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Term Paper on Cell Organisation
Term Paper Contents:
- Term Paper on the Introduction to Cell Organisation
- Term Paper on the Organells Seen in Fixed and Stained Cells
- Term Paper on the Experimental Evidence for Cell Organelles in Living Cells
- Term Paper on the Variation in Organisation of Animal Cells
- Term Paper on the Variation in Organisation of Plant Cells
- Term Paper on the Variation in Organisation of Bacterial Cells
- Term Paper on the PPLO (Pleuro-Pneumonia like Organisms)
- Term Paper on the Organisation of Viruses
- Term Paper on the Staining Technique for Living and Fixed Cells
- Term Paper on the Auto Radiography
- Term Paper on the Microscopical Techniques to See Stained Structures in Cells
Term Paper # 1. Introduction to Cell Organisation:
Although the term cell was first used in biological literature by Robert Hooke in 1665 to describe a compartment seen in the structure of cork, the cell as a unit of structure and function of all living things was not recognised until about 1839 when Schleiden and Schwann developed the cell doctrine. In modern terms the cell may be defined as “a unit of biological activity delimited by a selectively permeable membrane and capable of self-reproduction in a medium free of other living systems”.
It is common knowledge that the cell is differentiated into nucleus and cytoplasm, each of which is surrounded by a membrane, the nucleus by the nuclear envelope and the cytoplasm by the cell or plasma membrane.
The outer portion of the cytoplasm, called the ectoplasm, is generally more finely granular and of a stiffer consistency than the inner portion, called the endoplasm. It is familiar to all who have studied plant and animal cells that they are diverse in size and form.
A brief summary of the range in size of cells is given in Table 2.1. It is of moment, perhaps, to point out that cells are examples of a remarkable degree of miniaturization. Thus the human sperm carries in its minute mass half of all the genetic determinants of a mature individual.
Which might appear to be exceptions to miniaturisation, contain a vast store of nutrient for development of the embryo. The Valonia cells have a large central sap vacuole as well as a massive cell wall. In both cases the nucleus and cytoplasm represent a very small part of the total cell mass. Although diverse in size and form, cells have a similar intracellular organisation and usually possess the same formed structures or organelles.
The presence of most of the organelles in all cells suggests that each organelle performs some fundamental role in the economy of the cell. Determination of the functions of these organelles is one of the ultimate aims of the cell physiologist.
Term Paper # 2. Organells Seen in Fixed and Stained Cells:
Under the microscope, a nucleus is seen in every stained cell although in bacteria and blue-green algae it lacks a membrane and is less well organised than in true cells of higher forms of plant and animal life. Within the nucleus a nucleolus is commonly present and so is a linin network bearing the chromatin. Between these materials is the nucleoplasm.
The entire nucleus is surrounded by a nuclear envelope. Just outside the nucleus of animal cells is a centriole, a small granule which separates into two before mitosis (cell division). At mitosis the two centrioles move apart and form the poles of the spindles and the centers of the asters.
Scattered in the cytoplasm are mitochondria, or chondriosomes, which have been seen in many kinds of cells examined. The appearance of mitochondria depends upon the handling of the tissues before preparation; they may appear as long rods, short rods or small granules.
The Golgi complex, named after its discoverer, is particularly prominent in nerve and secretory cells of animals and seems to be closely associated with secretory activities. In fixed and stained preparations it appears to be a canalicular system closely associated with the mitochondria. It is absent from bacteria and blue-green algae. Its true structure and function has been the subject of long controversy which is not yet resolved.
Lysosomes are membrane-bound organelles containing hydrolytic enzymes. It is likely that the cell interior is protected from the enzymes by such packaging. The packaging may result from the formation of the enzymes on the surface of the endoplasmic reticulum and accumulation within it. Plastids are cytoplasmic organelles which may or may not contain pigment (for example, chlorophyll is the green pigment in chloroplasts, one of the kinds of plastids in green plants).
Vacuoles, such as the contractile vacuoles of protozoans, and the large central sap vacuoles which are characteristic of plant cells, are often seen in cells. When a cell is cleared by centrifugation of visible granules, oil globules and various organelles, a clear matrix material remains which is thought to represent the fundamental architecture of the cell.
In fixed and stained preparations under the ordinary microscope this substance is devoid of structure, but under the electron microscope, which resolves much finer detail, the matrix material appears to contain a network called the endoplasmic reticulum. Upon this reticulum can be seen fine granules called the ribosomes.
Intercellular cytoplasmic connections called plasmodesmata may be found between cells in some tissues. It is of interest to note that although a cell contains but one nucleus it may have about 500 mitochondria, 5 × 105 ribosomes and probably 5 × 108, enzyme molecules.
A green plant cell will probably have about 50 chloroplasts. This multiplicity of units is a characteristic feature of cell organisation. Even the nucleus of a diploid organism has two chromosomes of a kind and two genes of a kind, one in each of the members of a homologous pair of chromosomes.
The possibility was suggested that the fixing process, by precipitating the proteins, produces artificial structures, or artifacts, which do not represent the true structures of the cell.
At the turn of the century Hardy and Fisher showed that some of these organelles could be duplicated in gelatin by adding to it such fixing agents as tannic acid, chromic acid or the salts of heavy metals. Therefore, it became necessary to determine whether the structures observed in fixed cell preparations were actually present in living cells. Many new and ingenious techniques were devised to demonstrate them, and the evidence for each organelle follows.
Term Paper # 3. Experimental Evidence for Cell Organelles in Living Cells:
When a living cell is examined under an ordinary light microscope, its main organisation (nucleus, cytoplasm, plastids, granules, edges of membranes) is visible in outline because of differences in refraction of light by these structures.
When the living cell is centrifuged and observed during the process through the centrifuge microscope, the organelles, differing in specific gravity and size, are seen to move as discrete units.
For example, in centrifuged eggs of the sea urchin Arbacia, the red pigment granules are thrown to the bottom of the cell; above them the yolk is deposited and then a layer of mitochondria, and finally the oil droplets are collected at the top of the egg, forming an oil cap. Between the zone of the mitochondria and the oil cap is a clear zone of fine granular protoplasm called the ground substance, within which the nucleus of the egg comes to lie.
Clearly, the specific gravity of the nucleus is less than that of some of the other cellular constituents. The ground substance represents the cytoplasm cleared by centrifugation of the structures suspended in it. No structure in the ground substance of a living cell is visible under the ordinary light microscope, the ultraviolet microscope or the phase microscope.
However, if cells are first fragmented and then centrifuged, the fragments of higher specific gravity settle more rapidly than those of lower specific gravity, other factors being equal, and larger particles settle more rapidly than smaller ones of the same specific gravity. The various particulates within the cell can then be segregated from one another. Large structures such as nuclei are readily separated from other constituents, as are also mitochondria and large granules.
After all these structures have been removed, minute submicroscopic bodies remain which can be centrifuged down at very high speeds. These are called the microsomes, and some of them are ribosomes, the granules seen under the electron microscope in the endoplasmic reticulum. Others are fragments of the endoplasmic reticulum.
The Nucleus and Its Constituents:
The internal organisation of the resting nucleus in a living cell is difficult to discern by ordinary microscopy, and even during mitosis and meiosis the chromosomes are seen only under rare circumstances. However, under the phase microscope, where slight differences in refractive index between diverse cellular constituents are exaggerated, the chromosomes of some cells may be seen quite clearly.
In fact, time- lapse photography permits continual observation of a single chromosome preparation throughout a cycle of meiosis or mitosis, corroborating what was previously deduced from a random sampling of many fixed and stained preparations. Furthermore, chromosomes are seen without the distortion which usually attends fixation.
Time-lapse photography makes evident not only the sequence of events, but also the dynamic nature of the process-the movements of cell organelles such as mitochondria, and the changes in shape of the entire cells.
Chromosomes are also discernible in ultraviolet photomicrographs of cells in mitosis or meiosis, because the chromosomes absorb short wavelengths of this radiation more strongly than does the cytoplasm. Much has been learned by the use of this technique but it is not as generally useful as phase microscopy because cells are damaged by such radiation, and prolonged examination is thus impossible.
Chromosomes when visible may be reached by micromanipulator needles and moved about in the nucleus or in the cell, indicating that they are discrete units. Moreover, fragmentation and irregularities in the partition of chromosomes can be observed in cells irradiated with ionising radiation.
Chromosomes have also been released from a suspension of nuclei (obtained by fractional centrifugation) and they maintain their identity through a variety of manipulations. At present no doubt exists that chromosomes are real structural units, even though they are difficult to identify in the resting stages of cells.
Mitochondria are visible in living cells under the phase microscope. They are seen to undergo active movements during mitosis and meiosis. Mitochondria may also be seen in living cells stained with the vital dye Janus green B, which though not entirely harmless, permits the cell to remain alive for a long time so that one can observe how the mitochondria change in shape and size as the conditions vary.
Further evidence of the identity of mitochondria is provided by their movement as units during centrifugation of a whole cell and by their discreteness after cell fragmentation. They may be separated from other particles in fragmented cells by washing in salt solutions followed by differential centrifugation.
In suspension they maintain their identity if adequate conditions are provided. Such suspensions of mitochondria have been subjected to biochemical analyses, and many enzymes concerned with aerobic respiration, energy transfer and storage have been identified in them.
Perhaps for this reason mitochondria are concentrated in regions of greatest cellular activity- at the secretory surface of a glandular cell, near the nodes of a nerve cell at which propagation is believed to occur and in active muscle fibers. In these locations mitochondria might supply most readily the high energy compounds needed for the activities of the cells.
When cells divide, the mitochondria have been seen to orient themselves relatively symmetrically on each side of the division figure, partition between the two cells being fairly equal. In some cases mitochondria have been seen to fragment after division. It is possible that the fragments then increase in size, thus reconstituting the amount of mitochondrial material needed in each daughter cell.
However, it is thought that mitochondria may also develop from minute bodies in the cytoplasm. Mitochondria have been shown to contain deoxyribonucleic acid different from that found in the nucleus, which suggests that they may be self-duplicating systems.
b. The Golgi Complex:
In the living cell under favorable conditions the Golgi complex may be stained with methylene blue and observed for a limited time. It is also visible by means of phase microscopy. It seems to be closely connected to the endoplasmic reticulum but its outlines are smooth (ribosomes being lacking) and it is chemically somewhat different from the reticulum, containing much more lipid.
It can be separated from other constituents of the cell by differential centrifugation. Some question exists whether the Golgi complex really constitutes a true organelle. Cytologists are inclined to look upon the Golgi complex as an assembly area for different kinds of secretions, especially lipids, rather than as a structural unit. Into the Golgi area flow high energy compounds from the mitochondria and various secretions, probably including nucleic acids, from the nucleus.
By gently centrifuging certain kinds of cells lysosomes have been separated from other cell organelles and their enzymes studied, thus showing them to be true cell organelles.
The identity of chloroplasts as discrete structures has not been doubted because they are so readily seen in living cells of green plants. They have been separated by maceration of cells and fractional separation of the constituents. The internal structure of chloroplasts has also been studied by electron microscopy.
Chloroplasts have been found to contain deoxyribonucleic acid (DNA) different from that of the nucleus. Evidence has also accumulated that chloroplasts are self-replicating organelles, probably as a consequence of their extra nuclear DNA.
Other plastids, however, have been less thoroughly studied, perhaps because their functions in plant cells are not as clearly evident as those of the chloroplast. Leucoplasts, within which starch grains are formed, are organised bodies in the cytoplasm and they contain enzymes which synthesize starch from the glucose formed during photosynthesis. Perhaps other synthetic functions are also localised in the plastids of plant cells.
e. Microtubules and Micro Filaments:
Recently electron microscopists have discovered microtubules and microfilaments in the ground substance between the cisternae (larger membrane-bound cavities) and tubules of the endoplasmic reticulum.
Because the microtubules occur in abundance in cells maintaining an odd shape, as in the case of the vertebrate red cell, they are thought to be structural elements. But they also appear to be important in cell division. Microfilaments occur in motile cells such as the Plasmodium of the slime mold and may function in movement.
f. Miscellaneous Cell Inclusions:
Occasionally oil droplets and oil globules are seen in the protoplasm, particularly in cells in tissue culture, in marine eggs and in marine protozoans known as radiolarians. These oil droplets are probably stores of nutrient or in some cases devices for during the specific gravity of the cell, thus making floating easier. In some cells glycogen granules occur, presumably representing stores of nutrient for the cell.
At present, workers have accepted the ability of the major cell organelles and are now concerned mainly with determination of their chemical constitution, emicaliemical organisation and function.
Term Paper # 4. Variation in Organisation of Animal Cells:
In multicellular organisms, all of the cells are not exactly alike. They have become differentiated and adapted to perform some particular function of the organism.
A tissue is a mass of similar cells, usually continuous, held together in a supporting matrix (secreted by the cells), performing a common function and usually forming a part of an organ. Intercellular fluid is almost always found in tissues.
This is to be expected since every cell must have water frontage from which it obtains supplies and into which it pours its wastes. But because this intercellular fluid is not easily removed or modified, both plant and animal tissues, when used in experiments cannot be considered the equivalent of suspensions of cells.
Animal tissues are usually classified as:
d. Nervous and
Epithelial cells are generally closely packed, like bricks in a pavement, and form solid protective layers on the outside. They line external surfaces, cavities and tubules, produce secretions and proliferate cells. Epithelial cells may be isodiametric (having equal diameters in all directions), flattened, or elongated and columnar (often the columnar cells are glandular).
From the epithelial cells of glands exude various secretions. Germinal cells, which give rise to gametes, spring from the epithelial cells of gonads. In epithelial cells are found the typical organelles expected in a cell. In glandular epithelial cells the mitochondria and Golgi complex are likely to be particularly well developed. Although epithelial tissue consists primarily of cells, the external epithelium may secrete a cuticle, as in the earthworm, or a skeleton, as in arthropods.
Connective tissue, in contrast to epithelial tissue, is to a large extent composed of extracellular material and may contain relatively few cells. Some examples of connective tissues are cartilage, tendon, bone and adipose or fatty tissue.
During early development of the embryo the connective tissue cells lie close together, but during later development they secrete a gelatinous material (matrix) which separates the cells. In this gelatinous matrix are laid the fibers of connective tissue and the salts of bone. The chemical composition of connective tissue in tendon is diagrammatically represented in Figure 2.4.
In muscular tissue (smooth, cardiac or skeletal) the cells are considerably modified for the function of contraction. They are elongated cells and in all cases contain contractile fibrils, these being characteristic of muscle tissue and of no other tissue. The cells of the smooth or visceral musculature of vertebrates are spindle-shaped units containing a single nucleus in the center and have fine elongate fibrillae which represent the contractile elements.
The skeletal musculature of vertebrates is highly differentiated for its function. Its unit of organisation is the muscle fiber, consisting of the confluent bodies of numerous cells, each of which is indicated by a spindle-shaped nucleus. Elongate striated fibrils pack the cytoplasm of the skeletal muscle fiber, and large mitochondria are present; they are especially conspicuous in the highly active flight muscles of vertebrates and insects.
In the cardiac muscle of vertebrates, striated fibers are found and the unit is modified in organisation from that of skeletal muscle. Muscular tissues of invertebrates are very diverse, but in all cases contractile fibrils are found.
The cells of nervous tissue are highly specialised for conduction of nervous impulses. From the nerve cell body containing the nucleus extend the long fibers called axons and the short fibers called dendrites. The nerve cell with all of its processes is called a neuron. A well-developed Golgi complex appears in the cell body of a neuron, and mitochondria occur chiefly at the synapses.
Granular deposits of nucleic acids which probably serve as nutrient in the activities of the nerve cell are found in the fibers. The axon of a vertebrate nerve cell is called a myelinated fiber when it is covered with an envelope consisting of many layers of apposed cell membranes and enclosed by sheath cells.
The myelin sheath may add much bulk to the vertebrate nervous system and, because of it; nervous tissue like the brain contains a considerable amount of extracellular material. However, the outgoing fibers from autonomic nerve ganglia located outside the cord are not myelinated. Invertebrate nerve fibers have little if any myelin.
Blood also has cells that are highly specialised. For instance, in vertebrate blood the red cells (erythrocytes) are mainly composed of the oxygen-carrying pigment, hemoglobin. In mammals the mature erythrocytes even lose their nuclei and have none of the characteristic cell organelles.
The white cells of vertebrates and invertebrates-both granular amoeboid cells called leukocytes and the more or less spherical clear lymphocytes-are much more typical cells than the red cells since they have nuclei and the other cell organelles such as the mitochondria and the Golgi complex.
Term Paper # 5. Variation in Organisation of Plant Cells:
Unlike animal cells, plant cells usually possess plastids and vacuoles and are covered by a cell wall. Mitochondria, ribosomes and a nucleus are present as in animal cells. Plant cells have structures called dictyosomes which are similar to the Golgi complex of animal cells.
Little agreement exists on classification of plant tissues, but the following might be cited as one possible classification:
c. Vascular (xylem and phloem),
d. Sclerenchyma and
e. Supporting or bast fibers.
The cells of epidermal tissues of plants line external surfaces of plant structures. Embryonic cells of plants, e.g., the cambium, are made up of cells similar in appearance and function to epithelial cells of animals. Undifferentiated plant cells, called parenchyma, exist in large numbers in the cortex of the stem, filling the spaces between conducting or supporting tissues.
They also make up the mesophyll and the palisade cell layer of the leaf, in which photosynthesis occurs. Vascular tissue of plants consists of the phloem and the xylem. The phloem is constituted of elongated living cells which abut upon one another. It distributes the manufactured food throughout the entire plant. The xylem consists of tubular remains of cells; it distributes water and salts to the cells of the plant. In the adult plant the xylem ducts are entirely extracellular, the cellular material having disappeared.
Cells with thickened walls, like the gritty stone cells in the pear, are representatives of sclerenchyma tissue. Supporting “cells” or bast fibers, consisting largely of secretions of cellulose and lignin, occur along the vascular tissues and contribute to the skeleton of the plant. Supporting fibers in the adult plant are entirely extracellular.
As a result, an organ that contains an abundance of ducts and supporting fibers, like a mature stem, has much extracellular material. In contrast, a bud or a leaf is made up largely of relatively undifferentiated cells, and, except for the cell walls, consists of living cells.
Unfortunately, no convenient summary of the intracellular and extracellular constituents of plant tissue, comparable to that for animal tissues, is available, although it would be desirable since plant tissues are extensively used in cellular research.
Term Paper # 6. Variation in Organisation of Bacterial Cells:
Bacteria and blue-green algae, unlike all kinds of true cells of plants and animals, lack a distinct nucleus enclosed by a nuclear envelope, the nuclear material being scattered in the cytoplasm. The blue-green algae differ from bacteria in that they contain chlorophyll and a characteristic auxiliary pigment and differ from most bacteria in being photosynthetic.
Plant and animal cells with true nuclei are called eukaryotic cells and bacteria and cells of blue-green algae are called prokaryotic cells. Also, prokaryotic cells either lack all the other cell organelles (so characteristic of eukaryotic cells) or possess them in a primitive, undifferentiated condition. Because bacteria have been extensively investigated, they alone are considered here, but much of what is said is applicable to blue-green algae as well.
Bacterial cells are covered with a protective cell wall which usually consists of carbohydrates and polypeptides but may contain protein and lipids. The bacterial cell can be made to retract from this wall if it is placed in a concentrated solution of sugar or salt, so that the cell membrane (plasma membrane) now forms the outer boundary of the living material of the cell called the protoplast.
The cell wall may be digested with the enzyme lysozyme, exposing the protoplast which can now be made to swell or shrink like any other cell by decreasing or increasing the concentration of salts dissolved in the medium. The cell wall evidently plays some important role in addition to protection, since protoplasts divested of the cell walls do not divide or form new cell walls.
Some bacteria, e.g., Bacillus megatherium, called gram-positive bacteria, have ribonucleic acid present in the surface which takes up a stain made up of crystal violet and iodine. Bacteria which are gram- negative (e.g., Escherichia coli, the colon bacterium) differ in possessing cell walls heavily impregnated with lipids. Thick-walled resting stages called spores which resist high temperature and other unfavorable conditions are formed by some bacteria (bacilli).
Bacteria lack mitochondria and the Golgi complex although they generally are filled with bodies resembling the ribosomes of true cells. As in eukaryotic cells the ribosomes may cluster to form polyribosomes during the synthesis of proteins in bacterial cell homogenates.
The plasma membrane, which is rich in oxidative enzymes, probably performs the function usually carried out by the mitochondria of plant and animal cells. It is of interest that the plasma membrane of the large bacterium Thiovulvum majus sinks deep into the cytoplasm, forming a multilayered structure called the desmosome. In the tubercle bacillus, concentric membranes, suggesting incipient mitochondria, have been discovered in the cytoplasm with the aid of electron microscopy.
Many bacteria have been shown to possess large granules (round bodies) which grow and divide and are stained with the same dyes which affect chromosomes of true cells. These bodies appear to condense before division and become more diffuse thereafter.
Rickettsias, which cause Rocky Mountain spotted fever, endemic typhus and murine typhus, are of about the size of small bacteria and in general their organisation clearly places them among the bacteria. However, to some workers they appear to be escaped mitochondria and to others, a stage between viruses and bacteria. Like bacteria, they possess a number of respiratory enzymes which are lacking in viruses. They have not yet been cultured except in cells of a host they infect.
Term Paper # 7. The PPLO (Pleuro-Pneumonia Like Organisms):
The smallest bacteria, and the smallest cells known, are found in the group of organisms known as the pleuro-pneumonia like organisms, also known as the PPLO (Mycoplasmataceae). Mycoplasma laidlawii, a free-living strain growing in sewage, is the smallest cell so far studied. It shows a definite life cycle. In culture it exists in bodies of three sizes, minute elemental bodies 0.1 µ in diameter, intermediate size bodies and full-size cells about 1 p. in diameter.
Studies indicate that the small bodies develop into the large cells, which can also divide to form more of their kind. Mycoplasma gallisepticum, which causes chronic respiratory disease in poultry, is somewhat larger (about 0.25 p) than M. laidlawii and has been especially useful in biochemical studies. Some of the chemical constituents are similar to those found in bacteria; others are like those found in animal cells.
Of the nucleic acids found, some are linear and double-stranded (deoxyribonucleic acid), and others are in the form of spheroids combined with protein and resembling the ribosomes of other cells. Among the proteins are some 40 different kinds of enzymes, including those that catalyse the oxidation of pyruvic acid to carbon dioxide and water, reactions that liberate much energy and are found in a wide variety of cells, bacterial, plant and animal.
The lipids found include cholesterol and cholesterol esters, both typical of animal cells. The plasma membrane is flexible and electron micrographs disclose it to be a “unit membrane” similar in dimensions to the unit membrane of cells in general.
Term Paper # 8. Organisation of Viruses:
Some viruses are larger than bacteria; for example, the psittacosis virus measures 0.75 p in diameter whereas a pleuro-pneumonia bacterium measures only 0.25 ft in diameter. However, most virus particles are smaller than bacteria; the virus causing hoof and mouth disease is estimated to be only 0.01 p in diameter. The distinguishing character of viruses is that they are invariably intracellular parasites of specific hosts and none are known to be free living.
Viruses attack bacteria, plant and animal cells. This is probably a consequence of their lack of energy-yielding and synthetic enzyme systems. The only enzymes demonstrated in viruses enable the bacterial virus to enter a bacterium, presumably by digesting the cell wall.
Most viruses consist of a protein coat over a nucleic acid core, but lipids and lipoproteins have been claimed from some animal viruses. Viruses contain only one kind of nucleic acid, ribonucleic acid in plant and animal viruses, and deoxyribonucleic acid in bacterial viruses, whereas animal and plant cells have both.
In general, animal viruses appear to be of a more complex nature than viruses of plants and bacteria (bacteriophages), but less is known of them because they present so many experimental difficulties. All viruses lack cell membranes delimiting them from the medium.
They also lack the other organelles characteristic of bacteria, including cell walls, and thus viruses represent an entirely different category of organisation. Most extensively studied are the viruses which attack bacteria and are called phages, or more properly, bacteriophages (bacteria-eaters as the name implies).
Only the nucleic acid portion of a phage enters (infects) a bacterium, as can be shown by radioactive markers on the nucleic acid (32P) and on the protein (35S). After the phage nucleic acid enters the bacterium, the suspension of bacteria and phage can be agitated violently to detach the phage particles.
Infection occurs nonetheless, even though it can be demonstrated that only about 1 per cent of the labeled phage protein still adheres to the bacteria. Furthermore, nucleic acid-free “ghosts” containing all of the protein of bacteriophages are not infectious to bacteria although they attach themselves firmly.
The bacteriophage nucleic acid particle, once inside the bacterium, loses its identity (eclipse period), but after half an hour phages can again be identified (by electron microscopy). It can be demonstrated by various labeling techniques that, once inside, the nucleic acid of the phage takes command of the synthetic activities of the bacterium, suspending the normal synthetic activities of the bacterium, hydrolysing its nucleic acids and directing the synthesis of the virus type of nucleic acid and protein.
The protein coat of the virus is synthesized anew from materials taken in by the bacterial cell. About 10 minutes after infection, packaging of the virus nucleic acid in protein coats begins (as shown by electron micrography in disrupted virus-infected bacteria) and continues for another 20 minutes, when the process is complete. At this time the bacterium is ruptured (lysed), liberating between 40 and 100 or more complete phage particles.
The effect of a bacteriophage in a suspension of bacteria (in vitro) plated on nutrient medium can be detected by spots of lysis-clear areas produced by liquefaction of the layer of bacteria. This occurs only after a number of life cycles of the phage.
The number of particles or the titer in the initial suspension can thus be assayed by the number of lysed spots.
Phages may affect bacteria in either of two ways-they may grow and lyse the bacteria as just described, (phages that cause lysis are called virulent or lytic phages), or, they may become incorporated into the genetic material of the bacterium and remain in an inactive “masked” form.
That they have altered the hereditary characteristics of the bacteria is shown by the resistance (immunity) of the bacteria so infected to attacks by homologous phages which would otherwise lyse these bacteria. Phages that “hide” in this manner, dividing only when the bacterium divides, are spoken of as temperate or lysogenic phages (i.e., giving rise to lytic phages). An occasional masked lysogenic phage particle multiplies, perhaps because its host has become weakened and no longer holds it in check.
Damaging the bacteria infected with lysogenic phages by radiations or other unfavourable agents leads to a rapid development of the phage into an active lytic form. The particles are then released in infection form. Lysis of an occasional bacterium in an undamaged population can spread the infection to many previously uninfected members of the population, in this way maintaining a mild infection. Lysogeny, as the phenomenon is called, indicates the intimate relationship formed by the phage and the bacterium, a conception having great influence on microbial genetics.
Term Paper # 9. Staining Technique for Living and Fixed Cells:
Vital dyes penetrate living cells and colour certain structures without seriously injuring the cells. For example, neutral red may be used to stain the cytoplasm, although in some cells it accumulates in vacuoles instead. Janus green B stains mitochondria selectively. Methylene blue selectively stains the Golgi complex.
Vital dyes are not entirely harmless, but they kill only after cells have been exposed to them for a long time, thus allowing time for study. Sometimes the poisonous constituent (e.g., the heavy metals in Janus green B) may be removed from a vital dye solution, making the dye much less toxic than the original sample. Although helpful, the vital dye technique has only limited use because many of the cell organelles are not stained by such dyes and, in all cases, scattering of light obscures boundaries of structures.
However, dyes have been used to demonstrate that some cell organelles such as the mitochondria, Golgi complex and vacuoles are real structures present in the living cell. Most of the early work on the nature of cell organelles was done with fixed and stained preparations. Fixing agents, such as formalin, alcohol, acids, salts of heavy metals or mixtures of these, precipitate the proteins and render them insoluble.
Next, water is removed from the fixed tissues by dehydrating agents, such as, alcohol, and the tissues are embedded in paraffin and sectioned with a microtome. The sections are affixed to slides and the paraffin is removed with xylol and washed in xylol-alcohol. By washing in decreasing concentrations of alcohol the sections are partially hydrated and the proteinaceous material of the cell is then differentially stained to distinguish the structures present.
Natural dyes (e.g., hematoxylin) or basic anilin dyes (e.g., safranin and basic fuchsin) stain the nucleus selectively, and acid dyes (e.g., orange G, eosin and fast green) stain the cytoplasm. The sections are then dehydrated with alcohol. To reduce scattering of light, it is necessary to replace the alcoholic medium with a substance having the same refractive index as that of the protein particles.
This is accomplished with a clearing agent, such as xylol, which infiltrates among the protein particles. The preparation is then mounted in balsam, which has a refractive index about equal to that of the cell proteins. As a result, it is possible to look at the cell and see clearly the stained structures that are within.
Cytochemical techniques for identification by colour reactions of cell constituents and enzymes. Some cytochemical techniques have been developed at the electron microscope level.
Term Paper # 10. Auto Radiography:
If a cell is suspended in a solution containing a substance labeled by a radioactive isotope, which the cell is capable of incorporating, it is often possible to determine where in the cell incorporation occurs and to what extent. For example, 3H-uridine is presented to cells for a brief time and the cells are mounted on a slide and fixed. A layer of photographic emulsion is deposited over the preparation.
The radioactive emission from the 3H-uridine will affect the silver halide in the emulsion. On development, the grains affected by the radiation (beta particles of low energy value) are reduced and appear as black grains or traces over the structure into which the 3H-uridine had been incorporated. If the grains appear over the nucleus we know that the nucleus incorporates uridine into ribonucleic acid (RNA).
Similar experiments can be performed with 3H-thymidine, which is selectively incorporated into deoxyribonucleic acid (DNA), and with 3H-amino acids, which are incorporated into proteins. Autoradiography has been of inestimable value in localising the sites of a variety of syntheses in the cell.
Term Paper # 11. Microscopical Techniques to See Stained Structures in Cells:
The ordinary light microscope makes it possible to see stained structures so long as they are larger than the limit of resolution.
The smallest object, d, resolved (seen clearly and distinctly from similar sized objects separated by like small distances) by the light microscope may be determined from the following equation:
d = [λ/(NA + na)]…… (2.1)
In this equation A is the wavelength of light used, NA is the numerical aperture of the objective and na the numerical aperture of the condenser.
Since for critical study the numerical aperture of the condenser is chosen equal to that of the objective, the equation is often written:
d = λ/2NA ……….. (2.2)
The numerical aperture is given by the equation:
NA = n sin α .………. (2.3)
Here n is the refractive index of the light path in the formation of the microscopical image, and α is half the angular aperture of the light beam entering the condenser. When immersion oil is used between condenser and slide, and slide and objective, the numerical aperture may be as large as 1.6, although in practice it is seldom over 1.4.
The factor that limits resolution with the light microscope is therefore the wave length of light. Under optimal conditions the smallest object seen is theoretically about one-third the wavelength of visible light or about 0.13 p in blue light.
Usually the limit is set at about 0.2 p because the eye is most sensitive in the yellow green part of the spectrum (0.55 p) and a maximum numerical aperture of 1.6 is seldom achieved. An object of this size is visible only if the structure shows maximum contrast. In order to increase the contrast between unstained structures and to obtain greater resolution of details of cell organelles, various types of microscopes have been developed, each of which will be considered in turn.
The dark field microscope is an ordinary microscope with a special condenser. By use of a dark stop in the condenser the light from the center of the field is removed and the object is illuminated only by an oblique beam of light as seen in Figure 2.7.
The object is therefore seen by light reflected and scattered from interfaces in the object. With dark field microscopy the outlines of the cell, the nucleus, mitochondria, oil droplets, vacuoles and various inclusions may be readily identified. Moreover, in cells undergoing division, the centrosomes, asters, spindles and chromosomes may be observed. Objects smaller than those seen with the ordinary light microscope may be detected with dark field microscopy but not resolved.
The polarising microscope is like a light microscope except that the condenser and the ocular are equipped with polarising optics, each of which transmits only plane-polarised light. The condenser is then spoken of as the polariser and the ocular as the analyser.
If the optics of the condenser and ocular are crossed (at 90° to one another) no light is transmitted unless an interposed object rotates the plane of the light. The manner in which this occurs and the usefulness of the microscope in biology.
Light, passing through a doubly refracting crystal such as calcite, is separated into two rays which vibrate at right angles to one another. Such light is said to be polarised. The Nicol prism, one of the best known polarisers, is made by cutting a rhomb of Iceland spar (a pure, transparent form of calcite) in halves along the line of one of the optical faces, polishing the cut faces and cementing them together with Canada balsam.
As seen in Figure 2.8, unpolarised light entering one end is separated by the prism into two beams, one vibrating in the plane of the figure, the other at right angles to it. One of these beams is totally reflected by the layer of Canada balsam and is usually absorbed by the opaque case covering part of the rhomb.
The other beam is transmitted. Hence, a Nicol prism transmits plane-polarised light. If a second Nicol prism is used in series with the first, and in the same position, the polarised light is transmitted. If, however, the second prism is rotated through 90° in relation to the first prism, the light fails to pass.
But if an object like quartz, which polarizes light, is placed in the beam of light between the two prisms, and if the two Nicols are at right angles to each other (crossed), instead of a plain dark field, a brilliantly lit object is seen against a dark background, indicating that the object has polarised the light passing through it.
If the second prism is then rotated away from 90°, the field suddenly becomes dark. The angle required to rotate the second prism in order to extinguish the light is a measure of the degree to which the plane of polarised light has been rotated by the interposed object. The second prism is called an analyser because by moving it one can analyse the degree to which the plane of polarised light has been rotated by an object inserted in the light path.
Various natural substances are observed to be isotropic; that is, light passes through them equally well in all directions. Others, which affect the passage of light unequally in different directions, are anisotropic or birefringent. The ray passing directly through a crystal is called the ordinary ray; the ray which is deviated is called the extraordinary ray.
If the velocity of the extraordinary ray passing through such a material is less than the velocity of the ordinary ray, the crystal is said to be negative (e.g., quartz). If the extraordinary ray has a velocity greater than the ordinary, the crystal is said to be positive. Crystalline or intrinsic birefringence is found where the bonds between molecules (or ions) have a regular asymmetrical arrangement. The birefringence in this case is independent of the refractive index of the medium.
Dichroism occurs when the absorption of a given wavelength of polarised light changes with the orientation of the object. For example, the degree of absorption at wavelength 260 nm. by nucleic acids in cells depends upon whether the light is parallel to, or at right angles to, the oriented purine and pyrimidine residues. Dichroism is a sensitive indicator of orientation of molecules in a structure.
However, it is seldom seen in biological objects in the visible spectrum except in some stained preparations where it results from orientation of dye molecules. It may be seen at 260 nm. in the sperm nuclei where the concentrated nucleic acids appear to be oriented.
Birefringence of inanimate objects, then, is the result of their crystalline nature. Birefringence of biological materials indicates that their molecules are oriented with respect to one another much as in a crystal. Some particles-for example, nucleoprotein molecules-line up only if the solution is flowing; what they show is therefore called flow or streaming birefringence.
This indicates that the molecules are elongate and line up in the path of least resistance to a current, just as sticks line up in a brook. Organisms are more likely to show form birefringence, because of regular orientation of tiny particles (rods or planes) differing in refractive index from their surrounding medium. Birefringence in such cases disappears when the material is immersed in a medium of the same refractive index.
Oriented rods have different refractive indices for directions parallel to and at right angles to the major axis. Therefore, by making observations with polarised light one may determine in which direction in a given structure the rods lie.
Polarising optics has been especially useful in study of biological membranes and of the division figure in cells. They are also useful in studying fibrillar structures.
The phase contrast microscope, which has the same resolving power as the ordinary light microscope, enables one to see structures in the living cell by taking advantage of the slight differences in refractive index between any two structures to improve their visibility.
The phase contrast microscope does this by the use of a special diaphragm in the condenser and a phase plate in the objective. The annular diaphragm used in the condenser of the phase microscope permits light to pass through the condenser as a hollow cone, the remaining light being absorbed. This cone is focused on the object.
The phase plate placed at the back focal plane of the objective is a transparent disk containing a groove (or elevation) of such a size and shape as to coincide with the direct image of the sub stage annular diaphragm formed at the back focal plane when no object is viewed.
If an object is placed between the condenser and the objective, in addition to the direct image a number of overlapping diffracted images of the diaphragm then appear at the back focal plane of the objective.
The depth of the groove (or the elevation) in the phase plate of the objective is so made that the two sets of rays forming the direct image and the diffracted image differ in optical path by a quarter wavelength of the illuminating beam of light. Under these conditions the phase difference, which is not seen by the eye, is converted to an intensity difference which we see.
In the bright contrast phase optical system the two sets of rays are added to make a brighter image, whereas in dark contrast phase they partially cancel one another making a more contrasting darker image.
The phase contrast microscope (bright or dark contrast may be used), in which a small difference of refractive index is exaggerated, enabling one to distinguish adjacent structures, has made it possible to observe structures previously very difficult or impossible to see. The behavior of chromosomes of living cells during mitosis or meiosis can be followed with ease, and many of the other organelles may be studied.
The phase contrast microscope has furnished the most convincing evidence that many of the organelles identified in fixed and stained preparations of the cell are real and not artifacts. However, the images are often surrounded by a halo and the difference in intensity between two areas of unequal refractive index diminishes with the distance from their boundary, thus causing some distortion.
The interference microscope avoids the residual halo which accompanies the image seen under a phase microscope because the aperture of the objective of such a microscope is not limited by a phase plate and the same area of the objective is used for both interfering beams. The relative phase of the two beams is varied continuously so that any part of an object can be given maximum or minimum contrast at will.
A horizontal glass plate, L, nearly parallel to a similar plate, U, above the object, intercepts the illuminating cone of light from the condenser (both surfaces of this plate are partially aluminised).
A portion of the illuminating cone passes through the plate and is focused on the object, and a portion reflected downward at the upper surface plate L and is then reflected upward its lower surface. Although, only one beam traverses the object, two beams averse the object plane. The beam fail; to pass through the object, called the comparison beam, is internally reflected ice within the plate. It will be noted at it has a diameter much larger than at of the field.
A second horizontal plate, U, identical to the first and just about parallel to it, is located above the plane of the object. Both of its surfaces are also partially aluminised, but in addition its upper surface has an axial opaque spot somewhat larger in diameter than the maximum field of the microscope.
This spot prevents the direct vertical light beam from the source which has passed through the object from reaching the objective, but allows the light from the beam which has traversed the object and then enters the plate to the side of the spot to reach the objective.
Part of the illuminating beam which has traversed the object is reflected twice in plate U and part of the comparison beam traverses this plate without reflection; the two beams will emerge from the upper surface in coincidence when the plates are correctly aligned.
The observer above the upper plate sees two coincident fields of view, one an image of the light source only and the other an image of the light source upon which is superimposed an image of the object. Aside from the interference resulting from passage through the object, the phase difference between the two images will be zero.
The combined images may be viewed by a normal microscope objective. The interferometer plates L and U are movable so that the path difference between the two coincident fields can be varied at will over the whole area of vision. Maximum contrast cannot be obtained for objects much larger than the field of view, because the object is then traversed by a portion of the background beam.
Because an object such as a cell contains various substances, it has an optical path different from that of the surrounding water. For this reason when the background is adjusted to maximum light intensity (bright field), the cell appears darker and it is possible at a glance to note the distribution of structures in the cell much as if a stained preparation were being observed in an ordinary light microscope.
More than this, it is possible to determine the average amount of dry material (dry mass) present in a given area of the cell by the use of the interference microscope, because the greater the amount of material present in the cell, the greater will be the difference between its optical path (refractive index times thickness) and that of the surrounding fluid.
The difference in optical path is determined by the difference between interference bands in the surrounding fluid and in the object when the upper wedge plate is rotated so that the wedge axes are no longer parallel. The field is then crossed by parallel interference fringes.
These fringes are seen as lighter and darker bands of the same colour when monochromatic light is used or bands of colour on either side of white bands when white light is used. For example, one-tenth wavelength displacement for monochromatic green light (546 nm.) is equivalent to a dry mass of 10-13 gm/µ2.
The ultraviolet microscope looks like the ordinary light microscope but its lenses are made of quartz to permit transmission of wavelengths of invisible ultraviolet as short as 220 nm. (visible light covers the range from blue at 390 nm. to the far red at 780 nm.). The invisible ultraviolet light is then projected upon a screen which fluoresces in the visible spectrum.
Usually, however, it is recorded photographically. Because chromosomes absorb more short ultraviolet radiation than cytoplasm does, they are readily seen in photographs taken with ultraviolet light at 260 nm. Under the ultraviolet microscope resolution of fine structure is about twice that obtained with the ordinary light microscope, as may be judged from application of equations 2.1 and 2.2, the wavelength of short ultraviolet light being about half that of the visible. Unfortunately ultraviolet radiation damages living cells, so that prolonged observation of cells so illuminated is not possible.
An instrument which may serve as a valuable adjunct to all the microscopes considered here is the television camera attachment. Since the electron tube used in it “sees” at low light intensities and can be made sensitive to a restricted part of the spectrum (a narrow band in the green, blue or ultraviolet), much clearer pictures can be obtained than with ordinary microscopy.
Furthermore, a prolonged illumination is avoided since the image may be viewed on a phosphorescent screen which retains the image for a minute or two after exposure. Momentary illumination with light of low intensity suffices. This attachment, therefore, is especially useful for viewing living cells under ultraviolet light.
The electron microscope, which was developed on the eve of World War II, has proved a powerful tool for analysis of large chemical molecules, viruses and, to some extent, cellular structures. In the electron microscope the beams of electrons are focused by magnets which serve as lenses. The object must be viewed on a fluorescent screen or photographed.
The electron microscope has a resolving power 400 (theoretically 2000) times that of the light microscope. Although the wavelengths accompanying the electron beams are short (e.g., a 50,000 volt electron beam is accompanied by a wavelength of 0.05 Å), the numerical aperture of the electron microscope is only about 0.0005.
The image results from differential scattering of electrons from the molecular constituents of the cell. Scattering is greater the denser the material, regardless of its chemical composition. Even a gas scatters electrons; therefore, the biological object must be studied in a vacuum, since electron scattering from the gas molecules would obscure the differential scattering of the structures in the biological object.
The preparations must be dry because the water vapor in the evacuated preparation would have the same effect as a gas. A thin slice of the object must be used; otherwise the electrons would all be scattered and the electron micrograph would reveal no detail.
Since biological material varies little in density, it must be stained differentially, i.e., with some structures taking up more stain than others. Positive staining is obtained by fixing the cell in salts of heavy metals, e.g., osmium tetroxide or potassium permanganate.
In this case the electron-dense (i.e., opaque to electrons) metal adheres differentially to structures of the cell, causing them to scatter the electrons more than the surroundings. In other cases negative staining is used; that is, the object is infiltrated with a heavy metal compound such as phosphotungstic acid or uranyl acetate. These materials do not adhere to the biological structures but fill the interstices between them, making these areas electron-dense in contrast to the biological structures which are then seen as relatively electron-transparent structures.
The main disadvantages attendant upon use of the electron microscope is the required thinness of sections and the necessity of studying them dry (in a vacuum). The first difficulty has been overcome largely by the development of microtomes that can make sections 0.2 to 0.02 µ in thickness.
However, removal of water and salts dissolved in it as well as the use of stains is likely to alter structure; therefore, membranes, cytoplasm or formed structural components seen by electron microscopy must be interpreted with some caution. Notwithstanding these facts, it will become apparent in studies presented later that much has been learned about the cell by electron microscopy, and about structural elements such as the cuticle and the cell wall.
Because of the small contrast between some biological objects and the background even after staining, shadow casting with heavy metals is often used, especially to bring out three-dimensional topography. Heavy metals, especially gold, platinum and chromium are evaporated in a vacuum at an oblique angle to the object. The metal piles up like a windrow and outline the object. Shadowing may be performed from several directions by moving the object during the process of evaporation.
When the object is too thick for shadow-casting, a replica can be made. In this case the object, mounted on a grid containing holes, is subjected in vacuo to evaporation of platinum and carbon and becomes coated to the depth of several hundred angstroms. The preparation is then treated with hydroxide to remove the specimen and the replica is floated onto another grid and prepared for electron microscopic study.
Surface spreading has also been a useful technique for study of cell fine structure. The spread layer is “swept” in the trough to concentrate it, and mounted on a grid, dried and shadowed for electron microscopic study. In this manner a bacterial protoplast (bacterium from which the cell wall has been removed with the enzyme lysozyme) that is ruptured osmotically on the surface of a vessel filled with distilled water is found to liberate its chromosome as a single long fiber. The same technique has been used for a study of interphase chromosomes in cells of plants and animals and for a study of microtubules present in some cells.
The images of various cell organelles seen with the electron microscope have been criticised because they are structures seen in cells that have been fixed and stained. Are they real or are they artifacts introduced by the chemical procedures and drying?
It is disquieting also that different fixative sometimes gives different images; for example, osmium tetroxide fixation shows a plasma membrane consisting of a single line whereas permanganate fixation shows the plasma membrane consisting of two outer electron-dense layers and an inner less electron-dense layer.
It has proved possible to study the cell organelles by the use of unfixed cells for electron microscopy, in the method called freeze-etching. The object to be studied is placed in 20 per cent glycerol and is frozen at – 100° C. It is mounted on a chilled holder and splintered with a knife along natural cleavage planes, usually along surfaces of membranes. Occasionally the structure is cross-fractured, giving cross sections of organelles.
The splintered preparation is freeze-dried and covered with a platinum and carbon coating in the high vacuum of a freezing ultra-microtome. When the preparation is dry the vacuum is broken and the preparation is placed in water to float the replica off the carrier. The replica is then washed in basic solution to remove the cellular material and the replica is mounted on a grid and dried for electron microscopy.
Such replicas give the outlines of various cell structures and verify the structures seen in thin fixed and stained sections of the mitochondria, smooth and rough endoplasmic reticulum, double nuclear envelope with pores and plugs, unit plasma membrane of the cell, chloroplasts and so forth.
The views of structures are somewhat three- dimensional. Since some cells (e.g., yeast) subjected to these freezing and drying procedures revive and show no evident change in properties, it is assumed that electron micrographs of cells so prepared give images of cell structures in as nearly normal condition as is at present possible.
The fact that the structures compare favorably in size and appearance to those seen in fixed and stained cells is, in some measure, confirmation of the validity of the conventional method using fixatives.
In many cases considerable information has been obtained by time lapse photomicrography. Details that escape the observer on a single viewing become apparent after viewing the record repeatedly. Familiar examples are mitosis and meiosis in cells and irradiation of cells with a micro-beam. The components used in such studies are familiar.
Since, the end of the last century micromanipulators of one kind or another have been in use, first for handling single cells and later for microsurgery upon them. The pneumatic micromanipulator of De Fonbrunne has had wide use for microsurgery because of its ease of operation, speed and sensitivity of response.
Essentially it consists of three pumps two horizontal and one vertical, which connect by way of rubber tubes to the pneumatic capsules of the instrument holder (for needle or pipette). The movements in the horizontal plane of a single lever or joy stick are reflected at a ratio of anywhere from 1 : 50 to 1 : 2500, according to the setting decided upon. The vertical plane level is set by the screw on the lever.
An electric micromanipulator utilising thermal expansion wires has been described but has not had much use. Production of micro forges for the controlled manufacture of micro tools has also facilitated micro dissection. Important information on the function of the nucleus of cells has been gained by the use of such tools for transplantation of nuclei between cells.
The micromanipulator thus makes the microscope more than an observational tool, and properties of many of the cell organelles have been explored by its use. Even bacteria have been dissected under high power.