HISTOLOGY: THE FORM AND FUNCTION OF TISSUES

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1 Biology E-65C Lab #1 HISTOLOGY: THE FORM AND FUNCTION OF TISSUES Objectives: 1. To gain an appreciation of tissue types in terms of their functional significance 2. To gain experience with the use of a compound microscope Reading Lab Manual pages 4 43 INTRODUCTION Anatomy is the branch of science that addresses the external form and internal organization of organisms. The architectural principles upon which living organisms are built are of premier importance as is the understanding of the structural basis for the functioning of the various parts. Developmental mechanisms are also of fundamental importance. We will study two subdivisions of anatomy in lab; gross anatomy and microscopic anatomy or histology. You will become acquainted with gross anatomy in the two skeletal labs. In contrast to gross anatomy, histology involves the examination of the various structures too small to be seen with the naked eye. Obviously, the successful study of small structures requires refined optical systems and other ancillary techniques. In this lab, you will observe normal human tissue under the compound microscope. Your TF will provide a hands-on tutorial for the particular model of microscope you will find in your lab room. Each pair is provided a box of microscope slides. Please handle them with care. You should keep the cover on the slide box as much as possible; stains fade in the light and prolonged exposure will result in deterioration of the specimen. Remember as you study the slides that your goal is to be able to recognize and give a major characteristic for each of the various tissue types. You should make sketches or notes of your observations. You will then apply what you have learned about basic tissue types and will identify specific tissue types on organs provided in lab. Spend no more than 7-8 minutes per slide if you want to get through them all. Exercise 1 - Histology A helpful hint: Preparations of tissues can make determinations of cell shapes difficult. A good rule is to use the nucleus as the monitor of cell shape; nuclear morphology often reveals cellular morphology. An example: oval nucleus = columnar cell. TECHNIQUES OF PREPARATION This outline is provided strictly so you will gain an appreciation of what tissues are subjected to in the production of prepared slides. Preparation of Histological Specimens 1) Fixation. Chemical fixatives (such as various aldehydes) are applied to a specimen. Fixation causes denaturation of the cross-linkages of proteins, prevents autolysis by autosomes, and kills bacteria and viruses. 2) Dehydration. The sample is placed into successive baths of alcohol of increasing

2 concentration, then into an organic solvent such as xylene or benzene. 3) Embedment. The sample is embedded in paraffin or plastic. 4) Microtomy ("thin sectioning"). Specimens are cut into super-thin shavings a few microns in width by machines called microtomes or cryostats. 5) Mounting. Typically, a slide is coated with egg albumin and heated on a hot plate before the specimen is placed on it. This makes the specimen adhere. 6) Rehydration. Organic solvent is first used to wash out the paraffin, and then the specimen is successively treated with decreasing concentrations of alcohol. 7) Stain. (See discussion below.) Most stains are dyes from the textile industry. 8) Dehydration. Water --> alcohol --> xylene (which is a solvent for the organic cement found in tissue). 9) A Coverslip is permanently applied with organic cement. 10) Artifacts are mistakes substances or items in a slide that are not normal features of the tissue itself. A test to see if something is an artifact is a comparison of living vs. preserved tissue. Artifacts include shrinkage, tissue folds, knife marks, and waves. Staining At physiological ph, proteins have either a net negative or positive charge. It is this net charge of the various components of cells or tissues that allows them to pick up stain; dyes, too, are either negatively or positively charged and so bind with proteins. Dyes are divided into two classes: 1) Negatively charged dyes are called acid dyes (or anionic dyes). Acid dyes interact with proteins with a net positive charge. A commonly used acid dye is eosin. 2) Positively charged dyes are called basic dyes (or cationic dyes). Basic dyes interact with proteins with a net negative charge. Common basic dyes include hematoxylin, toluidine blue and alcian blue. Tissue components are referred to as "acidophilic" or "basophilic;" dyes are not. Acidophilic tissues will have a net positive charge; basophilic tissue components will have a net negative charge. The most commonly employed technique in animal histology is the use of hematoxylin and eosin together ("H&E"). The basic dye hematoxylin stains acidic structures a purplish blue; those structures are basophilic. Nuclei and rough endoplasmic reticulum, for example, both have a strong affinity for this dye owing to their high content of DNA and RNA respectively. In contrast, eosin is an acid dye that stains basic structures (therefore acidophilic) red or pink. Most cytoplasmic proteins are basic and hence cytoplasm generally stains pink or pinkish red. In general, when the H&E staining technique is applied to animal cells, nuclei stain blue and cytoplasm stains pink or red.

3 HUMAN TISSUE: FORM AND FUNCTION The human body has approximately 60 trillion living cells. Each day we lose 10,000 brain cells and 50 million skin cells. There are about 100 types of cells, which fall into 4 categories: epithelial tissue, connective tissue, muscle tissue, and nerve tissue. It should be mentioned that this classification is based on vertebrates, and that its application to other animals is often not relevant. The classification of animal tissues can be summarized as follows: I. Epithelium A. Simple epithelium (squamous, cuboidal, or columnar) B. Stratified epithelium (stratified squamous, stratified cuboidal, or stratified columnar) II. Connective Tissue A. Vascular tissue (blood, lymph) B. Connective tissue proper (loose connective tissue, dense connective tissue) C. Cartilage D. Bone III. Muscle A. Skeletal IV. Nerve B. Smooth C. Cardiac Epithelial Tissue Epithelial tissues form the covering or lining of all body surfaces, cavities and tubes, both internal and external, in animals. Epithelial tissues function in protection and absorption, forming sheets covering a surface; in secretion, forming glands; and in excretion. Examples include skin (epidermis), glands, and the linings of the digestive tract and blood vessels. The cells of epithelial tissues are tightly packed together with very little intercellular materials or spaces. Epithelial cells are divided into three principal types: flattened or squamous, cube-shaped or cuboidal, (when viewed in a section perpendicular to the tissue surface), and columnar. Epithelial tissue may be only one cell thick, in which case it is called simple epithelium, or it may be two or more cells thick and called stratified epithelium. The various types of epithelia are named on the basis of the cell type and the number of cell layers (it is the cells of the outermost layer that determine the name of the stratified epithelia). Thus we can recognize simple squamous epithelium, simple cuboidal epithelium, simple columnar epithelium, stratified squamous epithelium, etc. Epithelium, regardless of type, is usually separated from the underlying connective tissue by an extracellular basement membrane. The basement membrane is not penetrated by blood vessels; epithelia are thus dependent on the diffusion of oxygen and metabolites from underlying tissues. Both epithelial and connective tissue cells participate in the formation of the basement membrane. Epithelia involved in secretion are often arranged into glands. Glands are merely invaginations of epithelial surfaces. Those glands that maintain their continuity with the epithelial surface via a duct are called exocrine glands and secrete onto the free surface. Endocrine or ductless glands secrete directly into the blood stream and their secretions are known as hormones.

4 Lab Exercise #1 using your lab manual and the microscope become familiar with the different tissue types in the body, their location and their functions. Slide 1. Kidney: Simple squamous epithelial cells. Look for circular clumps of cells. Surrounding these aggregations is a single layer of squamous cells. (This is indeed epithelium because it is continuous with the urinary tract.) Simple cuboidal epithelium. This form represents an intermediate form between simple squamous and simple columnar epithelium. In cross sections, the cells appear square; on surface view, the cells are actually polygonal in shape. Simple cuboidal epithelium usually lines small ducts and tubules that may have excretory, secretory, or absorptive functions; examples are the small collecting ducts of the kidney, salivary glands and pancreas. Slide 2. Stratified squamous epithelium. The major function of this type of epithelium is protection against mechanical abrasion. This section shows several layers of epithelial cells and the basement membrane. Cells closest to the basement membrane appear cuboidal but the outermost layer of epithelia consists of squamous cells, so this epithelium is classified as stratified squamous. This type of tissue is found in the oral cavity, pharynx, esophagus, anal canal and vagina. Slide 3. Simple columnar epithelium [duodenum, x.s.]. At 10x magnification, search for an area of the slide that has finger-like projections surrounding a lumen these are the villi. Switch to 40x. Cells are tall and columnar, at right angles to the basement membrane. The nuclei are elongated and may be arranged toward the base, the center or occasionally the apex. Simple columnar epithelium is most often found on highly absorptive surfaces such as in the small intestine, although it may constitute the lining of highly secretory surfaces such as that of the stomach. Slide 4. Pseudostratified ciliated columnar epithelium gives one the erroneous impression that there is more than one layer of cells. However, all the cells rest on the basement membrane (although not all cells extend to the luminal surface), so it is a simple epithelium. The nuclei are disposed at different levels, thus creating the illusion of cellular stratification. Notice the border of cilia on the luminal side. This type of tissue is almost exclusively confined to the larger airways of the respiratory system (i.e., trachea) and therefore often is referred to as respiratory epithelium. What do you suppose is the function of this tissue? Slide 5. Stratified squamous keratinizing epithelium. The major means of epithelial tissue protection is through the production of the protein keratin. Cells have different amounts of this, which alters their characteristics. This specialized form of tissue constitutes the epithelial surface of the skin and is adapted to withstand constant abrasion and dessication; the cells of the skin become engorged with keratin, lack nuclei, appear scalelike and eventually die. Cells with lesser amounts exhibit "non"-keratinization they have nuclei, appear alive. The tough non-cellular surface of skin, then, is composed of the protein keratin and the remnants of degenerate epithelial cells.

5 Slide 6. Transitional epithelium [lining of a ureter]. This form of stratified epithelium is almost exclusively confined to the urinary tract in mammals, where it is highly specialized to accommodate a great degree of stretch and to withstand the toxicity of urine. It is sonamed because it exhibits features intermediate between stratified cuboidal and stratified squamous epithelium. In the relaxed state, this tissue appears to be approximately 4-5 cells thick; basal cells are roughly cuboidal, intermediate cells are more flattened or polygonal, and the surface cells are large and rounded. Notice that the plasma membranes of the surface cells are quite thick to provide a permeability barrier. Notice the two layers of smooth muscle surrounding the epithelia; the inner layer is longitudinal and the outer layer is circular. The next portion of the section is loose connective tissue and the outermost portion is adipose tissue (fat cells). Connective Tissue Connective tissue, with its many varieties, is the most widespread and abundant tissue in the body. It surrounds cells, encases internal organs, sheathes muscles, wraps bones, encloses joints, composes the blood, and forms the supportive framework for all organs. Structures made of connective tissue differ widely. Delicate tissue-paper webs, strong tough cords, rigid bones, and liquid blood all are made of connective tissue. Microscopically, connective tissue consists of three elements: cells, matrix (intercellular material), and fibers. One of the differences between connective and epithelial tissues is the proportionate relationship between cells and intercellular material. Cells predominate in epithelial tissues, with very little intercellular material. In connective tissues the reverse is true: there is a large amount of matrix secreted by relatively few cells. Cells of connective tissues, called fibroblasts, are responsible for the maintenance of the integrity of the connective tissues by continuous slow turnover of the intercellular elements. The fibroblast nuclei of loose connective tissue are condensed and elongated in the direction of the extracellular fibers. Indiscernible under the light microscope are cytoplasmic processes that extend into the matrix to meet up with those of other fibroblasts. Matrix (pl. matrices), also called ground substance, is composed of the intercellular substances secreted by the fibroblasts. The intercellular matrix allows for the diffusion of metabolites and is an important barrier to the spread of microorganisms. Matrices may be liquid, semisolid, or solid. The fibers of connective tissue are of three types: collagen, elastin, and glycoproteins. Collagen is the principal fiber type found in the matrix of most connective tissues and is the most abundant protein in the body. It is found in fibrous connective tissue, skin, tendons, ligaments and bone, in various arrangements from loose to dense. Parallel collagen fibers are arranged into strong bundles that confer great tensile strength to the tissue those bundles are visible under the light microscope. Collagen is secreted into the matrix in its precursor form of tropocollagen; in the matrix, the tropocollagen molecules polymerize to form collagen. Elastin is a rubber-like material that is arranged as fibers and discontinuous sheets in the matrix particularly of skin, lung, and blood vessels. Like collagen, elastin is synthesized by fibroblasts in a precursor form known as tropoelastin that undergoes polymerization after secretion. Glycoproteins are known to be associated with cell surface membranes where they appear to play a role in cell-to-cell interactions.

6 Connective Tissue with Liquid Matrices: Slide 7. Human blood. Blood and lymph are good examples of connective tissues with liquid matrices. In blood, the erythrocytes, or red blood cells, and the leukocytes, or white blood cells, are floating in the plasma, which is the liquid matrix. The fibers are the soluble protein fibrinogen; when clotting occurs, fibrinogen is converted into fibrin fibers that make up the clot. Notice the great predominance of erythrocytes with purple-stained leukocytes interspersed randomly throughout. (You'll notice that the leukocytes have different appearances there are several classes of them.) Also, small platelets can be seen, which appear as tiny purple dots. Question: why don't erythrocytes pick up the purple stain? You should be able to identify the erythrocytes as well as the different types of leukocytes. Connective Tissue with Semisolid Matrices: Slide 8. Loose fibrous connective tissue or areolar tissue is widespread throughout the body, functioning to bind together the individual cells of muscles and nerves, to bind organs together and hold them in place, etc. In other words, it acts as a biological packing material between other tissues of more specific functions. The matrix includes some large, tough collagen fibers (pink stained) and some thinner elastin fibers (blue stained); both types of fibers are produced by the purple stained fibroblasts. As you can see, in loose connective tissue the fibers are not packed tightly together and they are oriented in many different directions. Slide 9. Tendon, l.s. Tendons, which connect muscles to bones and ligaments, and bones to other bones, are composed of dense fibrous connective tissue. This tissue differs from loose fibrous connective tissue in that its elastin and collagen fibers are tightly packed together and are all oriented in the same direction, thus increasing strength and elasticity in that direction. These fibers are wavy in shape, and are closely packed in bundles, giving the tissue a characteristic wavy appearance. Between the bundles can be seen the flattened fibroblasts arranged in rows. Slide 10. [See Slide 7.] Adipose or fat tissue is a modified type of connective tissue. The stored fat occupies a large part of the contents of the cells so that only a small margin of cytoplasm and the nucleus can be noted around the cells. This gives the cells a ring-like shape. Fat tissue is important in nutrient storage, and also in protection of other tissues, insulation, and padding.

7 Connective Tissues with Solid Matrices: Slide 11. Hyaline cartilage. [Look at the center of the section.] This type of cartilage is the most abundant form in the body. It has a firm rubbery matrix consisting of many tightly packed collagen fibers. Cartilage fibroblast cells, called chondrocytes, are located in small spaces called lacunae, which are scattered throughout the matrix. Cartilage varies in its texture, color, and elasticity. It is found in the body in such places as the nose, larynx, trachea, ear, intervertebral discs, and many parts of the skeleton. From where in the body do you suppose this slide was taken? Slide 12. Elastic cartilage [from a monkey ear; focus in the center of the section]. This slide has been stained so that elastin fibers appear black. Your slide might contain hair follicles located on either side of the cartilage. In mammals, this type of cartilage is found in the external ear, in the walls of the external auditory and eustachian tubes, and in the epiglottis. Slide 13. Developing cartilage bone. The immature skeleton of the fetus is initially made up of mostly cartilage. Eventually, through a complex process known as ossification, the cartilage is calcified and replaced by bone. What you see in the slide is the interface between the shaft of this fetal bone and its end, or epiphysis. The shaft is comprised of marrow space surrounded by immature bone and is stained pink. The epiphysis is cartilagenous and is stained purple-gray. This area constitutes a growth plate where chondrocytes proliferate and eventually degenerate away from the epiphysis to give way to deposition of bone tissue; this zone of proliferation is responsible for the elongation of bone. Slide 14. Compact bone [from the human femur]. Bone has a hard, relatively rigid matrix comprised of numerous collagen fibers that are impregnated with calcium deposits. Compact bone consists of numerous structural units called Haversian systems. Each Haversian system is seen as a nearly round area, the central core of which is the Haversian canal, which runs lengthwise through the bone. Bone is living tissue; the blood vessels, lymph vessels, and nerves that supply the tissue run through the Haversian canal. Around the canal is the matrix, arranged in concentric layers called lamellae. The lamellae are perforated by lacunae where the osteocytes, the fibroblasts of bone cells, are located. (The lacunae are visible as flat dark spots.) Numerous fine canaliculi interconnect lacunae and Haversian canals to provide the resident osteocytes with tissue fluid and metabolites. Muscle or Contractile Tissue The cells of muscle tissue have greater capacity for contraction than do other cells, although all cells probably possess this property to some extent. The cells are usually quite elongate. Muscles are responsible for most movement in higher animals. Three principal types of muscle are recognized: skeletal muscle, which is responsible for most voluntary movement; smooth muscle, which is involved in most involuntary movements of internal organs; and cardiac muscle, the tissue of which the heart is composed. Muscle tissue accounts for 40% of the total human body weight.

8 Slide 15. Skeletal muscle (or striated muscle), x.s. Skeletal muscle fibers are extremely elongated unbranched cylindrical cells; they may be up to several centimeters in length, while their diameter is approximately μm. Each fiber is multinucleate, containing a great many flattened peripherally located nuclei at fairly regular intervals. The fibers are crossed by numerous alternating light and dark bands, or striations, providing this tissue with its name. These bands are the result of the arrangement of the contractile proteins, which are responsible for the peripheral displacement of the nuclei. The fibers of skeletal muscle are bound together into bundles by delicate connective tissue. Slide 16. Smooth muscle (or visceral muscle). [From the intestine; look around the periphery of the section.] Smooth muscle is found encapsulating the blood vessels and lines such structures as the intestinal tract, the uterus, and the bladder. Note the absence of striations. Each fiber is elongate, pointed at each end, and contains a single centrally located rod-shaped nucleus. Smooth muscle fibers interlace to form sheets of muscle rather than bundles. It might appear to you that smooth muscle looks like fish swimming. Slide 17. Cardiac muscle. The cells of cardiac muscle branch and interdigitate, forming a complex three-dimensional network. You'll see in the slide that where adjacent cardiac muscle cells meet are specialized intercellular junctions called intercalated discs; these not only provide points of anchorage for the myofibrils but also permit extremely rapid spread of contractile stimuli from one cell to another, resulting in nearly simultaneous contractions. Nuclei are round or ovoid and are centrally located within the cells. Note that cardiac muscle has striations but the striations are not as apparent as those of striated muscle. Nerve Tissue All protoplasm possesses the property of irritability to some extent, but nerve tissue (sometimes called conductile tissue) is highly specialized for this capacity. The nerve cells, or neurons, are easily stimulated and can transmit impulses very rapidly. Slide 18. Neurons [from spinal cord squash]. Each neuron consists of a cell body (the balloonlike structure) containing the nucleus, and long, thin fibrous extensions called dendrites and axons. The purple dots throughout the section are glial cells, the nutritive and support cells of the central nervous system. Slide 19. Small neurovascular bundle comprised of an artery, vein and nerve. The vessels supplying and draining a particular area of tissue tend to pass together, frequently accompanied by a peripheral nerve and surrounded by connective tissue that forms an illdefined protective sheath. This slide demonstrates that the three components lie in loose areolar connective tissue and are surrounded by a more condensed collagenous sheath. Notice that the artery has a rigid wall compared to the collapsed appearance of the vein. This is because the inner walls of the artery are largely made up of elastin to provide for the expansion and recoil necessary for the maintenance of the blood pressure. The diameter of the lumen is regulated by smooth muscles in the vessel walls and is under the control of the sympathetic nervous system.

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