CELLS AND TISSUES IN ROUTINE PREPARATIONS

Most of the microscope slides used in this course (and in the study of pathology) are tissue samples, which were embedded in paraffin wax. Six to 8 μm slices of these tissue samples were affixed to glass slides, stained with hematoxylin and eosin (H & E), and protected with a coverslip. In this laboratory period, you should become acquainted with the appearance of such routine preparations and be able to compare their staining characteristics with other procedures utilized (embedding in plastic, etc.). Get in the habit of looking at each slide at the lowest magnification to orient yourself prior to advancing to medium and high magnifications. You will not use the 100X oil immersion lens frequently, but you should learn how to use it properly during this lab period.

Slide B71 or B72, liver, pig and human (H&E). These slides are typical H&E-stained paraffin sections. After getting an overview with the low magnification objective, study the section more carefully with 10x and 40X lenses in place. Most of the blue color results from the staining of nucleic acids (nuclei, nucleoli, cytoplasmic RNA). Because these organelles stain with the cationic (basic) stain, hematoxylin, these are referred to as basophilic structures. Note that most liver cells (hepatocytes) have fairly large, rounded nuclei and a few cells contain two nuclei (binucleated). Smaller, more dense nuclei are seen clustered in some of the connective tissue spaces. These represent both fibroblasts and nucleated blood cells (lymphocytes, monocytes). Hepatocyte cytoplasm is pink (mildly acidophilic), and cell margins can be seen. This is not always the case, but in the liver, small canals and vascular channels serve to aid in delineating cells. Liver has very little connective tissue, or collagenous fibers, which are stained lightly pink. Note also the presence of red blood cells, which are light red in color. Their diameter (~7 μm) also serves as a guide for calculating the size of other tissue structures.

Slides A13 and B38, esophagus, monkey (H&E). These H & E sections show somewhat greater variety of cell and tissue types than seen in sections of liver. They have a multi-layered lining of epithelial cells. Note that the most apical of these layers stains somewhat differently than the deepest layer of cells. Beneath the epithelium is connective tissue (mostly fibers with small numbers of cells) containing some smooth muscle and blood vessels. Peripheral to the connective tissue are thick layers of skeletal muscle. Both skeletal and smooth muscle fibers are densely packed with filaments (actin and myosin), which stain pink-to-red in these sections.

Slide A45, skeletal muscle, longitudinal section, plastic, (H&E). This tissue was embedded in plastic rather than paraffin, and sections can be cut much thinner (1-2 μm) due to the hardness of the plastic. Compare the appearance of skeletal muscle seen in this slide to that observed around the upper esophagus in slides Slide A13 or B38. Note how clearly the striations in muscle fibers can be seen in Slide A45.

Slide A92, coronary artery, cross-section, pig, (Trichrome). In addition to H & E stains, you will examine some slides stained to highlight specific cellular components (e.g., PAS staining for carbohydrates, elastic tissue stains, and immunostained preparations for specific proteins). This slide was stained with a trichrome stain to allow distinction to be drawn between connective tissue fibers and muscle fibers. In most trichromes, muscle fibers will be stained red and collagenous tissue green (shown here) or blue. Nuclei are usually purple, and nerve fibers have a gray color. The latter are scattered in the outer green layer of connective tissue in the coronary artery slide.

Cells

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.

Slide C39, ovary, cat, (Trichrome). Most of the ova are located at the periphery of the ovary right beneath the capsule or germinal epithelial layer. They contain large nuclei in which the chromatin is often clumped. Nucleoli are prominent, round, and dark staining. Each nucleus may not contain a nucleolus in the section you are studying. This occurs when the section is cut through the nucleus at a point not occupied by the nucleolus. Under high dry or oil immersion, focus up and down with the fine adjustment and note the granular appearance of the cytoplasm. The coarse granules are yolk granules, which accumulate in the cytoplasm of the ovum during the early stages of follicular growth.

Slide B72, liver, human, (H&E). Study the liver (hepatic) parenchymal cells and observe some of their features. The cells appear to be arranged in "cords" separated by spaces, but three-dimensional re-constructions of the liver have shown that the cells are actually aligned in broad plates or sheets. The spaces are the liver sinusoids, and red blood cells can be seen in many of them. The hepatic cells are polyhedral in shape and have round nuclei. Some cells are binucleated. The cytoplasm is granular and may contain small clear areas previously occupied by glycogen or fat droplets, which were dissolved during tissue processing. Cytoplasmic pigment is unusually abundant in this preparation. Distinct cell boundaries are present between some of the cells.

Slide A53/54, blood smear, human, (Wright's). Examine, under high dry and oil immersion, the cells present in adult peripheral blood, which has been spread on a slide and stained with Wright's stain. Distinguish between red blood cells (erythrocytes) and white blood cells (leukocytes), but do not attempt to identify the various types of white blood cells at this time. The size of blood cells in a smear is larger than the size of blood cells observed in sections of tissue processed by conventional techniques for light microscopy. One reason for their larger size is that the cells spread when they contact the surface of the slide. Another reason is that the alcohols used in tissue processing dehydrate and shrink the blood cells in tissue sections.

CELLULAR ORGANELLES, INCLUSIONS & SURFACE SPECIALIZATIONS

The basic unit of tissues and organs is the cell. It can be seen in a wide variety of shapes and sizes, all of which contain an array of inclusions and organelles. Some of these are readily visible by light microscopy.

1. Nuclei, Interphase: 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.

Slide A53 or A54, blood smear, human (Wright stain). Observe the variations in size (relative to the cell), location and shape of the nuclei of the white blood cells. The white cells are larger and staining more intensely (especially the nuclei) than the sea of surrounding red blood cells (which are enucleate).

Slide B73, liver, monkey (H&E). Nuclei are staining a deep blue. Note both eu- and heterochromatin and nucleoli (staining much darker blue than the surrounding nucleoplasm). Some cells contain two nuclei.

Slide A21, fibroblast, human, (H&E). Fibroblasts are the main cell type found in connective tissue. They are stellate shaped cells containing an oval nucleus. The cytoplasm of these cells extends out from the nucleus as wispy processes and sometimes isn’t very visible. Note the prominent nucleoli present inside the nucleus of each fibroblast.

Figure 2: Diagram of the basic architecture of a cell. Note the location of the Rough Endoplasmic Reticulum and the Golgi apparatus within the cell. Taken from: Junqueira and Carneiro, Basic Histology, Text and Atlas, page 42, Figure 2-27.

2. Golgi apparatus: This organelle is usually located in a “juxta-nuclear” position or right next to the nucleus. See Figure 2. In H&E preps, the Golgi apparatus would appear as a pale unstained region (negative image) in the cytoplasm above or around the nucleus. Special stains are required to visualize this organelle with the light microscope, but it can be readily seen with EM.

Slide A3, Golgi apparatus, (silver stain). The epithelial cells lining the epididymal tubule have a prominent Golgi apparatus. Here they are stained a deep brown or black using a silver stain. The intensity of the staining varies across the slide, some areas being too black and others a pale brown.

3. Rough endoplasmic reticulum: 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. See Figure 2.

Slide B84, pancreas, human, (H&E). The exocrine portion of this gland (majority of the gland) is comprised of acinar cells, which are in the shape of round clusters (grapes). If you look for the nuclei, which are usually found in the basal aspect of the cells, you will see that they are surrounded by deep blue or purple cytoplasm. The basophilia of the cytoplasm is due to the presence of RER in this area of the acinar cell.

Slide B87, pancreas, alloxan and control rat, (H&E). Another example of basophilic staining near the basal aspect of the pancreatic acinar cell.

Slide B51, intestine, monkey (H&E). Next to the cross section of the duodenum on the far left of the slide, is a wedge of pancreatic tissue. Note the intensity of the staining (deep purple nuclei and nucleoli, bright pink cytoplasm) of the acinar cells. Note the basophilia in the basal cytoplasm, due to the abundance of RER.

4. Secretory granules: Usually seen as large acidophilic (e.g. pink in H&E preparations) granules located in the apical portions of secretory cells. See Figure 3 below. They stain intensely because of their high protein content.

Slide A19, lacrimal gland, human (H&E). Look for acidophilic granules in the apical portions of the glandular profiles. They are staining intensely pink in comparison to the basophilic nucleus and pale pink cytoplasm.

Slide A27, mast cells, mesentery, (T. blue and fuchsin). The tissue on this slide is a wedge of ovary and its surrounding mesentery (loose areolar tissue). On low power, identify the dark purple staining cells dispersed throughout the mesentery surrounding numerous tubular profiles of the oviduct. The dark purple staining cells are mast cells. On high power, look at the cytoplasm of these cells and identify the granules. They almost completely mask the basophilic nucleus and therefore, quite frequently look like a purple blob and mistaken as a staining artifact.

Figure 3: Diagram of a cell with secretory granules in the apical cytoplasm. Taken from: Junqueira and Carneiro, Basic Histology, Text and Atlas, page 87, Figure 4-26.

Slide B32, submandibular gland, monkey, plastic (H&E). This tissue is a composite gland consisting of both serous and mucous portions. On low power (10X), find the dark pink-purple staining acinar profiles (serous portions of the gland). Then at 40X, identify the secretory granules in the apical cytoplasm that are staining intensely acidophilic.

5. Intracellular fibrils and filaments: 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.

Slide A46, skeletal muscle, diaphragm, longitudinal and cross-sections (Fe Hem). Myofibrils can be seen in the cross section of muscle. The striations are formed due to the interactions between the actin and myosin filaments and are easily visualized in this preparation.

Slide BB40, skin, human (Fe Hem). Tonofibrils are a prominent feature in certain cells of stratified squamous epithelia. Look for these in the deep layers of the epidermis. See photomicrograph on the front cover of the lab manual.

6. Other organelles: These structures will be encountered and studied during the semester, and include lysosomes, mitochondria, smooth endoplasmic reticulum, peroxisomes, lipid droplets 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 Best’s Carmine. Ideally, control slides treated with salivary amylase should accompany such tissues stained for glycogen since a number of other substances stain positively with these procedures.

Slide BB37, heart, purkinje fibers, ox (Best’s Carmine). The cardiac muscle is staining a reddish-orange color. In contrast, the Purkinje fibers are staining intensely pink due to the large quantity of glycogen in their sarcoplasm. Focus up and down with the 40X objective to see the abundance of granules in the cytoplasm.

Slide B74, liver (PAS). Study the hepatic parenchymal cells under low power and then under high power to observe the small, red staining, glycogen granules in the cytoplasm. Glycogen, a polymer of glucose, is stored in the liver cells and serves as a depot from which glucose can be released when needed for the metabolic activities of cells. Large amounts of glycogen are stored in the liver and in skeletal muscle.

Slide B73, liver (H&E). Compare with the above slide. The cytoplasm of the hepatocytes appears clear and vacuolated due to the loss of glycogen during the fixation process.

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.

Slide A97, thin skin, human, pigmented & non-pigmented, (H&E). This slide contains two sections of skin, unpigmented with relatively few melanin-containing cells (left) and the pigmented, with much melanin (right). Under low power, find the epidermis of the section on the right. With high power, study the granules of melanin pigment that are present in the epidermal cells and note that the pigment is most abundant in the keratinocytes located near the dermis. Pigment decreases in the keratinocytes as the stratum corneum is approached. The stratum corneum is the layer of dead cells at the free surface of the skin. Compare to the same area on the unpigmented section of skin.

Surface specializations

1. Cilia. Slide B10, trachea, human, (H&E). Find the lumen of the trachea and observe the ciliated pseudostratified columnar epithelium, which lines the luminal surface. In some areas, the epithelium has been lost during tissue processing. Under high power, note the fine, thread-like cilia, which extend from the cellular apices into the tracheal lumen. In life, the cilia beat toward the pharynx to help expel mucus and foreign material. 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.

Figure 4: Pseudostratified columnar epithelia with cilia (C) on the apical surface of the cells (left photograph). EM of Cilia in the right photograph. Taken from: Wheater’s Functional Histology, a text and colour atlas, p. 90, Figure 5.15.

2. Stereocilia. Slide C22, epididymis, monkey, (H&E). Several cross sections of the duct are present in this section. Find the lumen in one of the cross sections and note the stereocilia, which project into the lumen from the epithelial cells. 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. The thin, dark line at the base of the stereocilia represents an area where secretory material has collected.

Figure 5: Stereocilia on the apical surface of cells from the epididymis. Taken from: Wheater’s Functional Histology, a text and colour atlas, p. 91, Figure 5.17

3. Striated or brush border. Slide B59, ileum, monkey, plastic, (H&E). Under low power, find the epithelium lining the rounded villous projections of the ileum. With high power, note the cells, which have large pale unstained areas within their cytoplasm that appear to open into the lumen. These are goblet cells and will be studied later. Focus up and down with the fine adjustment and study the prominent pale pink, striated border at the free surface. The striated border is found throughout the small intestine (as well as several other locations) and is referred to as striated because it appears to have vertical striations under oil immersion of the light microscope. Electron micrographs show that the border is formed by cytoplasmic extensions called microvilli. Microvilli of the small intestine are regularly arranged and consistent in height (about 2μm), which resembles the bristles on a brush, hence the other name, brush border. They greatly increase the surface area of the cells and function in absorption. 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.

Figure 6: Multiple microvilli form a striated border (SB) on the apical surface of simple columnar epithelial cells of the GI tract. These cells are specialized for the process of absorption and the microvilli increase the surface area of the cell several fold. The bottom two photographs are EMs. Taken from: Wheater’s Functional Histology, a text and colour atlas, p. 91, Figure 5.16.