VM8054 Veterinary Histology

Exercise 3

Cells, Organelles, and Inclusions

Author: Dr. Thomas Caceci
What am I, Life? A thing of watery salt
Held in cohesion by unresting cells,
Which work they know not why, which never halt,
Myself unwitting where their Master dwells?

--John Masefield (1878-1967)
Sonnets 14
Objectives for this Exercise

The Cell Theory

Nowadays we take the concept of "cells" as the basis for living organisms so much for granted that it's hard to realize it wasn't always this way. The cell theory enunciated in 1838 and 1839 by Theodore Schwann (1810-1882) holds that all living things are composed of cells, and that cells can only arise from pre-existing cells. Although the truth of this view is undoubted today, the concept of "spontaneous generation" of life from nonmoving precursors died very hard, and for its day, the cell theory was a daring and avant-garde concept: what today we'd call "cutting edge" science. This laboratory exercise will provide you an opportunity to examine the structure of animal cells and some of their subunits.


The Nucleus and Cytoplasm

Any slide in the set will serve to demonstrate the nucleus and the cytoplasm of typical cells. Nuclear morphology varies greatly from one cell type to another, and can be used as a rough guide to cell type and activity. If you examine a cell specialized for the synthesis of proteins, for example, its nucleus will differ in appearance from that of a cell with a different function. Cells with a mechanically supportive function will have nuclei nothing like secretory cells, and so on. It is worth your while to get a feel for the variations of nuclear appearance in different tissues and organs, as this will be of importance in diagnosing pathologic conditions. The nucleus is one of the first of the subcellular structures to undergo changes when cellular death is imminent.


Basophilia

One pretty much universal characteristic of nuclei is that they tend to take up basic stains i.e, those which are alkaline in nature. Since nuclei contain comparatively large amounts of acidic material (DNA and RNA) there tends to be a very strong affinity between the nucleus and this sort of stain. This is called basophilia or a basophilic reaction. In the case of H&E preparations, nuclei typically stain blue to dark purple, because the hematoxylin is a basic stain. Cresyl violet is another basic stain, usually used in nervous system preparations, and binds strongly to RNA and DNA.

Cytoplasm tends to be much poorer in acidic materials than nuclei (there are some exceptions to this statement) and the cytoplasmic reaction with hematoxylin (or other basic stains) tends to be markedly less than that of the nucleus. Frequently, cytoplasm is termed acidophilic or (in the case of H&E slides) eosinophilic in its staining reaction. Of course, the degree of basophilia and/or acidophilia will vary depending on cell type and physiologic state, and the terms are relative, not absolute.

To see an example of basophilic and eosinophilic staining, click here.

A good example of this principle can be found on slide 34. This is a preparation from the pancreas. Most of the cells have eosinophilic granules/cytoplasm at one end, while that below the nucleus is moderately basophilic. This cytoplasmic basophilia results from the presence of large amounts of RNA in the RER, which is clumped at the basal region of the cell. The apical eosinophilic areas are relatively free of RNA, and contain the secretory granules of peptides that will eventually be released.


Effect of Cellular Activity on Nuclear and Cytoplasmic Appearance

Some types of cells are specialized as large scale protein producers (the pancreatic cells just examined are a classic example) and their cytoplasmic staining and nuclear morphology reflect this. Since the DNA of these cells is uncoiled (for access during protein synthesis) the staining reaction in these regions of the nucleus is less pronounced. The DNA still absorbs the stain, but the stained material is dispersed throughout the entire nuclear volume.

A certain amount of DNA will bind a certain amount of any given basic dye. But as a general rule, the actual appearance of the stained material or structure in microscope sections depends on how thick the stain is. The reason for this is physical: stains impart color by absorbing light, and the more stain there is between you and the light source, the more heavily stained a structure will appear.

Because uncoiling of the DNA to be read disperses the stain molecules over a larger area, they aren't "stacked up" on top of each other. Hence they don't absorb so much light per unit thickness of the section, and thus the apparent intensity of staining is reduced.

Nuclei with uncoiled DNA and with spotty or absent staining are described as "vesicular." You also expect to see dense nucleoli in these vesicular nuclei. The nucleolus is very strongly basophilic, because it's made almost entirely of RNA.

As a first approximation when you are attempting to identify a cell type, the combination of a basophilic cytoplasm and a vesicular nucleus sporting prominent nucleoli is a pretty good clue that the cell in question is secretory, and more to the point, it is secreting proteins.

Secretory nuclear morphology can be seen on slide 552 in neuron cell bodies. Neurons are very active secretory cells, producing proteins for transport out of the cell.

To see an example of this type of nuclear morphology, click here.


Other Organelles

Most subcellular organelles and inclusions are visible with the light microscope, but unfortunately, routine H&E staining is usually insufficient to visualize these structures. Special procedures must be used, but don't expect them to look like H&E stained material; the purpose of such staining routines is not to reveal general morphology, but to take advantage of some chemical peculiarity to make a particular structure or structures visible. Even with special routines, the level of detail visible in the LM is not too high, and the electron microscope is needed to get resolution high enough to make out the internal structure of, say, mitochondria.


Mitochondria

Much to many people's surprise, mitochondria are visible in the light microscope. It requires a special stain to make them visible. Mitochondria are very small, on the order of 0.1 to 0.2 µm in diameter, actually about the size of bacteria. Hence they are at the limit of resolution of even very good light microscopes. We are much more used to seeing images of them as they appear in the electron microscope. The mitochondrion was first described in 1890, but until fairly recently mitochondria were "black boxes" that were known to consume oxygen and produce ATP. Until the mid-1950's, when instruments capable of showing internal details became available, and it became possible to formulate and test hypotheses as to their function, no one had a clue as to how the mitochondrion was built and how it might work.

To see an example of mitochondria, click here.

Ribosomes

Ribosomes and rough endoplasmic reticulum (RER) are not individually visible in the light microscope, but large aggregations of them show up in the form of basophilic regions of cytoplasm. They are most easily seen in nervous tissue, in the large cell bodies of neurons. The ribosomes of the rough endoplasmic reticulum in neurons take up basic stains like cresyl violet quite readily, and as the RER of these cells is in large flakes or patches, the staining renders the RER visible. The basal basophilia of the pancreatic acinar cell is another example of this phenomenon.

To see an example of RER in the form of Nissl bodies, click here.

You should see this on slide 552. The flaky purplish material inside neurons is the RER with ribosomes on it.


Cilia

Cilia are very common in mammalian systems, and are most easily seen in slides of the respiratory system. Of course, ciliated cells aren't restricted to mammals or to the respiratory system. They were first described from the reproductive tracts of birds (by Purkinje and Valentin in 1831) and are present in all of the metazoa.

To see an example of cilia, examine slide 26, the trachea from a dog. This organ is lined with ciliated cells. The cilia can be seen projecting into the lumen as a sort of "fringe" whose function is to trap inhaled dirt particles and sweep them back up to the pharyngeal region. Similar cells can be seen in some areas of slide 115.

To see an example of ciliated cells, click here.


The Plasma Membrane

The plasma membrane, the limiting barrier between a cell and the outside world, is not directly visualizable with the LM, but its presence can be deduced from the fact that there is demonstrably an "inside" and an "outside" for any given cell. Although the concept of the membrane was accepted from the mid 19th century onwards, it was not in fact until the 1950's that the EM made it possible to demonstrate it as a physical entity.


Plasma Membrane Specializations

Plasma membrane structural specializations are often visible in the LM. One such specialization is that for absorption, the microvilli.

To see examples of microvilli, click here.

Examine slide 40. This is the duodenum of a monkey, and at high power, you will easily a refractile band along the luminal surface of the cells lining this organ. This is the brush border or striated border. It's made up of innumerable microvilli, packed close together and in orderly array. Individual microvilli are too small to be resolved, but in the aggregate they present the appearance of a differently stained band along the edge of the epithelium. Microvilli are to be expected wherever it's necessary to increase surface area without increasing cell size, especially in regions of absorption, such as the intestine and kidney.


The Glycocalyx or Cell Surface Coat

The PAS stain reveals a cellular component which strictly speaking is neither an organelle nor an inclusion, but falls into the category of plasma membrane specializations. This is the glycocalyx or cell surface coat. All cells are covered on their outside surfaces by a polysaccharide material they secrete, which is bound to the outer leaflet of the plasma membrane. This layer acts as an ion trap, and helps the cells maintain normal internal levels of ions; it also serves a protective function. A demonstration of this is available.

View examples of the glycocalyx by clicking here.

Microtubules

Microtubules are organelles. One place they're found is in the core in cilia and flagellae, but while cilia and flagellae are easily visible in the LM, the microtubules inside unfortunately are not. An electron microscope is used demonstrate their basic structure.

Slide 54 provides a good example of another microtubular structure. This slide shows mitosis in whitefish eggs. In the mitotic process, spindle fibers are formed connecting condensed anaphase chromosomes to anchor points in each forming daughter cell. These spindle fibers are microtubular in nature. The whitefish embryo grows rapidly and mitotic figures are numerous.

Microtubular Construction

Microtubules are composed of polymerized subunits of a special protein, tubulin. In the case of spindle fibers, the fibers are formed, and as division proceeds, they are depolymerized by removal of subunits at the end furthest from the chromosomes. Eventually the entire set of tubules has been dismantled. When division occurs, the tubules are reassembled and disassembled again. If you locate a cell whose chromosomes are lined up in the center, or just beginning to separate, you should be able to make out the microtubules of the spindle fibers. These appear to radiate from two points, one on each side of the about-to-divide cell.

To see an example of microtubules, click here.


The Golgi Apparatus

One organelle which was the source of much controversy is the Golgi apparatus, named for its discoverer, Camillo Golgi. The Golgi apparatus (out of respect for the man, not "golgi" with a lower-case "g") is intimately involved in the chemical modification of proteins synthesized in the RER (usually by adding sugars to them) and with their "packaging" into membrane bound vesicles for transport to the surface of the cell and release.

The Golgi apparatus, as you might expect, is very active and most easily visible in cells that are secreting materials rapidly. The appearance of this organelle is another clue that the cell is secretory in nature. One place it's relatively easy to see this organelle is in the cells lining the epididymis, an excurrent duct of the male reproductive system.

On slide 241 you'll see the Golgi apparatus as a clear area near the nucleus of the cells lining the organ. These slides are stained with H&E, so the unstained Golgi apparatus stands out from the background. The Golgi apparatus stains poorly with H&E because it has neither an acidic or a basic nature; but because of its high content of sugars, it can frequently be rendered visible with the PAS stain for carbohydrates.

For many years, the existence of this organelle was controversial, and it was considered by some people to be an artifact of preparation; but its demonstrability in unstained preparations (using phase contrast microscopy) and in the transmission electron microscope settled the question. The Golgi apparatus is found in most cell types, though it's sometimes inconspicuous.

To see examples of the Golgi apparatus in the light and electron microscopes, click here.


Inclusions

Subcellular inclusions are nonmoving material that are usually the result of metabolic activity of the cells in which they're found. In most cases they require special stains to be seen clearly. They may be pigments produced in the cell, or they may be accumulations of nutritive materials such as fat or carbohydrates.

Lipofuscin

Lipofuscin pigment, often called "wear-and-tear" pigment, is easily seen in nervous tissue and in areas of high macrophage activity. Lipofuscin is the undigested residue of subcellular lytic reactions. As organelles become aged and useless, they are broken down for their components, and what is left that can't be salvaged is the pigment you see here. It tends to be greater in older animals. Since neurons do not divide, they tend to accumulate the pigment over the years. Hence the name "wear-and-tear" pigment. Lipofuscin is also found in fat cells, and it's the material which turns the fat of older animals yellow.

Macrophages are scavenger cells, and their job is to pick up and destroy potentiallu harmful materials, such as bacteria, dust particles, etc. They also are called in to clean up an area of infection after other immune system cells have dealt with it. Macrophages can contain lipofuscin from almost any source: dead cells, their own organelles, killed bacteria, and so forth. They may also contain the pigment that's produced by the breakdown of old red blood cells, hemosiderin (see below). To see examples of lipofuscin, click here.


Glycogen

Glycogen is another type of pigment inclusion. Glycogen is a polymer of glucose, used as a storage material when glucose supplies are high. The liver and the muscles store large quantities of it to release on demand by depolymerizing it back to glucose. You'll see it on slide 500. This is stained with the periodic acid-Schiff's reaction (PAS). The PAS reaction turns carbohydrate constituents a magenta color. Unlike lipofuscin, glycogen is not exclusively located in one part of the cell. In those cells which accumulate it, it will be found scattered through the cytoplasm.


Melanin

Melanin is yet another pigment inclusion. It's the most common of the biological pigments, and is found, so far as I'm aware, in virtually every phylum of the animal kingdom.

You can see melanin on slide 24; in the deep regions of the skin of the footpad, it's visible as brown granular material in cells at the junction of the epidermis and the underlying connective tissue. You may encounter melanin in neurons as well.

To see an example of melanin, click here.


Hemosiderin

Hemosiderin is the indigestible residue of blood cell destruction, and hence the "hemo" in the word refers to hemoglobin, the blood's oxygen carrying pigment. The heme moiety of hemoglobin contains an iron atom at its core, and so staining routines for hemosiderin rely on detection of iron. Hemosiderin is most easily demonstrated in the spleen, where aged erythrocytes are phagocytosed; but it can also be found in hemal nodes and in the liver.

Hemosiderin can be seen on slides 673 and 674. Because reclamation of erythrocytes takes place in the spleen, hemosiderin tends to accumulate in large amounts in the resident splenic macrophages. You can compare the staining reaction on slide 673 (stained with H&E) to that on slide 674, stained with the Prussian blue reaction. The hemosiderin is visible in the former slide as as a deep brown pigment, more or less indistinguishable from lipofuscin (or melanin, for that matter). On slide 674, it has been stained a deep blue color that reflects the presence of iron. This is a classic example of differentiating morphologically similar structures by their chemical differences, i.e., histochemistry in action.


Lipid

Lipid is usually a nutritive inclusion, one that has a role as an energy source in the metabolism of the cell. The time of residence of lipid in a cell may be very short (minutes to hours, as in absorptive cells of the gut) or it may be much longer (days to months, as in adipose tissue). Inside the cell it's stored in vacuoles that are usually easily seen with the light microscope, but are much better appreciated in the EM. If lipid metabolism is interfered with--there are some genetic defects in which this occurs--a lipid storage disease may result, in which lipid accumulates in abnormal locations or in abnormal amounts.

To see an example of lipid in intestinal cells, click here.


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