Lab 1: Cytology

Objectives:

General considerations
1. Acquire a visual representation of the major morphologic features of the cell (at both light microscopic and electron microscopic levels).
2. Correlate light microscopic images with ultrastructural features.
The cell
1. Identify the following organelles:
  1. Plasma membrane
  2. Nucleus and nuclear membrane
  3. Golgi apparatus
  4. Mitochondria
  5. Endoplasmic reticulum (rough and smooth)
  6. Lysosomes
2. Identify the following inclusions:
  1. Glycogen
  2. Lipid
  3. Melanin
  4. Protein secretion granules
3. Relate morphologic features of cells to physiologic function.
  1. Examples include:
    1. Basophilic staining with nucleic acids and protein synthesis.
    2. Mitochondria with high energy requirement for ion transport.
    3. Uniform cytoplasmic particles with granules.
4. Review mitosis.
5. Identify the following stages of mitosis on tissue specimens:
  1. Interphase
  2. Prophase
  3. Metaphase
  4. Anaphase
  5. Telophase

Slide List:

2. Liver (also #166)

33. Lymph node

3. Epididymis

4. Liver - mitochondrial staining

32. Uterus

6. Pancreas

7. Adrenal

8. Liver

10. Adipose Tissue

11. Root tip

160. Intestine

The Cell : Glass Slide #2, The Liver

Cell Schematic:

This is a highly schematized artist's rendition of a eukaryotic cell as viewed with a light microscope. The boundary of the cell is sharply defined by a plasma membrane. In the middle a large round body, the nucleus, is prominent. Around the nucleus lies a transparent substance that fills the rest of the cell's interior, the cytoplasm. This space is packed with what seems at first to be a jumble of tiny miscellaneous objects. These consist of organelles, granules, fibers, vacuoles and other components that are discussed in more detail below. Familiarize yourself with the terminology of cellular components and relate the terms used to the cells shown on glass slide 2.

From 2D to 3D:

Cells vary widely in size and shape. In the body they are generally continuous and have extensive membrane contacts, so you will not usually see cells in their entirety. Most of our slides are 6 um thick plane sections cut at random orientations through various portions of such cells. As you study, try to develop three-dimensional concepts of cells and tissues from sectioned specimens. This example is intended to represent cytoplasmic contents both deep to and arising above the imaginary cut surface.

Hepatocytes 1:

This is an area from a section of dog liver taken with oil immersion optics. Locate similar areas on glass slide 2 and look for cells with distinct nuclear profiles. The light open spaces are normally filled with blood. Identify the round, heavily stained nucleoli within the nuclei of the cells. There may be more than one nucleolus per nucleus. Chromatin is the darkly stained, strand-like material at the edge as well as the thin strands that extend to the interior of the nucleus.

Multiple cell types are present. The isolated purple clump near the center contains one hepatocyte (H) and one very slender endothelial cell with a dark oval nucleus (E).

Hepatocytes 2:

The light microscope does not resolve cell membranes. However, there are often membrane-bound structures, intra- and extra-cellular proteins, and structural specializations that help distinguish adjacent cells. In the liver there are small channels (bile canaliculi) that do not stain. When oriented correctly, they indicate the edges of single cells. The center hepatocyte is one such example.

Try to find hepatocyte boundaries in this image made at 100X and on your slide.

Pig Liver: Glass Slide #166

Sinusoids

This example of pig liver is stained less intensely, allowing different cellular details to be appreciated. Cell margins (some with bile channels called canaliculi) and nuclear morphology are evident. In much of this section the blood sinusoids (S) are collapsed and red blood cells have been rinsed from the organ.

Topic: Sinusoids - zoom

The yellow lines indicate the approximate locations of cell boundaries. S = Blood Sinusoids. Triple stain, 25X original

Membrane Structure

Membrane Strux:

Schematic drawing of the molecular structure of the plasma membrane. Note the one-pass and multipass transmembrane proteins. The drawing shows a peripheral protein in the external face of the membrane, but the proteins are present mainly in the cytoplasmic face, as shown in the next figure. (Junqueira and Carneiro, used with permission)

Lipid Model:

A cytomembrane is a fluid bimolecular lipid layer with proteins floating in the lipid. The lipid molecules have polar ends (purple) apposed to the aqueous environment of the cytoplasm and extracellular space, and non-polar ends (yellow) thermodynamically excluded from a polar environment. Globular portions of membrane proteins are confined to the lipid layer of the membrane while the polar or charged portions of the molecule protrude into the cytoplasm or the extracellular space.

Assoc. Molecules:

The fluid mosaic model of membrane structure. The membrane consists of a phospholipid double layer with proteins inserted in it (integral proteins) or bound to the cytoplasmic surface (peripheral proteins). Integral membrane proteins are firmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are called transmembrane proteins, whereas others are embedded in either the outer or inner leaflet of the lipid bilayer. Integral membrane proteins have specialized regions where hydrophobic amino acids interact with the hydrophobic portions of the membrane. Many of the proteins and lipids have externally exposed oligosaccharide chains.

Freeze Fracture:

Membrane cleavage or fracture (lower right) occurs when a cell is frozen and fractured (cryofracture). Most of the membrane particles (1) are proteins or aggregates of proteins that remain attached to the half of the membrane adjacent to the cytoplasm (P, or protoplasmic, face of the membrane). Fewer particles are found attached to the outer half of the membrane (E, or extracellular, face). For every protein particle that bulges on one surface, a corresponding depression (2) appears in the opposite surface. Membrane splitting occurs along the line of weakness formed by the fatty acid tails of membrane phospholipids, since only weak hydrophobic interactions bind the halves of the membrane along this line.

Endoplasmic Reticulum - Lymph Node; Glass Slide #33

Endoplasmic Retic.:

In many cells a significant fraction of the cytoplasm is filled with aggregates of cytomembranes called the endoplasmic reticulum (ER). The ER, involved in the synthetic and secretory activity of the cell, is divided into two broad categories, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER). RER stains basophilically due to its ribosomal content and thus its presence is apparent with the light microscope (LM). SER can only be visualized directly with an electron microscope or special stains.

Topic: Endoplasmic Retic.

Schematic of a plasma cell. RER lumen indicated by pink. Note that the RER is excluded from the nucleus and Golgi apparatus (described on next page).

Plasma Cells:

Plasma cells contain a round, eccentric, heavily stained nucleus. The cytoplasm, except for an area adjacent to the nucleus, is very basophilic. The unstained area is filled with smooth cytomembranes called the Golgi vacuole. Glass slide 33 is a section of a lymph node. Find an area containing plasma cells. Note the position of the nucleus with its heavily stained chromatin which is characteristic of plasma cells. Observe the basophilia of the cytoplasm and note the Golgi vacuole (Golgi apparatus) adjacent to the nucleus in many cells. A few plasma cells have pale staining cytoplasmic granules that replace the basophilia. Such cells contain large amounts of protein in the cisternae of the RER and are no longer forming protein.

Topic: Plasma Cells

The plasma cells are ubiquitous in active lymph nodes, mixed with macrophages and supporting cells.

Plasma Cell TEM:

Electron micrograph of a plasma cell showing an abundance of rough endoplasmic reticulum (R). Note that many cisternae are dilated. Four profiles of the Golgi complex (G) are observed near the nucleus (N). M, mitochondria.

Topic: Plasma Cell TEM

N = nucleus, G = Golgi, M = mitochondria, R = RER

Epididymis - Glass slide #3

Golgi morphology:

The epididymis is a single coiled tube that may be 60 meters in length in the boar. This organ, associated with the testicle, is the site for maturation and storage of sperm. Locate one of the two small sections of the epididymis on glass slide 3 and find an area similar to the micrograph on this page. The epididymal lumen is lined by a layer of tall, columnar shaped cells. They are polarized from the base to the lumen (or apex). With silver staining, a pale nucleus can be discerned at the base of each cell. The darker brown stained structure beside the nucleus in each cell is the Golgi apparatus, which consists of a series of lamellae of smooth cytomembranes involved in the secretory activity of the cell. Secretory material synthesized by other cytoplasmic organelles passes through the 'Golgi' for molecular modification prior to being extruded by the cell. Thus, the Golgi apparatus contributes to the functional and morphologic polarity of the cell.

Topic: Golgi morphology

Left side - silver stain; right side - triple stain. The nucleus (N) of epididymal cells is located basally, while the Golgi apparatus (G) is located more apically in the cytoplasm. The lumen of the tubule is indicated (L). Note that the Golgi is indicated in triple stained cells only by an unstained (or lighter-staining) region.

Golgi closeup:

The Golgi apparatus has two key functions: 1) it houses the enzymes responsible for the synthesis of complex carbohydrate structures found on many proteins and lipids; and 2) it is an important site for the sorting of proteins and lipids for delivery to specific subcellular destinations. The 'input' area is called the 'cis' face and the 'output' region is called the 'trans' face.

Topic: Golgi closeup

Artist's schematic of a typical Golgi apparatus. Transport vesicles shuttle between adjacent lamellae.

Golgi closeup:

Transport through the Golgi apparatus occurs via pools of recycling vesicles. These bud from the margins of flattened membranous lamellae and carry their contents to more distal regions of the organelle. Eventually, other specialized vesicles carry the modified cell products to their next destination.

Topic: Golgi closeup

Vesicle transport within the Golgi apparatus. The trans- face of the Golgi apparatus produces multiple vesicle types, each with its own function or destination.

Liver Mitochondria - Glass slide #4

Liver mitochondria:

Mitochondria are cellular organelles involved in oxidative phosphorylation processes. Mitochondria are best viewed with LM after staining with supravital dyes, as in glass slide #4. Special purpose stains such as used in this section demonstrate little cytologic detail beyond the particular component of interest. This image is a group of hepatocytes (liver cells) that have been stained with a mitochondrial stain and imaged using a 100X oil immersion objective. The numerous small purple dots (less than 1um in size) scattered randomly in the cytoplasm are mitochondria. Nuclei stain pink-fuscia; red blood cells stain light blue. On glass slide 4, examine the distribution of mitochondria within the hepatocytes. The number and shape of the mitochondria is all that can be seen with LM. By TEM (left), mitochondrial cristae are easily identified.

Topic: Liver mitochondria

TEM of mitochondria - In this image the round structures with parallel channels of membrane are mitochondria. The membrane provides an interface for the enzyme systems that allow mitochondria to produce cellular chemical energy in the form of ATP.

Liver mitochondria:

Additional area with well demonstrated mitochondria, by LM.

Topic: Liver mitochondria

Cristae are foldings of the mitochondrial inner membrane. Located on the membranes are complexes of enzymes that are responsible for the organelle's function.

Uterine Endometrium - Glass Slide #32

Macrophages:

In this section of a uterus from a bitch, the area just deep to the lumen contains many active macrophages. Find an area on your glass slide #32 that is similar. These macrophages are large cells containing red blood cells in phagocytic vacuoles. On glass slide 32, identify similar appearing cells in the tissue. The blood cells are contained in secondary lysosomes (digestive vacuoles), which appear as brown/black granules within the cells. The ingested cells may be intact or more likely in varying stages of cytolysis. Eventually they form pigment granules representing the end product of hemoglobin degradation. Cytolysis of red blood cells is a very rapid process; 20 minutes following ingestion by a macrophage, the rbc can no longer be identified as a cell.

Topic: Macrophages

Red arrows indicate active macrophages. These contain phagocytized red blood cells, but almost any kind of cellular/organic 'waste' may be encapsulated, degraded, and recycled. Macrophages have diverse morphologies and roles, depending on their location.

Lysosomes:

Lysosomes are membrane-bound capsules of hydrolytic enzymes. Definitive identification of lysosomes can be accomplished only by histochemical demonstration of one or more of the acid hydrolases which they contain. The presence of lysosomes can be inferred from cytoplasmic vacuoles that contain phagocytized cells or particulate matter.

Topic: Lysosomes

In transmission electron micrographs, primary lysozomes are smaller and more electron-dense. The upper dark sphere is a primary lysosome. Two secondary lysosomes are below the primary lysosome.

Calf Pancreas - Glass slide #6

Zymogen granules:

Zymogens are protein molecules that are inactive forms of a digestive enzyme. The zymogen granules seen here are membrane-bound droplets of concentrated proenzyme molecules. They are stored in the apex of pyramidal-shaped cells. These cells are arranged in roughly spherical structures called acinin, hence the name 'acinar cells'. Acinar cell aggregates comprise the exocrine portion of the pancreas, a gland that secretes several digestive enzymes into the lumen of the small intestine. Acini are the functional subunit of the exocrine (zymogen-secreting) pancreas.

Topic: Zymogen granules

Pyramidal cells (yellow outline) of a pancreatic acinus (green outline). Acini are the primary functional subunit of the ducted (blue arrow) exocrine or zymogen-secreting pancreas. In tissue sections acini often appear irregular in shape, though boundaries between adjacent acini are easily identified. Within acinar cells, secretory granules are stored in the apical cytoplasm (red arrows) until needed.

Granule ID:

In these acini, the zymogen granules stain intensely red. Examine the apical regions of the acinar cells in glass slide #6 at high power.

Topic: Granule ID

Electron micrograph of a pancreatic acinar cell from the rat. Numerous mature secretory granules (S) are seen in association with condensing vacuoles (C) and the Golgi complex (G).

Granule variation:

Scanning around the slide, notice that there are regional variations in the amounts of stored (intracellular) zymogen granules (intense red) within different pancreatic acini.

Topic: Granule variation

Pyramidal cells of a pancreatic acinus can appear triangular, trapezoidal or rectangular in shape. Zymogen granules are apical - adjacent to the lumen of the acinus, which drains via ducts.

Melanin - Glass slide #7

Melanin in Adrenal:

Melanin is a pigment found in various tissues of the body; skin, hair, parts of the brain, and the adrenal gland are examples. In this example melanin producing cells are found under the capsule of the adrenal gland. The capsule of an organ is the outermost layer comprised of connective tissue. The granules are so numerous in the cytoplasm that the nucleus is often totally obscured. Albino animals lack the enzyme tyrosinase, which is involved in the production of melanin, therefore these animals lack melanin pigment.

The section of the adrenal gland on glass slide #7 was stained with saffranin O which does not bind to melanin. How do you account for the black appearance of the melanin granules?

Topic: Melanin in Adrenal

Blue arrows indicate adrenal cells containing melanin.

Liver glycogen - Glass slide #8

Glycogen Staining:

The large image is a rabbit liver section stained with a special dye that indicates the presence of glycogen. The brilliant red stained clumps in the cytoplasm of the hepatocytes represent aggregates of glycogen. On your slide note the glycogen can appear on only one side of any given cell. This is 'glycogen flight,' an artifact due to a wave of chemical fixative washing the glycogen into large aggregates as it penetrated the tissue.

Topic: Glycogen Staining

Blue arrows indicate hepatocytes with significant glycogen deposits. Stain = PAS, 25X objective

Adipocytes - Glass Slide #10

Common Adipose:

Lipids present in cells as cytoplasmic inclusions are non-polar molecules such as the triglycerides of body or milk fat and the cholesterol derived lipids such as steroid hormones. Since these bimolecules lack polar groups they are excluded from the aqueous environment of the cytoplasm and are sequestered in varying sized droplets in the cell. A consequence of the lack of ionic groups on lipids is that ordinary histologic stains will not attach to them. Without special stains fat cells look like ‘cellular ghosts’ with a distinct plasma membrane surrounding an empty cytoplasm. The adipocyte contains a thin rim of cytoplasm surrounding a single fat droplet. The lipid contents are extracted by the tissue processing procedure. The nucleus of the cell is displaced to one side giving the cell a signet ring appearance. Locate an aggregate of large, pale staining fat cells on glass slide #10. They are typically found near the edges of the section.

Topic: Common Adipose

Common adipocytes are present near the top of this image. Asterisks in lipid droplet. Multilocular adipocytes (M) have much smaller lipid inclusions and make up the tissue commonly known as 'brown fat'.

Multilocular Adipose:

Multilocular adipose tissue contains many small lipid droplets rather than a single large droplet. Small lipid droplets are also found in other cell types.

Topic: Multilocular Adipose

Multilocular adipose tissue. Note the central nucleus, multiple fat droplets, and abundant mitochondria. A sympathetic nerve ending is shown at the lower right.

Most of glass slide #10 consists of multilocular adipose tissue.

Nucleus and Chromatin - Glass slide #2

Hepatocyte nuclei:

On glass slide #2 examine several nuclei. Note the pattern of heterochromatin as a rim of stained material which extends into the nucleoplasm. The unstained areas contain euchromatin. Nuclei with this appearance are 'leptochromatic' (lepto-, thin) and the cells that contain such nuclei are typically synthetically active. [H=hepatocyte, E=endothelial cell]

Hepatocyte nucleoli:

The nucleolus of eukaryotic nuclei contain the nuclear DNA that serves as a template for the formation of ribosomal RNA. The nucleolus may contain segments of DNA from several chromosomes; thus, young recently divided cells may have several nucleoli in a nucleus. The multiple nucleoli coalesce into a single structure with time. Examine the nuclei of the hepatocytes on glass slide 2 and distinguish the more round nucleoli from the irregular clumps of heterochromatin.

Schematic:

Three-dimensional representation of a cell nucleus to show the distribution of the nuclear pores, the heterochromatin (dark regions), the euchromatin (light regions), and a nucleolus. Note that there is no heterochromatin closing the pores. The number of nuclear pores varies greatly from cell to cell.

TEM of nucleus:

Electron micrograph of a nucleus, showing the heterochromatin (HC) and euchromatin (EC). Unlabeled arrows indicate the nucleolus-associated chromatin around the nucleolus (NU). Arrowheads indicate the perinuclear cisterna. Underneath the cisterna is a layer of heterochromatin, the main component of the so-called nuclear membrane seen under the light microscope.

TEM of nucleolus:

Electron micrograph of a nucleolus. The nucleolar organizer DNA (NO), pars fibrosa (PF), pars granulosa (PG), nucleolus-associated chromatin (NAC), nuclear envelope (NE), and cytoplasm (C) are shown.

Nuclear Morphology - Glass slide #33

Nuclear morphology:

On glass slide 33 evaluate the pattern of heterochromatin in the plasma cells. Nuclei with heavy patches of heterochromatin are termed 'pachychromatic' (pachy= thick). Notice that the nucleus and cytoplasm are both basophilic due to the presence of nucleic acids - in the cytoplasm as ribosomes and messenger RNA. Although the cells are very active in protein synthesis, the nuclear chromatin is relatively condensed because only a few genes are responsible for the large amount of mRNA in the cytoplasm.

100X objective

Topic: nuclear morphology

Plasma cells, which are common in lymph nodes, contain highly patterned nuclei that help in the cell's identification. Euchromatin 'spokes' and 'rim' around the nuclear membrane can be suggestive of a wagon wheel.

Phases of Mitosis - Glass slide #11

Micrographs:

Images obtained with a confocal laser scanning microscope from cultured cells. An interphase nucleus and several nuclei are in several phases of mitosis. DNA appears red, and microtubules in the cytoplasm are blue. Medium magnification. A: Interphase. A nondividing cell. B: Prophase. The blue structure over the nucleus is the centrosome. Note that the chromosomes are becoming visible because of their condensation. The cytoplasm is acquiring a round shape typical of cells in mitosis. C: Metaphase. The chromosomes are organized in an equatorial plane. D: Anaphase. The chromosomes are pulled to the cell poles through the activity of microtubules. E: Early telophase. The two sets of chromosomes have arrived at the cell poles to originate the two daughter cells, which will contain sets of chromosomes similar to those in the mother cell. F: Telophase. The cytoplasm is being divided by a constriction in the cell equator. Note that the daughter cells are round and smaller than the mother cell. Soon they will increase in size and become elongated. (Courtesy of R Manelli-Oliveira, R Cabado, and G Machado-Santelli.)

Schematic:

Schematic emphasizing differences between phases of mitosis.

Mitotic Figures in Situ - Glass slides 160 & 163

Intestinal glands:

This image is a photomicrograph of part of the wall of the intestine. Select an area on glass slide 160 or 163 similar to this. Attempt to find metaphase configurations in the epithelial cells of the intestine. If you have difficulty in finding mitotic figures on glass slide 160/163 try glass slide 1, which is a section of ovary. It is valuable to learn to recognize mitotic figures now because you will encounter them on many of the tissue sections that you examine later in the course. If time permits you can view additional slides with good mitotic figures. These include: glass slide 1 - ovary, glass slide 74 - lymph node, glass slide 76 - palatine tonsil, and glass slide 80 - nasopharyngeal tonsil.

Topic: Intestinal glands

Yellow arrows indicate chromatin of mitotic figures in this rapidly replaced region of the intestine.

Intestinal glands:

Topic: Intestinal glands

Anaphase (black pointer) and metaphase (red arrows) mitotic figures in intestinal glands. Source: Lindsay Bush - iPhone camera through student microscope ocular.