. The two pairs of centrioles begin to separate; (onion root cells lack centrioles). Around the centrioles fine fibrils appear forming aster rays. The whole structure is called an amphiaster and some of the aster rays elongate to form the spindle fibers. The nucleolus and the nuclear membrane begin to disappear. Compare prophase cells with cells in interphase where the chromosomes are indistinguishable.

2. At METAPHASE, the chromosomes become arranged at the equatorial plate which is midway between the two pairs of centrioles. The spindle is now fully formed. It does not stain (achromatic) and consists of fine microtubules, some of which are attached to the chromosomes. During this period the chromosomes begin to show indications of splitting.

3. At ANAPHASE, each chromosome pair completes its splitting, and the two daughter chromosomes move toward opposite poles. Spindle microtubules may be seen between the retreating chromosomes.

4. At TELOPHASE, the chromosomes have reached the spindle poles and appear as a dense, basophilic mass within which individual chromosomes cannot be defined. The nuclear membrane reappears, and the outlines of the chromosomes disappear leaving scattered chromatin granules connected by a pale-staining network. Nucleoli reappear and seem to be associated with a particular chromosome. Cytokinesis, the division of the cytoplasm, usually occurs during the telophase, but the synchrony between nuclear and cytoplasmic telophase, but the synchrony between nuclear and cytoplasmic events is not constant; cytokinesis may begin as early as late anaphase or be delayed beyond the nuclear reconstruction of telophase.

Mitosis occurs very frequently in cells of the bone marrow, gonads, epidermis, and epithelium of the intestine. However, slides of these structures will often show very few mitotic figures. This is due in part to marked diurnal variation in the rate of mitosis in different tissues; for example, sections of the skin will seldom show mitotic figures unless the biopsies are taken at night when cell division is most frequent. The methods used to obtain and prepare the cells for examination can also influence the number of mitotic figures seen. This involves not only the fixing and staining procedures, but also the treatment of the cells prior to fixing. For example, bone marrow slides are usually prepared by smearing the cells on a slide. This treatment causes most of the cells in mitosis to be disrupted, thereby greatly reducing the number actually seen. Mitosis is also affected by many factors in vivo, such as hormones, irradiation and drugs. Colchicine prevents the formation of spindle fibers, thereby holding the cell in prolonged metaphase and preventing it from proceeding into anaphase.

Next, examine a section of intestine (slide # 3). The simple columnar epithelium is folded to form deep crypts. Look at the base of these crypts for mitotic figures. The cytoplasm of the mitotic cells stains less intensely, and nuclei of mitotic cells stain more intensely than those of interphase cells and change their position relative to the basement membrane. The mitotic figures will not be as distinct as in the onion root. However, you may be able to find examples of prophase, metaphase and telophase. Cell division occurs in the crypt epithelium and the cells push out over the villus where they slough off into the lumen of the intenstine. The process from division to sloughing takes from two days to seven days depending on the segment of the G.I. tract. Mitotic figures can be found in most epithelia though less frequently than in the intestine. Pathologists measure the proportion of cells in mitosis to obtain an index of the rate of cell division in a biopsy specimen. This can often provide diagnostic information as to the degree of malignancy of a tumor.