____________________________________________________ Objectives

When you have finished this lab you should be able to: Identify the parts of a microscope, demonstrate proper care in handling the microscope, and use it correctly. Describe the general structure of a cell and explain the functions of the major organelles. Describe the process and explain the significance of cellular replication, or mitosis. Draw a sketch illustrating and indicating the location of the structures mentioned above.

LAB 2:THE CELL

Introduction
Cells are the smallest living unit that can reproduce, metabolize, respirate etc... Although diverse, cells have basic similarities and requirements. Similar cells are frequently aggregated into tissues. The cytoplasm, nucleus, and nucleolus are the most obvious parts of a cell, and a major part of histology is concerned with the variation in appearance of these parts in the different cells of the body. As you study different cell types, keep in mind that sectioned material is being observed and that the appearance of the cells may vary, depending upon the plane of the section. Cellular Organelles 1. Nucleus: The cell nucleus, which controls all cellular reactions, is surrounded by a bilayer nuclear envelope with communicating nuclear pores. Most nuclei you will see are in interphase stage of cell division. They contain variable amounts of lightly stained (eu-) and heavily stained (hetero-) chromatin as well as one or more nucleoli (usually). Observe their size, shape, and location within the cells. 2. Golgi apparatus: The Golgi apparatus is an organelle consisting of membranous cisternae that form layered stacks organelle. This organelle is usually located in a juxta-nuclear position or right next to the nucleus. Special stains are required to visualize this organelle with the light microscope, but it can be readily seen with EM.

3. Endoplasmic reticulum (ER): The ER is part of an extensive transporting system within the cell associated with transport of proteins. On its outer surface the ER may be lined with ribosomes, which give the ER a granular appearance; or the surface of the ER may be free of ribosomes, giving the ER a smooth appearance.The Secretory cells synthesizing protein typically contain large amounts of rough endoplasmic reticulum (RER). This material is seen at the light microscopic level as cytoplasmic basophilia since the abundant RNA has an affinity for basic dyes (stains blue in H&E preparations). This organelle is usually located in the basal portion of the cell. 4. Mitochondria: Mitochondria are double-membrane organells with an outer, smooth membrane and an inner, extensively folded membrane that forms cristae. Mitochondria can be visualized at the light microscopic level using a variety of special techniques. 5. Secretory granules: Usually seen as large acidophilic (e.g. pink in H&E preparations) granules located in the apical portions of secretory cells. They stain intensely because of their high protein content. 6. Cytoskeletal elements: Actin filaments, intermediate filaments, and microtubules are examples of cytoskeletal elements. The best examples of filaments composing a major component of the cytoplasm are the actin and myosin filaments in muscle. Large numbers of non-muscular cells contain smaller quantities of actin, and possibly myosin filaments, but these can only be visualized either at the EM level or by using certain cytochemical or immunochemical methods. 7. Other organelles: These structures will be encountered and studied during the semester, and include lysosomes, peroxisomes, and others. These are all discernable at the ultrastructural level and can be visualized at the light microscopic level using a variety of special techniques. Cellular Inclusions 1. Glycogen: Glycogen is a prominent inclusion in certain cells, particularly muscle and hepatocytes. It is dissolved by routine histologic fixatives, but can be retained by using alcoholic preservative solutions. If properly preserved, glycogen can be stained using the PAS technique or Bests Carmine. 2. Lipid droplets: Fat droplets are usually nutritive inclusions that provide energy for cellular metabolism. What is seen as a fat droplet in light microscopy is actually a hole in the cytoplasm that represents the site from which the lipid is extracted (lipid itself is usually removed by embedding solvents).

2. Melanin: Melanin is produced by specialized cells called melanocytes, which are located between the cells of the lower epidermis. Melanocytes have elongated processes which course between the epidermal cells and release their pigment to the keratinocytes. Melanin is largely responsible for the color of the skin. Since the number of melanocytes is about the same in all races, the differences in skin color can be attributed to variation in the amount of pigment produced by the melanocytes.

Surface Specializations 1. Cilia: Cilia are motile cytoplasmic structures capable of moving fluid and particles along epithelial surfaces. Dark lines where the cilia attach to the epithelial cells are occasionally visible. It has been shown with the electron microscope that the dark line is composed of basal bodies and that each basal body gives rise to a cilium. 2. Stereocilia: Unlike cilia, stereocilia are non-motile and are not attached to basal bodies. They are long cytoplasmic processes of the free surface of the cell, which function in resorption of secretory material. Stereocilia often adhere to each other and form tufts. 3. Microvilli: Microvilli are cytoplasmic processes that extend from the apical surfaces of most epithelial cells. Microvilli of the small intestine are regularly arranged and consistent in height (about 2m); they resemble the bristles on a brush, hence the other name, brush border. Certain enzymes, which facilitate absorption, are present in the microvilli. Electron micrographs show that a surface coat (glycocalyx or fuzzy coat) is present on the microvilli. The surface coat contains carbohydrates, which stains vividly pink when using PAS as the stain. Junctional Complexes. A cell junction is a structure within a tissue of a multicellular organism. Cell junctions are especially abundant in epithelial tissues. They consist of protein complexes and provide contact between neighbouring cells, between a cell and the extracellular matrix, or they build up the paracellular barrier of epithelia and control the paracellular transport. Tight Junctions (zonula occluden) Zonula Adherens Macula Adherens (Desmosome)

Hemidesmosomes Gap Junction

1. Slide 106, mammalian ovum in ovary of monkey, H&E, X400.

This illustrates a fixed, paraffin embedded and hematoxylin-eosin (H&E) stained section of a round cell. Look for it under the smooth capsule. Not all cells show nuclei if, while cutting, the knife does not hit them. 2. Slide 29, multipolar nerve cells, medulla oblongata of cat (cresyl violet:Nissl stain), X400.

This illustrates a type of cell with several processes. Notice clumped basophilic granular endoplasmic reticulum around the pale nucleus; nucleolus is dark.

3. Slide 1, mitochondria, kidney of rat which is stained with luxol fast blue and periodic acid-schiff + Hematoxylin (LFB-PAS-H), X400.

The mitochondria appear as green granules of rods in the cells lining the proximal convoluted tubules. Note that the cells have a PAS +ve brush border on their luminal aspect and PAS +ve basement membrane. 4. Slide 83, liver stained for glycogen with the PAS technique(pinkish), X400

This is within cells of liver. The congregation of granules (glycogen) to one side of cells is an artifact of fixation. The hematoxylin counterstain gives the background nuclear blue color. 5. Slide 113, melanin granules,eye, H&E, X400.

This is in pigment connective tissue cells of choroids of eye. Highly irregular branched cells full of dark brown pigment. Nuclei are apparent (Hematoxylin +ve). Outer pigment epithelial cells of the retina contain fewer discrete granules. 6. Slide 73, small intestine, H&E, X400.

Mitotic figures in crypts (tubular glands) of mucosa.

7. Slide 11, Golgi apparatus, dorsal root ganglion, (silver impregnation technique + hematoxylin), X400.

Large and small neurons with black comma-like structures

Electron micrographs of the cell

The Cell Cytoplasm of Purkinje cell of cerebellum shows numerous conglomerations of granular endoplasmic reticulum (seen as nissl bodies by L.M.). Mit = Mitochondria Ri = Free ribosomes. X15,200

The Cell Nu = Nucleus D = Desmosome IC = Interfacial canal M = Melanin Fi = Filaments (tonofilaments)

Electron micrograph of a platinum replica of a cell which had been frozen without fixation and then fractured to enable visualization of the cell interioir, especially membrane faces. After freeze fracturing pores in the otherwise smooth nuclear envelope (n), sheets of fenestrated endoplasmic reticulum (er), the cytoplasmic matrix (cm) and a variety of cytoplasmioc vacuoles all are clearly visible. Onion root tip. X36,000.

Electron micrograph of a portion of a elta cell. The granules are homogenous and tend to fill their limiting membrane, but they vary considerably in density. It is not clear whether these cells represent a distinct type or are altered alpha cells. X21,000. (courtesy of A. Like)

Photomicrographs of successive changes in a lily endosperm cell during mitosis, as seen in the Normarski optical system. This system has the advantage of revealing spindel fibers in living cells. Time after A:B, 14 min.; C, 1 hr. 4 min.; D, 1 hr. 14 min.; E, 1hr. 33 min.; and F, 1 hr. 47 min.